Characterisation of the Potential of Probiotics Or Their Extracts As Therapy for Skin

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Characterisation of the potential of probiotics or their extracts as therapy for skin

A thesis submitted to the University of Manchester for the Degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences

2014

Walaa Mohammedsaeed, Master of Science (MSc)

School of Medicine

Table of Contents

Contents Table of Contents ...... 2 Table of Figures ...... 5 List of Tables ...... 8 List of Abbreviations ...... 9 1 Abstract ...... 11 2 Declaration ...... 12 3 Copyright Statement ...... 13 4 Acknowledgements ...... 14 5 The author ...... 15 6 Publications arising from this Thesis ...... 15 Chapter One ...... 16 Introduction ...... 16 1.1Skin structure and barrier function ...... 16 1.2 Wounding and the immune response ...... 26 1.3 Probiotics ...... 40 1.4 Aims and Hypotheis ...... 55 Chapter Two ...... 57 Methods and Materials ...... 57 2.0 Reagents and materials ...... ….………..57 2.1 Bacterial cell culture ...... 57 2.2 Screening of inhibitory activity of L. rhamnosus GG ...... 60 2.3 Fractionation of the L. rhamnosus GG lysate by Reverse Phase Liquid Chromatography ...... 62 2.4 Mammalian cell culture ...... 68 2.5 Wound healing study in vitro and ex vivo models ...... 69 2.6 Microarray study ...... 75 2.7 Enzyme Linked Immunosorbent Assays (ELISA) ...... 77 2.8Statisticalanalysis...... 77 Chapter Three ...... 78 An in vitro investigation into the effects of species of Lactobacilli on toxicity of S. aureus to primary human keratinocytes ...... 78 3.0 Bacterial growth curves ...... 79 3.1 The effect of S. aureus on the viability of keratinocyte monolayers ...... 81 3.2 The effect of probiotic species on the viability of keratinocytes ...... 82

3.3 Specific species of Lactobacilli protect keratinocyte from the pathogenic effects of S. aureus...... 84 3.4 The effect of L. rhamnosus GG lysates and spent culture fluid on keratinocyte monolayers infected with S. aureus...... 86 3.5 The protective effect of L. rhamnosus GG or lysate is not time-dependent.. 88 3.6 Mechanisms of protection by L. rhamnosus GG ...... 91 3.7 Discussion ...... 101 Chapter Four ...... 106 Fractionation of Lactobacillus rhamnosus GG lysate ...... 107 4.0 Heat-killed or protease-treated lysate does not protect keratinocytes from S. aureus...... 107 4.1 Separation of L. rhamnosus GG lysate proteins by Reverse Phase Liquid Chromatography (RP-LC) and analysis by SDS-PAGE ...... 109 4.2 Specific L. rhamnosus GG lysate fractions protected keratinocyte from S. aureus infection ...... 112 4.3 Specific fractions of of L. rhamnosus GG lysate inhibit the growth of S. aureus ...... 113 4.4 Specific fractions of of L. rhamnosus GG lysate have anti-adhesive action against S. aureus ...... 114 4.5 Anti-adhesive activity of the 50% acetonitrile fraction from L. rhamnosus GG lysate ...... 116 4.6 Further purification of the 50% and 60% acetonitrile fractions from L. rhamnosus GG lysate using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) ...... 117 4.7 Identification of proteins from the 50% and 60% acetonitrile fractions using Tandem Mass spectroscopy (MS/MS) ...... 125 4.8 Discussion ...... 128 Chapter Five ...... 133 An in vitro study to investigate the potential use of probiotic lysates as a therapy for wound healing ...... 133 5.0 Probiotic lysates modulate the re-epithelialisation of keratinocyte monolayers in a species-dependent manner ...... 134 5.1Effect of L. rhamnosus GG and L. reuteri lysates on the rate of keratinocyte migration ...... 136 5.2 Effect of L. rhamnosus GG lysate and L. reuteri lysate on the rate of keratinocyte proliferation ...... 138 5.3 Effect of the L. rhamnosus GG lysate and L. reuteri lysate on keratinocyte re- epithelialisation in the presence of Mitomycin C ...... 139 5.4 Genome-wide Affymetrix microarray study of the effects of the L. rhamnosus GG lysate on keratinocyte gene expression and quantitative RT-PCR studies...... 142

5.5 Direct measurement of CXCL2 and FGF7 secretion by keratinocyte cultures ...... 148 5.6 L. rhamnosus GG lysate alters the expression of CXCR2 and FGFR2 protein receptors in keratinocyte cultures ...... 150 5.7 Effect of CXCR2& FGFR2 neutralizing antibodies on the healing of scratched keratinocyte cultures ...... 151 5.8 Discussion ...... 155 Chapter Six ...... 161 An investigation into the effects of Lactobacillus rhamnosus GG lysate on the re-epithelialisation of ex vivo wounded skin ...... 161 6.0 L. rhamnosus GG lysate stimulated re-epithelialisation of human skin maintained in organ culture ...... 162 6.1 L. rhamnosus GG lysate increases thickness of the epidermis in wounded skin ...... 164 6.2 L. rhamnosus GG lysate stimulates cell migration in wounded skin ...... 167 6.3 L. rhamnosus GG lysate stimulates cell proliferation in wounded skin ..... 169 6.4 L. rhamnosus GG lysate did not affect cell apoptosis in wounded skin ..... 171 6.5 L. rhamnosus GG lysate up-regulated wound healing-associated differentiation in human skin ...... 172 6.6 CXCL2 and FGF7 secretion in wounded skin cultures ...... 175 6.7 Discussion ...... 176 Chapter Seven ...... 181 Conclusions and Future Work ...... 181 7.0 Overview ...... 181 7.1 Future work...... 184 Bibliography...... 187 Appendixes……………………………………………………………………...227

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Table of Figures

Figure 1: A representation of the skin layers ...... 17 Figure 2: A representation of the epidermal layers ...... 20 Figure 3: Distribution of skin microbiota and its associated microenvironments (dry, sebaceous, moist) ...... 24 Figure 4: Phases of the wound healing process in normal skin ...... 31 Figure 5: Chromatographic peak of the eluted compound ...... 64 Figure 6: A standard protein curve ...... 65 Figure 7: Schematic representation of the measurement scales for scratch wound healing ...... 70 Figure 8: Schematic representation of the measurement scales for full-thickness wound biopsies ...... 73 Figure 9: Growth curves for probiotic species and S. aureus in batch culture ...... 80 Figure 10: The effect of S. aureus numbers on keratinocyte viability ...... 82 Figure 11: The effect of specific Lactobacilli species of keratinocyte viability ...... 83 Figure 12: Effect of Lactobacilli on the viability of keratinocyte monolayers infected with S. aureus ...... 85 Figure 13: Lysate or spent culture fluids from L. rhamnosus GG protect keratinocyte from the effects of S. aureus ...... 88 Figure 14: L. rhamnosus GG or lysate and sepnt culture fluids protect keratinocytes from S . aureus ...... 89 Figure 15: L. rhamnosus GG or lysate, but not its spent culture fluid, rescue keratinocytes from S. aureus mediated toxicity ...... 91 Figure 16: The effect of L. rhamnosus GG or L. reuteri lysates and L. rhamnosus GG spent culture fluid on S. aureus growth in competition assay ...... 93 Figure 17: The effect of L. rhamnosus GG lysate and spent culture fluid on Staphylococcal viable count...... 94 Figure 18: Zone of inhibition (mm) produced by L. rhamnosus GG bacteria or lysate on pathogenic bacteria using a spot on lawn assay ...... 95 Figure 19: Mean pH values of S. aureus cultures after treating with L. rhamnosus GG or its derived material ...... 97 Figure 20: Live L. rhamnosus GG lysate or spent culture fluids inhibit S. aureus from adhering to keratinocytes ...... 99 Figure 21: L. rhamnosus GG lysate or spent culture fluid inhibit S. aureus from adhering to keratinocytes by competetive exclusion ...... 100 Figure 22: L. rhamnosus GG inhibited S. aureus from adhering to keratinocytes by competetive displacement ...... 101 Figure 23: Heat or protease treatment of the L. rhamnosus GG lysate removed its ability to protect keratinocytes from the effects of S. aureus ...... 108 Figure 24: SDS-PAGE separation fro L. rhamnosus GG lysate ...... 110 Figure 25: Extraction and fraction scheme of the L. rhamnosus GG lysate ...... 111 Figure 26: The protective efect of the ysate is contained within specific fractions of the L. rhamnosus GG lysate ...... 112 Figure 27: Specific fractions of the L. rhamnosus GG lysate inhibit the growth of S. aureus in aspot-on lawn assay...... 113 Figure 28: Specific fractions from L. rhamnosus GG lysate inhibit S. aureus adhesion to keratinocytes ...... 115

Figure 29: The 50% acetonitrile fraction frm L. rhamnosus GG lysate reduces S. aureus adhesion to keratinocytes by competetive displacement ...... 116 Figure 30: The UV chromatograph and SDS-PAGE of 50% acetontrile fraction from L. rhamnosus GG lysate...... 119 Figure 31: The protective effect of the 50% acetontrile fractions is contained within specific HPLC fractions ...... 120 Figure 32: Specfic HPLC fraction from 50% acetontrile fractions reduced S. aureus adhesion to keratinocytes ...... 121 Figure 33: The UV chromatograph and SDS-PAGE of 60% acetontrile fraction from L. rhamnosus GG lysate...... 122 Figure 34: Spot-on Lawn assay ...... 123 Figure 35: Scheme of activities of L. rhamnosus GG lysate fractions against S. aureus ...... 124 Figure 36: SDS-PAGE for protein profile of 50% acetontrile fraction and its HPLC fraction F4 ...... 127 Figure 37: Specific probiotic lysates stimulated keratinocytes re-epithelialisation in vitro ...... 136 Figure 38: L. rhamnosus GG lysate and L. reuteri lysate increased the rate of keratinocyte migration ...... 138 Figure 39: L. rhamnosus GG lysate and L. reuteri lysate increased keratinocyte proliferation ...... 139 Figure 40: Efect of L. rhamnosus GG lysate and L. reuteri lysate on keratinocytes treated with Mitomycin C ...... 141 Figure 41: Microarray analysis of scratched keratinocytes treated vs untreated with L. rhamnosus GG lysate ...... 143 Figure 42: Network of genes associated with cell migration ...... 146 Figure 43: Network of genes associated with cell proliferation ...... 147 Figure 44: L. rhamnosus GG lysate increased the level of CXCL2 and FGF7 production in scratched keratinocyte cultures ...... 149 Figure 45: Western blot analysis confirmed altrations in the level of CXCR2 and FGFR2 protein receptors post-treatment with L. rhamnosus GG lysate ...... 151 Figure 46: Representative images of the effect of CXCR2 and FGFR2 antibodies on cells treated with L. rhamnosus GG lysate ...... 153 Figure 47: CXCR2 and FGFR2 blocking antibodies reduced the keratinoyte re- epithelialisation in the presence of L. rhamnosus GG lysate ...... 154 Figure 48: Wound-healing assay design ...... 162 Figure 49: Increased re-epithelialisation in human skin in response to L. rhamnosus GG lysate ...... 163 Figure 50: Epidermis thickness increases in human skin following L. rhamnosus GG lysate treatment with increasing keratinocyte numbers ...... 165 Figure 51: Epidermis thickness increment in human skin following L. rhamnosus GG lysate treatment ...... 166 Figure 52: Increased keratinocyte numbers in wounded skin epidermis following L. rhamnosus GG lysate treatment ...... 167 Figure 53: Phosphorylated cortactin expression increased in wounded skin following L. rhamnosus GG lysate treatment ...... 169 Figure 54: L. rhamnosus GG lysate increased cell proliferation in human skin punch wounds ...... 170 Figure 55: L. rhamnosus GG lysate did not affect cell apoptosis in human skin punch wounds ...... 171

Figure 56: L. rhamnosus GG lysate increased cytokeratin10 (CK10) expression on human skin punch wounds ...... 173 Figure 57: Effect of L. rhamnosus GG lysate on Involucrin expression in human skin punch wounds ...... 174 Figure 58: CXCL2 and FGF7 levels in ex vivo wounded skin culture ...... 176

List of Tables

Table 1: Probiotics and their recognised health benefits in humna clinical trials within the gastrointestinal tract diseases ...... 43 Table 2: Organisms used in this study ...... 58 Table 3: Standard protein solutions used to make a standard protein curve ...... 65 Table 4: Western blot buffer recipes ...... 67 Table 5: Protocol for histological staining with haematoxylin and eosin ...... 72 Table 6: A general protocol for immuno-staining of ex vivo wounded skin ...... 75 Table 7: Gene signature plates ...... 76 Table 8: Diameters of inhibition zone (ZOI) for spot-on lawn assays (n=3)...... 96 Table 9: Diameters of inhibition zones fro L. rhamnosus GG lyaste and its fractions by spot-on lawn assays (n=3) ...... 113 Table 10: Proteins were identified from L. rhamnosus GG lysate fraction 50% acetontrile and HPLC fraction F4 by mass spectroscopy (MS) ...... 126 Table 11: Proteins were identified from L. rhamnosus GG lysate fraction 60% acetontrile by tandem mass spectroscopy ...... 128 Table 12: Bioinformatic analysi of L. rhamnosus GG lysate effects on cell activities ...... 145 Table 13: Uniprot function of some genes following L. rhamnosus GG lysate treatment ...... 146 Table 14: Uniprot function of some genes following L. rhamnosus GG lysate treatment ...... 147

List of Abbreviations

ADAM17 Metallopeptidase domain 17 AMP Antimicrobial peptide AMS Junction adhesion molecules ANOVA Analysis of variance ATCC American Type Culture BCA Bicinchoninic Acid Assay BMP2 Bone morphogenetic protein 2 BV Bacterial vaginosis CAMP Cathelicidin antimicrobial peptide CD Crohn’s disease CDKN1B Cyclin-dependent kinase inhibitor 1B CE Cornified envelope CFS Cell-free supernatant/ Spent culture fluid CFU Colony forming units CM Conditioned medium CnBP Collagen binding proteins CXCL C-X-C chemokine Ligand CXCR C-X-C chemokine Receptor DAPI 4',6-diamidino-2-phenylindole DNA Deoxyribonucleic acid EaP Extracellular adherence protein EDN2 Endothelin 2 EDTA Ethylenediaminetetraacetic acid EF-Tu Elongation factor thermo unstable EGF Epidermal growth factor ELISA Enzyme linked immunosorbent assay EPS Extracellular polysaccharide FGF Fibroblast growth factor FITC Fluorescein isothiocyanate FnBP Fibronectin binding protein GAPDH Glyceraldehyde 3-phosphate dehydrogenase GF Growth Factor GMH Glucomannanhydrolysates GRAS Generally recognised as safe HA Hyaluronic acid Hbd Human beta defensin HPLC High performance liquid chromatography IBD Inflammatory bowel disease IFN Interferon IGFBP Insulin-like growth factor-binding protein IgG Immunoglobulin G IPA Ingenuity Pathway Analysis’ program IRF Interferon regulatory factors JNK1 c-Jun N-terminal kinases LAB Lactic acid bacteria LEP Leptin LF L. fermentum LGG L. rhamnosus Goldin and Gorbach LP L. plantarum LPS Lipopolysaccharide

LR L. reuteri LS L. salivarius LTA Lipoteichoic acid MAMP Microbial associated molecular pattern MAPK Mitogen activated protein kinase MEK1 Mitogen-activated protein kinase kinase MRS Man -Rogosa Sharpe MRSA Methicillin resistant S. aureus MS/MS Mass Spectrometry MSA Mannitol salt agar NF-κB Nuclear factor-κ -B NHEK Normal human epidermal keratinocytes PBMC Peripheral blood mononuclear cell PBS Peptone buffered saline PCR Polymerase chain reaction PDGF Platelet-derived growth factor receptors PGN Peptidoglycan PRR Pattern recognition receptor RNA Ribonucleic acid SA S. aureus SB Stratum basale SD-Ag Silver sulphadiazine SEM Standard error of the mean SG Stratum granulosum SSP Stratum spinosum STAT3 Signal transducer and activator of transcription TEER Trans-epithelial electrical resistance TEWL Trans-epithelial water loss TFA Trifluoroacetic acid TGF Transforming growth factor TJ Tight junction TLR Toll -like receptor TNF-α Tumour necrosis factor-α TPI Triosephosphateisomerase TRAF-6 TNF receptor associated factor-6 UC Ulcerative colitis VEC Vaginal epithelial cells VGF Vascular endothelial growth factor WC Whole cell WCA Wilkins -Chalgren Agar WCB Wilkins-Chalgren Broth ZO-1 Zona-Occludens-1

1 Abstract

Characterisation of the potential of probiotics or their extracts as therapy for skin The University of Manchester, Walaa Mohammedsaeed, Degree of PhD, 2014 A limited number of studies have investigated the concept of using enteric probiotic bacteria to alter microbial communities in areas other than the gut. The use of enteric probiotics is attractive because they are considered generally as non-pathogenic and safe. The aim of this thesis was to explore the possibility that probiotic bacteria, or material derived from them, have utility for skin in health and disease. Primarily, the thesis investigates whether probiotics can protect skin cells from the effects of the common pathogen, S. aureus, and whether probiotics can accelerate the wound healing process. Furthermore, some of the underlying mechanisms were investigated. In the first study, the potential of probiotics to protect primary human keratinocytes from the effects of S, aureus was investigated. When primary human keratinocytes were exposed to S. aureus, only 25% remained viable following 24h incubation. However, in the presence of 108cfu/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57%. Interestingly, L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes (P=0.006, P=0.01) following the same period of incubation. Keratinocyte survival was enhanced significantly, regardless of whether the probiotic was applied in the viable form, or as cell lysates, 2h before or simultaneously (P=0.005) or 12h after (P=0.01) to S. aureus infection. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion; however, only viable bacteria or the lysate could displace S. aureus (P=0.04 and 0.01 respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid (Chapter 3). Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. This idea has been confirmed by partial purification of L. rhamnosus GG lysate. Fractionation of the lysate demonstrated that the protective effect of L. rhamnosus GG lysate depends not only on inhibitory substances, but also on anti- adhesion substances present in the lysate (Chapter 4). The L. rhamnosus GG lysate increased the re-epithelialisation rate of model wounds in vitro in a scratch assay(P=0.02) (Chapter 5). Furthermore, L. rhamnosus GG lysate stimulated the re-epithelialisation of ex-vivo skin cultures (Chapter 6). In vitro and ex- vivo proliferation and migration assays demonstrated that L. rhamnosus GG lysate significantly increased keratinocyte proliferation and migration relative to controls; however, the dominant mechanism was migration (Chapter 5). Therefore, the pathways underlying these effects were explored by doing Affymetrix analysis for genes expressed in response to treatment with L. rhamnosus GG lysate. The results highlight that the CXCR2/CXCL2 and FGF7/FGFR2 are up-regulated, which may mediate the acceleration of re-epithelialisation of keratinocytes through stimulation of cell migration under the effect of L. rhamnosus GG lysate. In summary, these data suggest that L. rhamnosus GG and its extract may be a useful method of counteracting S. aureus infection and reducing the toxicity of pathogens. At the same time, specific probiotic lysates may be useful for improving the wound healing process and ultimately reduce the severity of impaired wounds in the community. Thus, the use of safe probiotic bacteria or lysates presents new options for the development of a new treatment that could improve wound healing while simultaneously reducing infection.

Declaration Author Name: Walaa Mohammedsaeed Faculty: Faculty of Medical and Human Sciences Higher Doctorate Title: Characterisation of the potential of probiotics or their extracts as therapy for skin.

 Dr. O’Neill (Main supervisor) and Walaa Mohammedsaeed identified the main aims for the project. The experiments were designed by Walaa Mohammedsaeed, Dr O’Neill, Dr McBain and Dr Cruickshank. Walaa Mohammedsaeed carried out all the project experiments and also carried out the analysis of data with some input from Dr Rawshan Choudray for the expeirments described in chapter 4. The thesis was written by Walaa Mohammedsaeed and critically reviewed and proofread by Dr. O’Neill, Dr. McBain and Dr. Cruickshank.  No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

I confirm that this is a true statement and that, subject to any comments above, the submission is my own original work.

Signed: Walaa Mohammedsaeed Date: 3/12/2014

2 Copyright Statement

i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electroniccopy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of any patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses.

3 Acknowledgements

“All praises to Almighty Allah, the most Beneficent and the Merciful”

I would like to give my heartiest gratitude to my supervisor, Dr. Catherine O’Neill who has been incredibly patient and supportive. It is hard to express how thankful I am. I would not have come to this stage without here kind support, guidance, and advice. It has been an honour to me to be PhD student and to receive here full attention thoroughly.

I am very grateful to my beloved parents and brother who gave me love and moral support during my PhD. I do not know how to express my appreciation to my beloved husband without whose supportive and encouragement, it would not have been possible for me to carry on my PhD. His love and encouragement has been invaluable to me over the last four years.

Finally, thanks go to Taibah University and Saudi Arabia cultural bureau for their financial support.

4 The author

In 2008 the author graduated from the Saudi University, King Abdul-Aziz University in Jeddah with a BSc in Clinical Biochemistry with first class honours. In 2010 the author graduated from the University of Manchester with an MSc in Clinical Biochemistry. In April 2011 the author joined the School of Medicine at the University of Manchester on registration for the PhD programme that was completed in 2014.

5 Publications arising from this Thesis

Publication paper

Mohammedsaeed W, McBain AJ, Cruickshank SM, O'Neill CA. (2014). Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes. Applied and Environmental Microbiology, 80(18):5773-81. (Appendix 1)

Conference contributions

Mohammedsaeed W, McBain AJ, Cruickshank SM, O'Neill CA. (2012). Lactobacillus rhamnosus GG can inhibit cytotoxic effects of S. aureus on keratinocytes in vitro. British Society of Investigative Dermatology Annual Meeting, Exeter, UK.

Mohammedsaeed W, McBain AJ, Cruickshank SM, O'Neill CA. (2012). Lactobacillus rhamnosus GG increases the re-epithelialisation rate of model wounds by stimulating keratinocyte migration. European Society of Dermatological Research Annual Meeting, Venice, Italy.

Mohammedsaeed W, McBain AJ, Cruickshank SM, O'Neill CA. (2013).Lactobacillus reuteri increases the re-epithelisation rate of model wounds by stimulating keratinocyte proliferation. The Symposium on Advanced Wound Care and Wound Healing American Society. Denver, CO, USA.

Chapter One

Introduction

1.1Skin structure and barrier function The skin is a complex sensory organ with a unique structure. It has the capacity to grow and restore itself continuously. Among the important functions of the skin is control of body temperature by regulating liquid evaporation from the surface and the excretion of toxic materials, such as urea from sweat glands located in the dermis of skin. In addition, the skin synthesises vitamin D3 (using sunlight), which is essential for normal growth of teeth and bones (Mackie et al., 1997b). However, by far the most essential function is the role of skin as a physical barrier between the body and the external environment. It provides protection against pathogenic invasion, chemical agents, Ultra Violet radiation and physiological assaults, such as extreme temperatures and dehydration (Proksch et al., 2008). The skin barrier is formed by the epidermis, which acts as an “inside-out” barrier to prevent excessive trans-epidermal water loss (TEWL), and an “outside-in” barrier that protects against pathogenic invasion (Brandner et al., 2002). This ability of the skin can be attributed to the fact that skin not only has unique structural components (Section 1.1.1), but also has an innate immune system (Section 1.1.2) that can respond quickly and efficiently to microbial challenges posed by the environment. The importance of this protective barrier can be observed in people who have defective barrier function; for example, in burn victims, who are more at risk of infection, and in skin conditions such as epidermal blistering and dehydration where fluid imbalances occur (Mackie et al., 1997b).

1.1.1 Skin structure: Skin is composed of three main layers. From inside to outside these are: the subcutaneous tissue (or hypodermis); the dermis; and the epidermis (Figure 1). Each layer has a specific, unique structure and function (Mackie et al., 1997b).

Figure 1: A representation of the skin layers. Diagram showing the epidermis, dermis and subcutaneous (hypodermis) layer of skin (Taken and adapted from http://www.nlm.nih.gov/medlineplus/ency/imagepages/8912.htm).

The subcutaneous (hypodermis) tissue is the innermost of the three layers of skin and is composed mainly of fat that acts as an energy reserve. This layer contains a large number of blood vessels and nerves, which pass upwards to the dermis, thereby providing the skin’s vascular supply (Mackie et al., 1997b).

The dermis is the second major layer of the skin, comprising mostly of connective tissue. It is rich in extracellular matrix components, such as collagen and elastin. Fibroblasts are the main cell type in the dermis, secreting matrix components that provide the skin with mechanical strength and elasticity (Mackie et al., 1997b). Moreover, immune cells are present in this layer, such as mast cells and dermal dendritic cells that are involved in defence against invading pathogens.

The epidermis is the outermost layer of the skin and, as such, is exposed to the external environment. For this reason, the epidermis plays an important role in the skin’s barrier function. Keratinocytes are the main cell type within the epidermis but other types are also found here; for example, melanocytes (which generate melanin pigment), dendritic cells (which have an important function in the skins immune system) and Merkel cells (a type of sensory cell, Mackie et al., 1997b). The epidermis is a multi-layered structure with the ability to renew itself continuously through cell division (Figure 2). The four separate layers of theepidermis are produced by the differing stages of keratinocyte maturation. These cells move upwards from the lower

17 layers to the surface and undergo a process of terminal differentiation as they migrate which gives rise to the four layers of the epidermis that are:

•Stratum basale The innermost layer of the epidermis sits directly on the top of the dermis, and is a single layer of keratinocytes that differentiate from epidermal stem cells residing in this layer. The keratinocytes divide continuously in the stratum basale and for reasons that are incompletely understood; some of them leave this layer and migrate upwards. In the stratum basale keratinocytes are tall cuboidal phenotype that are characterised by expression of the structural proteins Keratins 5 and 14. However, once they begin to migrate, their phenotype changes as does the expression of keratins (see below) (Simpson et al., 2011).

•Stratum spinosum The second layer of the epidermis is the stratum spinosum. This comprises 8-10 layers of polygonal keratinocytes. In this layer, keratinocytes are beginning to become flattened and the cells also connect with each other by “desmosomes” which give the layer a ‘spiky’ appearance under the microscope (Simpson et al., 2011). In the stratum spinosum, the keratinocytes are characterised by the expression of keratins 1 and 10 (Simpson et al., 2011).

•Stratum granulosum The third layer is called the stratum granulosum layer and comprises of 3-5 layers of flattened, nucleated keratinocytes. The cytoplasm of these cells contains many granules of keratohyalin, a protein involved in keratinisation. These granules give this layer its name (McEwan et al., 1993; MacKieet al., 1997b). The keratinocytes are also characterised by the expression of involucrin, ﬁlaggrin and loricrin (Simpson et al., 2011). In this layer lamellar bodies also found that are responsible for the ultimate release of lipids into the stratum corneum and have a role in the prevention of water loss (Alibardi et al., 2006).

•Stratum corneum The outermost layer is the stratum corneum or “horny layer” and is the end result of the keratinocyte terminal differentiation programme. It has 25-30 layers of flattened, dead keratinocytes that are known as Corneocytes (Stephen and Steinert, 1994;

Nemesand Steinert, 1999; Alibardi et al.,2006). Dead Keratinocytes in the stratum corneum are continuously replaced by the cells formed in the deeper layer of the epidermis. The Corneocytes in the stratum corneum are surrounded by epidermal lipids (ceramides, fatty acids, and lipids) that perform as a ‘mortar’ between the skin cells that can be considered ‘bricks’ since the structure has a bricks and mortar type of appearance. This combination of keratinocytes embedded within lipids acts as a waterproof barrier that reduces trans-epidermal water loss (TEWL) to preserve the moisture of the skin. The Corneocytes are enclosed by cornified envelopes are (CE) composed of proteins such as filaggrin, profilaggrin, involucrin and loricrin, and junctional proteins such as desmoplakin. Aggregates of these proteins with keratin create a toughened “cornified envelope” which supports the skin's barrier function (Stephen and Steinert, 1994; Nemes and Steinert, 1999; Alibardi et al., 2006). This barrier protects against invading microorganisms, chemical irritants, and allergens.

The epidermis has different mechanisms in place to create a barrier. For example, the tough, waterproof stratum corneum with its dead cells embedded in lipid and the stratum spinosum which contains tight junctions (Ehrhardt et al.,2002). Tight junctions are cell-cell junctions having a role in barrier function and regulation of the paracellular movement of molecules through the skin (Brandner et al.,2009). Recently, the role of tight junctions in maintaining the epidermal barrier was demonstrated in a study conducted by Furuse et al in 2002. They found that a knockout mouse lacking the tight junction protein, claudin-1 died within 24 hours of birth due to abnormal skin permeability and dehydration (Furuse et al., 2002). The arrangement of corneocytes and lipids in the stratum corneum and the presence of tight junctions act as a barrier to prevent movement of water. Consequently, the lack of water on the skin’s surface helps prevent colonisation by pathogens and the reserve water in the skin helps keep it hydrated. Furthermore, the lipid bilayers may prevent bacterial translocation similar to the effect of mucins in the intestinal epithelial barrier (Alibardi et al., 2006). In addition to the physical barrier function of the skin, an innate immune system provides an essential defense against injury and infection.

Figure 2: A representation of the epidermal layers. The diagram illustrates the epidermal layers from the deepest to the most superficial: Stratum basale, Stratum spinosum, Stratum granulose and Stratum corneum) (Taken and adapted fromhttps://www.worldwidewounds.com%2F2009%2FSeptember%2FFlour%2Fvuln erableskin1.html&ei=kn4xVKOYNIbgarfogZAI&psig=AFQjCNGmZ0TmxneTQIZ2 488rta3Zf8Ciug&ust=1412616182565090).

1.1.2 Skin innate immune system: The skin’s immune system consists of several cell types and multiple molecules. Combined, these form the skin’s immunological barrier. Indeed, langerhans cells, macrophages and keratinocytes are all involved in the immune system’s response to the pathogens. However, here, keratinocytes will be the main focus for discussion as, in the epidermis, they act as a first barrier against the pathogenic invasion by activating their own innate immune responses.

1.1.2.1 Keratinocytes Keratinocytes are the major cell type of the epidermis. Although not traditionally thought of as immune cells, keratinocytes contribute to the immune response of skin when it is challenged by microbes. Keratinocytes interact with bacteria through Toll- like receptors (TLRs) and, subsequently, produce cytokine/chemokines in addition to antimicrobial peptides (AMPs). Keratinocytes express multiple receptors in the category of “pattern recognition receptors”, such as TLRS. These receptors recognise specific pathogen-associated molecular patterns (PAMPs), which are usually cell wall or membrane components such as Lipopolysaccharide (LPS) or specific ligands for either bacteria or viruses. Activation of TLRs stimulates the host cell signalling pathways that promote the skin’s innate immune response i.e. the production of cytokines and AMPs. Different types of TLRs are expressed by keratinocytes (TLR 1,

2, 3, 4, 5, 6, 9) (Hornef and Bogdan, 2005; Sumikawa et al., 2006; Lebre et al., 2006) and these recognise different types of PAMPs. For example, Lebre and colleagues (2006) found that gram negative LPS interact with TLR4 whereas flagellin interacts with TLR5 (Lebre et al., 2006). Once TLRs have interacted with microbes, a complex signalling cascade is initiated, whereby chemokines and cytokines are produced (Lai and Gallo, 2009). Cytokines and chemokines produced by TLR signalling include Interleukins (IL-1α, IL-1β, Il-6, IL-8), Interferons (IFN-α, IFN-β), growth factors, Tumour Necrosis Factors (TNF-α), and Transforming Growth Factors (TGF-α, TGF- β). Some of these, such as IL-8, are chemoattractants for other cells of the innate immune response. They attract these cells to the site of microbial attack to help clear the infection (Lai and Gallo, 2009). One study has demonstrated that, in tissue injury, the release of damage-associated molecular pattern molecules (DAMPs) from dead and dying cells results in the activation of TLRs (especially TLR3) in “sterile inflammation”. The release of endogenous TLR ligands occurs predominantly in the aftermath of massive tissue injury, especially in circumstances where a significant percentage of cells undergo necrosis, as in ischemia- reperfusion injury (Lai et al., 2009).

Crucially, the interaction of keratinocyte recognition receptors with PAMPs leads to a range of cytokines and chemokines being released, thereby providing a link between the innate and the adaptive immune response ( Lai and Gallo, 2009). Several studies extol the benefit of pro-inflammatory cytokines produced by keratinocytes, such as IL-8 and TNF-α. These cytokine/chemokines play an important role as chemoattractants for immune cells from the skins dermis, such as neutrophils and T- cells (Larsen et al., 1989; Griffiths et al., 1991; Guilloteau et al., 2010). In chronic inflammatory diseases of the skin, such as chronic plaque psoriasis, the uses of cytokine inhibitors have been evaluated as treatments. It is thought that a relationship exists between a reduction in inflammation of the skin and that of TNF-α and IL-8 levels produced by keratinocytes (Chaudhari et al., 2001; Krueger and Callis, 2004). Furthermore, some studies report the effect of keratinocyte TLRs on the wound healing process. For example, one study found that increased TLR2 and TLR4 expression in non-healing venous leg ulcers causes activation of pro-inflammatory cytokine secretion that impairs healing in cutaneous wounds in animal models (Pukstadet al., 2010). However, increased TLR3 expression and activation of TLR3

21 using poly IC (TLR3 agonist polyriboinosinic-polyribocytidylic acid) improved healing in acute skin wounds in mice and humans through stimulation of CXCL2. This, in turn, stimulated the recruitment of neutrophils, macrophages and keratinocytes (Lin et al., 2011a & b & 2012).

The keratinocytes of the epidermis are the first cells capable of fighting microbes through the production of AMPs (Niyonsaba et al., 2006). AMPs are small, “cationic, amphipathic” molecules that disrupt microbial membranes and assist in killing microbes. For example, TLR3 ligation stimulates keratinocyte production of human ß-defensin-2 (hBD-2) and hBD-2 has been shown to inhibit gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa (Bals et al., 2000).Moreover, some pro-inflammatory cytokines, such as IL-1, IL-12, TNF-α and IL-22, activate TLR3-ligand, which subsequently, stimulates the production of hBD-2 from keratinocytes.

Another important AMP produced by keratinocytes is hBD-3 (human ß-defensin-3), which is active against Staphylococcus aureus (S. aureus) and some fungi, such as Candida albicans (Schroder et al., 2010). It has been demonstrated that some AMPs, such as hBD-3 and psoriasin, can stimulate the inflammatory response, thus influencing processes such as cytokine release, wound healing, and cell proliferation/migration (Brown and Hancock, 2006; Lai and Gallo, 2009).

Cathelicidins are another example of AMPs produced by keratinocytes that play a critical role in the skin’s defence against bacterial infection. One study demonstrates that cathelicidins may inhibit the growth of S. aureusin vivo, whereas keratinocytes obtained from mice lacking any expression of cathelicidin had limited ability to inhibit the growth of S. aureus (Braff et al., 2005c). Several studies revealed that during infection or injury, cathelicidin production increases strongly from keratinocytes. This enables them to boost cytokine and chemokine secretions that act to stimulate a great number of immune cells, enhance innate immune responses and increase cell proliferation and angiogenesis in wounded skin (Wolk et al., 2006).

Briefly, the main components of the skin barrier are its unique structure and the innate immune response from keratinocytes that produce cytokines/chemokines and

AMPs. Alongside these components, exogenous bacteria (micro-flora) create an additional barrier against the pathogens and prevent transient pathogens from colonizing the skin.

1.1.3 Commensal microbiota of the skin: Historically, the skin microbiota was characterized by their cultivation from swabs of the skins surface. This approach led to the identification of only a small subset of the microbiota present since most skin bacteria are uncultureable. The detection and characterization of skin microbiota has more recently been revealed by studies that used gene sequencing analysis and molecular analysis (Grice et al., 2008; Grice et al., 2009; Grice et al., 2014). Similar studies by the Segre group analysed bacterial diversity using 16S rRNA sequencing at 20 different skin sites in 10 healthy humans. Nineteen bacterial phyla were identified, but most skin bacteria fall into four phyla; Actinobacteria (51.8% e.g. Corynebacteri aand Propionibacteria), Firmicutes (24.4%e.g. Staphylococci), Proteobacteria (16.5%), and Bacteroidetes (6.3%) (Grice et al., 2008 & 2009). These are the same principle four phyla found in the oral cavity and the gut but the proportions differ in skin. Where the Firmicutes and Bacteroidetes are more abundant in the gut, it is the Actinobacteria that are most abundant on the skin.

Skin is different depending on its anatomical location and can be described as moist, sebaceous, and dry (Figure 1.3, Grice et al., 2008 & 2009; Gallo et al., 2013a & 2013b). The bacteria inhabiting these sites are very different because each site represents a distinct ecological niche with its own characteristic nutrient availability. For example, sebaceous areas, such as the back, face, behind the ears, are colonized by high quantities of the “lipophilic” bacteria Propionibacterium, while moist areas, such as axilla, are colonized by bacteria such as Staphylococcus and Corynebacterium that prefer high humidity environments. Dry areas of the skin, such as forearm, have massive amounts of bacterial diversity, such as Proteobacteria and Bacteroidetes but in less quantity than sebaceous and moist areas (Figure 3, Grice et al., 2008 & 2009).

Figure 3: Distribution of skin microbiota and its associated microenvironments (dry, sebaceous, moist). Classification of bacterial colonizing an individual is shown with the phyla in bold. Skin bacterial is grouped as sebaceous (blue circles), moist (green circles) and dry (red circles). Figure and data are adapted from Grice et al., 2009.

Several studies have demonstrated that the skin’s physical barrier and innate immune system are not the only the defense mechanisms against external insults. Normal microbiota on healthy human skin is also a major component of the barrier against colonization of potentially pathogenic microbes or against excessive growth of “opportunistic” pathogens (Cogen et al., 2009; Cogen et al., 2010). Resident commensal microbes generate their own AMPs, which stimulates their production from keratinocytes to combat infection (Cogen et al., 2010). For example, commensal microbes, such as Propionibacteria have the ability to inhibit the growth of S. pyogenesby producing free fatty acids from triglycerides (Hentgeset al., 1993). Another species present on the skin is S. epidermidis, which is capable of producing antimicrobial factors, such as phenol that affect the colonization of pathogens (Cogen et al., 2009, Cogen et al., 2010). In addition, S. epidermidis produces Lipoteichoic acid (LTA) that is recognised by TLR2 and activation of TLR2 has been revealed to increase the phosphorylation of tight junction proteins such as Occludin in cultured keratinocytes by activating phosphor-atypical PKCα, suggesting a role for commensal

24 microbes in preserving barrier homeostasis by enhancement of tight junction function (Yuki et al., 2011).

Skin microbiota are also able to regulate the skin’s inflammatory response to minor epidermal injury by reducing and regulating cytokine production in order to maintain healthy skin (Kong et al., 2012). A number of studies highlight the roles of skin microbiota in the wound healing process. Certain bacteria appear to assist healing by interacting with the host immune system (Tenorioet al., 1976). For example, species such as S. epidermidis may be able to stimulate the wound healing process by regulating the inflammatory response from the cells. A unique Lipoteichoic acid (LTA) produced by S. epidermidis reduces uncontrolled skin inflammation during injury by inhibiting the TLR3-driven inflammatory cytokine production in cultured keratinocytes and reducing the levels of inflammation in vivo following wounding (Lai et al., 2009).

The relationship between the host innate immune system and the normal skin microbiota to maintain healthy skin has been studied in skin diseases, such as acne, atopic dermatitis and psoriasis, where an imbalance of the microbiota was observed (Gaoet al., 2008; Scharschmidt et al., 2009). Several studies demonstrate that disturbance of the microbiota, or ‘dysbiosis’, is associated with diseases such as acne vulgaris, a common human skin disease defined by areas of red skin with comedones (blackheads and whiteheads), papules (pinheads), nodules (larger papules), and probably scarring. Acne may be attributed to a combination of hormonal changes during puberty and bacterial infection such as overgrowth of Propionibacterium acnes (Leyden et al., 1998; Bek-Thomsen et al., 2008). Another example, analysis of skin microbiome of Atopic dermatitis (AD), the most common form of eczema, demonstrated an alteration in bacterial populations, with overgrowth of Corynebacterium, Streptococci and over colonization of S. aureus and S. epidermidis in addition to reduction in Pseudomonas species compared with normal skin (Williams et al., 1990; Scharschmidt et al., 2009; Kong et al., 2012). Furthermore, another study investigated the skin microbiota of children with Atopic dermatitis pre and post antibiotic treatment and compared it to that of healthy children. The study demonstrated a significant loss of bacterial diversity in the AD cohort particularly increases in Staphylococcus species such as S. aureus and S.

25 epidermidis. This loss of diversity correlated with disease severity (Segre et al., 2012). However, in the antibiotic treated patients S. aureus proportions were lower, there was an overall restoration of microbial diversity and this correlated with improvement in clinical symptoms of disease. These data suggest that the AD treatments can alter microbial diversity and reduce Staphylococcus proportions that decrease the disease severity (Segre et al., 2012).These findings provide a link between common skin diseases and changes in the skin microbiota. Therefore, the notion of restoring the natural balance of bacteria in the skin could be useful to maintain healthy skin and manage skin diseases related to dysbiosis (Ouwehand et al., 2002; Dunne et al., 2001).

1.2 Wounding and the immune response: The barrier function of the epidermis prevents potential pathogens from accessing the body. However, in some circumstances, this barrier is broken and a wound is formed. According to the Wound Healing Society, a wound is defined as the “disruption of normal anatomic structure and function” (Frances et al., 2001) and can be categorized into two main groups - acute and chronic - according to the nature of the healing process. Acute wounds are typically tissue injuries resulting from cuts or surgical incisions with obvious signs of healing without complication. In most individuals with an efficient immune system, acute wounds heal within a reasonable time frame; usually a month (Falanga et al., 1993). In contrast, chronic wounds are defined as those that fail to complete the healing stages and have a prolonged inflammatory phase (Usuiet al., 2008). Non-healing wounds are sometimes associated with underlying conditions, such as diabetes, age, obesity and poor nutrition (more details in Section 2.2.2). Chronic wounds include pressure ulcers, diabetic foot ulcers and venous leg ulcers (Persoon et al., 2004).

1.2.1 The normal wound healing process and the innate immune system response: In normal healthy skin, a physiological process begins within minutes of sustaining an injury and sets of biochemical events occur to support the healing mechanism. Healing is defined by the Wound Healing Society as “a complex dynamic process that results in the restoration of anatomic continuity and function” (Frances et al.,

2001). The healing process consists of a series of overlapping stages, a summary of which is provided in Figure 4.

Haemostasis is the first response of the body following injury in order to control bleeding. Haemostasis starts with vasoconstriction in the injured blood vessels and platelet aggregation in the wound to form a clot. Platelets play an important role in the coagulation cascade as they release different types of growth factors, cytokines and inflammatory proteins that work in parallel to complete the coagulation pathway. These include: platelet-mediated growth factors; epidermal growth factors (EGF); fibroblast growth factor (FGF); insulin-like growth factors (IGF-1, IGF-2); keratinocyte growth factor (KGF); platelet-derived growth factor (PDGF); transforming growth factor-α (TGF-α); and transforming growth factor-β (TGF-β). These growth factors orchestrate the influx of fibroblasts and keratinocytes, the synthesis of collagen and other Extracellular Matrix (ECM) proteins, the regulation of cell movement within the wound microenvironment, the directed growth of new capillaries and vascular beds (angiogenesis), and the production of enzymes that remodel the newly formed connective tissue (Rodrigues et al., 2006; Deborah et al.,2007). Furthermore, haemostasis is critical to successful wound healing because the formation of the fibrin clot provides a provisional matrix composed of fibrin and fibronectin. Fibronectin contributes to the wound healing mechanism because it acts as a chemoattractant for cell migration and as a template for depositing collagen (Clark et al., 1995). Studies indicate that deficiency of growth factors or coagulation mediators, such as factor XIII (Fibrin-stabilizing factor), can lead to impaired wound healing (Frances et al., 2001).

The inflammation phase commences once the bleeding is under control and the inflammatory cells migrate to the wound area. Platelets and endothelial cells produce mediators, such as histamine and Cyclooxygenase (COX-2)enzyme, which stimulate prostaglandin synthesis. All these cause vasodilatation of blood vessels near the injury in order to increase vascular permeability and porosity. Consequently, proteins transfer from the bloodstream to the extracellular space, which results in tissue oedema. Moreover, inflammatory cells, such as neutrophils and macrophages, infiltrate the injured area in order to phagocytose bacteria and produce bactericidal substances, such as oxygen free radicals. Proteases are also released to remove and

27 clean damaged tissue in the wounded area. Furthermore, neutrophils initiate wound repair by activating a variety of growth factors to promote the revascularization and repair of injured tissue, such as Interleukin 8 (IL-8) and Vascular endothelial growth factor (VEGF) (Rodrigues et al., 2006; Deborah et al., 2007). Neutrophils are the main cells present for the first few days following wounding and then disappear unless the wound becomes infected. In this case, neutrophils infiltration continues until the infection is controlled (Rodrigues et al., 2006; Deborah et al., 2007). Macrophages play an essential role in the wound healing process. They migrate to the wound area as monocytes and then mature into macrophages that have the ability to phagocytize bacteria and ingest dead neutrophils, the fibrin clot, and other cellular debris. The recent findings of Khallou-Laschet et al (2010) state that macrophages can alter phenotypically from inflammatory macrophages (M1) to anti-inflammatory macrophages (M2 phenotype) due to environmental changes in cytokine expression (Khallou-Laschet et al.,2010). The activation of macrophages in response to pathogenic stimuli or cytokines such as tumour necrosis factor (TNF), has been defined as “classical activation”, and the resulting macrophages identified as M1 macrophages. Activated M1 macrophages improve host defense against a variety of bacteria, protozoa and viruses, promote the secretion of pro-inflammatory mediators such as TNF, nitric oxide (ΝΟ) and IL-1 that contribute to activation of different types of antimicrobial mechanisms such as enhanced the oxidative processes in order to kill invading organisms (Khallou-Laschet et al.,2010). In contrast, the activation of macrophages in response to large amounts of IL-10 and TGF-β has been defined as “alternative activation “, and the resulting macrophages identified as M2 macrophages. Activated M2 macrophages promote the anti-inflammatory response, which plays an important role in wound healing (Gordon et al., 2003).Infiltrating macrophages in the wound display mainly a M1 phenotype in wound models (Daley et al., 2010). However, M2 play also a pivotal role in the transition of the inflammatory phase to the proliferative phase in which they coordinate and sustain the wound healing events (Mantovani et al., 2002). Macrophages M2 release cytokines such as Interleukins (IL-1α, IL-1β), Interferon (IFN-α) that stimulate the inflammatory response and growth factors, which are involved in the migration, proliferation and organisation of new connective tissue and vascular beds within the wound. The macrophages can produce and secrete cytokines over time, causing continuation of the process of tissue repair (McQuibban et al.,2002).

In addition, macrophages synthesize nitric oxide (ΝΟ) that has a function in the wound healing process that has not been fully elucidated. Animal studies indicate an important role of nitric oxide (Mantovani et al., 2002) in the regulation of keratinocytes because low concentrations of nitric oxide could cause an increase in keratinocyte proliferation, whereas high concentrations lead to increased cell differentiation (Stall et al., 1999; Weller et al., 1999). Macrophages also synthesise special enzymes called matrix metallo-proteinases (MMPs) such as collagenase that play a crucial role in wound debridement and the shaping of new connective tissue. Moreover, macrophages stimulate different types of cells such as keratinocytes, fibroblasts in order to push the wound healing process into the proliferative phase (Madlener et al.,1998). At the conclusion of the inflammation stage of wound healing, bleeding is controlled and the wound bed is clean. This generates the perfect environment for the next stage of healing; cell proliferation and repair.

The proliferation phase is characterised by three processes: re-epithelialisation, where keratinocytes cover the wound surface, neovascularisation, rebuilding vascular integrity to the region, and granulation where the structural integrity of the tissue is restored by filling the defect with new connective tissue. The proliferation phase is triggered when fibroblasts and keratinocytes migrate to the injured site in order to re- establish the protective barrier. These are the main proliferating cells in this phase. Critical parts of any repair process include the availability of a constant supply of nutrients and oxygen. The process of rebuilding the vascular network, neovascularization, is stimulated by growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), which are released from platelets and macrophages (Madlener et al., 1998). Tissue hypoxia is also thought to stimulate macrophages into secreting angiogenic growth factors (Madlener et al., 1998). Re-epithelialisation phase of a wound occurs when keratinocytes completely cover the surface of the wound. The re-epithelialisation process is stimulated by locally- released growth factors to start keratinocyte migration across the wound bed within 12 to 24 hours following injury. Keratinocytes migrate from surrounding tissue to the wound area and begin proliferation and differentiation to form the new epidermis of the skin. The initial step of migration involves keratinocytes moving from the wound edges to the central area in response to paracrine signals sent from surrounding cells

(Clark et al.,1995). Then, the migrating keratinocytes begin their transformation by elongating themselves in the direction of the required growth. Next, the cells contract, and draw themselves forward across the wound surface. This process continues until the migrating cells from the opposing side of the wound meet in the centre, when the cells make contact at specific points, the cell migration is impeded by a process known as contact inhibition (Kirstein et al., 2002; Krull et al., 2001).

Some studies highlight the possibility that keratinocytes initiate re-epithelialisation in an autocrine manner. Keratinocytes secrete chemokines, which are able to bind to receptors already present on their cell surface. This autocrine loop initiates migration and/or proliferation, but does not influence epidermal differentiation (Florin et al., 2005; Fujimoto et al., 2008). Other vitro studies highlighted that IL-8 has the ability to stimulate human keratinocyte migration and proliferation through expression of CXCR2, the receptor for IL-8 on keratinocyte surfaces (Nanney et al., 2003; Devalaraja et al., 2005). Once migration is complete, the keratinocytes stabilize by creating firm connections to each other and cells start proliferation through mitotic processes to produce the new basement membrane (Kerstein et al., 1997). Proliferation process is activated by growth factors such as and FGF7 that are produced from platelets, macrophages and keratinocytes (Florin et al., 2005; Fujimoto et al., 2008). During the proliferation phase granulation tissue, a transitional substance that replaces the fibrin/fibronectin matrix is generated which includes new blood vessels. Fibroblasts are the predominant cells during this stage; however, inflammatory cells, endothelial cells and new components of the Extracellular matrix (ECM), also have important functions. ECM is composed of substances, such as fibronectin, which can improve and promote cell migration and adhesion; glycos-aminoglycan that stimulate tissue hydration (hyaluronan); proteoglycan that can help to regulate cells migration and glycoproteins (collagens, elastin) that provide tissue strength. Fibroblasts produce collagen III that is laid down and deposited in the wound area in order to increase the strength of wound and provide more resistance for any traumatic injury. This process is stimulated by growth factors such as PDGF, TGF, FGF that are produced from platelets and macrophages (Florin et al., 2005; Fujimoto et al., 2008). The size of wound is decreased because it fills with granulation tissue and the margins of wound start contraction that is associated with fibroblast differentiating into myo-fibroblasts

(Florin et al., 2005; Fujimoto et al., 2008). At the end of the granulation process the fibroblasts begin to apoptose in order to convert the granulation tissue from a cell-rich environment to a collagen-rich environment (Ortiz-Urda et al., 2005).

Remodelling phase or maturation phase is triggered by the production of collagen I, which is stronger than collagen III. Collagen I is organised in a network to provide the final shape of the tissue. This process can take one year or less depending on the type and size of wound, and also on the health and diet of the affected individual (Waaijman et al., 2010; Florin et al., 2005; Fujimoto et al., 2008). A summary of the wound healing process is presented in Figure 4. In addition to the keratinocytes, fibroblasts, endothelial cells, macrophages, platelets interactions, several intercellular signals, such as growth factors, cytokines and chemokines, have been shown to play important roles in all stages of the wound repair. These are described in next section.

Figure 4: Phases of the wound healing process in normal skin. Haemostasis is the initial step of wound healing where the platelets aggregate immediately in the injury area. A) The inflammation response is usually completed within the first 24 to72 hours after injury when cells infiltrate (White blood cells) into injury site and release inflammatory mediators after the coagulation process. B) Proliferation and repair is typically occurring 1 to 3 weeks after injury, the cells start division, angiogenesis, re- epithelialisation, granulation tissue formed and collagen deposition. C) The final stage, Maturation, begins approximately 3 weeks after injury and may take months. The Maturation occurs when the wound contracts and collagen is remodeled and a scar formed (Taken and adapted from http://cnx.org/contents/14fb4ad7-39a1-4eee [email protected]:29/Anatomy&Physiology).

1.2.2 The role of growth factors, cytokines and chemokines in normal wound healing: In brief, the wound healing process is initiated instantaneously following injury when several growth factors, cytokines, and chemokines from different types of cells are released. Examples of common growth factors are; Platelet-derived growth factor(PDGF), the initial growth factor revealed to be chemotactic for cells such as neutrophils, monocytes, and fibroblasts, migrating into the wound area and stimulating their proliferation (Embil et al., 2002). Indeed, several experimental and clinical studies have characterized a beneficial effect of PDGF as treatment for wound healing disorders (Edmonds et al., 2000; Embil et al., 2002).

Additionally, a sequence of studies (Shukla et al., 1998; Kibe et al., 2002) demonstrates an effective unique role of growth factors, such as Fibroblast growth factor (FGF7) in repair processes. They suggested that increasing FGF7 levels appears to be an essential mechanism for stimulating the repair of damaged epithelia, as observed in the skin through the stimulation of epithelial cell proliferation and migration. FGFs apply their functions in an autocrine, paracrine, or endocrine manner that are mediated through their specific receptors on cell surfaces (FGFR2, Kibe et al., 2002). Certainly, the effects of FGF7 have been demonstrated in many experimental studies that suggest a function of this growth factor in wound repair through stimulation of angiogenesis (Breuing et al., 1997) and proliferation of several cells types at wound sites (Abraham et al., 1997). Consequently, FGF7 is candidates to be used as topical treatment for stimulation of wound repair (Karvinen et al., 2003; Auf et al., 2004).

Vascular endothelial growth factors and their receptors (VEGF/ VEGFRs) play roles in regulating keratinocyte proliferation and migration and promoting epidermal regeneration (Traci et al., 2012). The direct effect of VEGF/VEGFRs on keratinocytes was investigated by utilizing neutralising antibodies against the VEGFRs in an vitro study. Results have shown the neutralising antibody against VEGFR has the ability to inhibit VEGF stimulatory effect on keratinocyte migration and this correlates with reduction in numbers of migratory keratinocytes (Traci et al., 2012).

Multiple studies have demonstrated a valuable effect of other growth factors including epidermal growth factor (EGF) and transforming growth factor (TGF-α or β) that are apparently present at high levels in wound areas. Expression of these factors suggested their roles in re-epithelialisation of wounds through stimulation of cell proliferation and migration (Ashcroft et al., 1997; Babic et al., 1999). The benefits of EGF factors have been demonstrated in vitro scratch wounding assays of keratinocyte monolayers using neutralizing antibodies against the EGFR receptors. Blocking the receptor inhibited EGF functions and this in turn suppressed keratinocyte proliferation (Tokumaru et al., 2000).

Other than the growth factors, a number of cytokines have been revealed as having vital functions in wound repair. These include chemokines such as CXCL2, CXCL5, CXCL4 and CXCL10, interleukins such as IL-8 and TNF-α. It is noticeable that some chemokines occur in normal wounds in order to stimulate keratinocyte proliferation or migration but in chronic wounds (that fail to heal) the level of these factors is very low, suggesting that specific chemokines play a crucial role in wound repair such as CXCL2, CXCL3 and CXCL4 (Gillitzer et al., 2001; Satish et al., 2003; Yates et al., 2008& 2009). Indeed, CXCL2 and it receptor (CXCR2) are necessary to stimulate migration and proliferation of keratinocytes to enhance the wounds re-epithelialisation (Nanney et al., 1995; Devalaraja et al., 2000) and CXCR2 knock-out mice showed impaired in re-epithelialisation after wounding caused by a decrease in recruitment of neutrophils and reduced keratinocyte migration and proliferation during re- epithelialisation with a significant delay in the neovascularisation process (Devalaraja et al., 2000; Gibbs et al., 2011). Some studies found a stimulatory effect of IL-8 on keratinocyte proliferation and migrations, which suggests that elevated levels of such cytokines may contribute directly to the stimulation of wound repair in vitro and vivo (Rennekampff et al., 2000). The cytokine, IL-8 is known to bind to two receptors on keratinocyte surfaces: CXCR1 and CXCR2, which in turn bind with other chemokines, especially with CXCL1-2 and CXCL5- 7. They interact with CXCR1 and CXCR2 to stimulate cell migration and improve the wound healing process in mouse models (Pastore et al., 2005). A topical application of IL-8 on human skin grafts in a mouse model stimulated re-epithelialisation by increasing keratinocyte proliferation and migration (Rennekampff et al., 2000).

Other studies have demonstrated the roles of pro-inflammatory cytokines, such as interleukins IL-1, IL-6, and TNF-α in wound repair. These cytokines are likely to have an influence on different processes at the wound site including stimulation of keratinocyte and fibroblast proliferation or migration and regulation of the immune response (Jacinto et al., 2001; Gurtner et al., 2008). The expression of these cytokines was revealed to be significantly enhanced during the inflammatory phase of normal healing; whereas, expression of these cytokines decreased strongly in impaired wounds in models, such as gluco-corticoid-treated mice (Hubner et al.,1996) and “genetically diabetic” mice (Wetzler et al., 2000). Furthermore, anti-inflammatory cytokines, such as IL-10, have been shown to be essential regulators for wound healing process through termination of the inflammatory responses and regulation of the proliferation and differentiation of a range of immune cells and keratinocytes (Moore et al., 2001). However, augmentation in IL-10 expression may be associated with impaired healing because the levels of this cytokine were considerably raised in human chronic venous ulcers compared with wounded tissue that is healing normally (Jacinto et al., 2001;Gurtner et al., 2008). A multitude of growth factors, cytokines and chemokines present at the wound site interact with each other and with cells to improve the healing process. Any alterations in the expression or production of these factors are expected to affect the healing process. Hence it has been revealed that pro- inflammatory cytokines and growth factors that are released during the early phase of wound healing are effective stimulators of the expression of various growth factors and chemokines from different types of cells.

1.2.3 Factors affecting wound healing: There are many factors affecting the wound healing process that might have positive or negative effects. These are classified as “local” factors – impacting directly on the wound itself; or “systemic” factors - based on the individual’s overall health, which may affect their healing capability. Local factors, such as, the level of oxygen in the wound area is critical to correct healing (Bishop et al.,2008). Oxygen works to induce angiogenesis, improves keratinocyte differentiation, increases fibroblast proliferation and collagen synthesis, and enhances wound contraction (Bishop et al., 2008). Furthermore, oxygen is a fundamental substance to protect the wound from infection that leukocytes require it in order to produce superoxide that can kill pathogens (Bishop et al., 2008). Temporary reduction in the oxygen level in the wound area is

34 useful initially, as it acts as a signal to stimulate cytokine and growth factor production from macrophages, keratinocytes and fibroblasts. However, prolonged reduction in the oxygen level can cause defects and delays to healing (Bishop et al., 2008). The wound area can be an ideal place for microorganisms to colonize and access deep tissues. This may lead to a failure in healing because bacteria produce endotoxins that promote the production of pro-inflammatory cytokines, such as IL-1 and TNF-α, resulting in a prolonged inflammation phase (Diegelmann et al., 2003). Wound infection is classified as contamination, colonization, local infection and spreading invasive infection depending on the stage of infection and the replication of microorganism (Section 1.2.4).

The development of infection in the wound area is based on the bacterial count per gram of tissue and the immune response of host. In severe cases, some wounds develop a biofilm. This occurs when wounds suffer from increases in bacterial number and the bacterial interaction occurring between two or more types produce a biofilm community that is resistant to the host immune system and antibiotic treatments. S. aureus and P. aeruginosa are common types of biofilm-forming bacteria (Diegelmann et al., 2003).

Systemic factors: Multiple factors impact on the wound healing process; for example, obesity, sex hormones, medication (immuno-suppression drugs and anti- inflammation drugs) and alcohol consumption (Campos et al.,2008). Many human and animal studies have been conducted to examine wound healing at different ages. They found an alteration in the wound healing process according to age such that wound healing in the young is faster than in the elderly group (Hardman and Ashcroft et al.,2008). Increasing age can lead to delayed wound healing because the ageing process is associated with delays in the inflammatory response, delayed infiltration of macrophages and lymphocytes, impaired macrophage function, decreased secretion of growth factors and collagen synthesis and wound contraction. Although ageing causes a temporal delay in wound healing, it does not necessarily affect the quality of healing (Hardman and Ashcroft et al.,2008).Furthermore, sex hormones could affect age- related wound healing deficits. Compared with aged females, aged males have been shown to delay healing of acute wounds. An explanation for this is that the female

35 oestrogens (oestrone and 17β-estradiol), male androgens (testosterone and 5α- dihydrotestosterone, DHT) and their steroid precursor dehydroepiandrosterone (DHEA) which appear to have significant effects on the wound healing process (Hardman and Ashcroft et al.,2008). Hardman and Ashcroft (2008) discovered that the variances in gene expression between elderly male and young human wounds are almost exclusively oestrogen regulated. Oestrogen affects wound healing by regulating a variety of genes associated with regeneration, matrix production, protease inhibition, epidermal function, and the genes primarily associated with inflammation (Hardman and Ashcroft et al.,2008).

An individual’s state of health can impact on the wound healing process. For example, 15% of people with diabetes have chronic, non-healing diabetic foot ulcers, which are caused by three factors; prolonged hypoxia, hyper-glycaemia and high levels of metallo-protease production. Moreover, these patients suffer from dysregulation and dysfunctions in inflammatory cells, such as imperfection in macro- phagocytosis (Hardman and Ashcroft et al., 2008).

1.2.4 Non-healing wounds: Non-healing wounds are a significant cause of morbidity and mortality for a large proportion of the population (Hunt et al., 1997; Schultz et al., 2003). If the mechanisms responsible for the failure of chronic wounds to heal could be identified, that might help to identify therapies, which could cause reduction in wound healing times. Chronic wounds have delayed uncompleted healing processes, which results in poor anatomical and functional outcome (Waldrop et al., 1991). Chronic wounds differ significantly from an acute healing wound in the inflammatory reaction (Simpson et al., 2009). The inflammation stage in an acute wound healing process is established normally in order to prepare the wound bed for healing by eliminating necrotic tissue, debris, and bacterial contaminants. Under normal conditions, the inflammation phase is a self-limiting process (Simpson et al., 2009). During this phase, the activated neutrophils are the main cells but are nearly non-existent after the first 72 hours. Conversely, chronic wounds have a prolonged inflammation phase that causes more injury, and neutrophils are present throughout the healing process. The presence of activated neutrophils could be caused by bacterial overgrowth, leukocyte trapping, or ischemic reperfusion injury (Diegelmann et al., 2003). The up-regulation

36 of the inflammatory cascade causes a significantly abnormal inflammatory profile for chronic wounds and the large number of activated neutrophils leads to excessive amounts of degradative matrix metallo-proteinases (MMPs). In a normal wound, all of the MMPs can be inhibited by the non-specific proteinase inhibitor such as α-2- macroglobulin known as a common tissue inhibitor of MMPs. However, in impaired wounds, the MMPs and its inhibitor enzyme are not balanced by an equal amount of both enzymes that could lead to an abnormal ratio between degradative and protective process (Yager et al., 1997). The excessive number of inflammatory cells also disturbs the cytokine profile in the wound by causing reduction in growth factors that promote proliferation, such as platelet-derived growth factors, keratinocytes growth factors and transforming growth factors (Frances et al., 2001).

1.2.4.1 Infected wounds: There is debate in the literature regarding whether infection prevents healing, or whether it is a consequence of non-healing (Davies et al., 2001). However, regardless of the driver, wound infection is a significant cause of morbidity and mortality (Diegelmann et al., 2003). Overuse of antibiotics has given rise to many antibiotic resistant bacterial strains that are often associated with poor outcomes following surgery (Falanga et al., 1993; Dow et al., 1999; Wysock et al., 2002).

Wound infection is classified according to the state of infection and the replication of microorganisms. “Contamination” is the development of infection in the wound area and is based on the bacterial count per gram of tissue and the immune response of the host. “Colonization” is defined as the occurrence of replicating micro-organisms adherent to the wound area. “Local infection” is defined as an increase in the number of microorganisms per gram of tissue, such as in wounds have microbial counts of more than 105 organisms per gram of tissue are considered to be infected, and the wound has an unhealthy appearance and delayed healing and it is an intermediate between colonization and “invasive infection” (Garrett et al., 1998; Dow and Browne, 1999; Frances et al., 2001). Invasive infection occurs when bacteria invade the deep tissue and cause septicaemia (Dow and Browne, 1999). The impact of bacterial infection on the wound healing process is elevations in pro-inflammatory cytokines and in matrix metalloproteinase (MMPs) levels, which leads to prolonged inflammation and delays in the healing process (Diegelmann et al., 2003). The classic

37 signs of infection are redness, swelling, heat and pain, but may also include increased exudates, odour, contact bleeding, delayed healing and abnormal granulation of tissue (Diegelmann et al., 2003). Many species of bacteria are implicated in wound infection. However, the most common is Staphylococcus aureus (Diegelmann et al., 2003).

1.2.4.1.1 Staphylococcus aureus: Staphylococcus aureus is gram-positive cocci that normally colonize human skin surfaces and nares. Colony counts of less than 106S. aureus per cm2 of skin are supposed to characterize colonisation while an amount equalling or more than 106 suggests an infection (Leyden et al., 1987). S. aureus is responsible for many types of infection such as infected skin wounds and invasion infections that directly cause organ failure and death (Iwatsuk et al., 2006). S. aureus infections occur commonly in 20%-30% of wounded skin, following antibiotic or surgical therapy (Peacock et al., 2001). However, the type of infection can be divided into two categories; skin and soft tissue infections, for instance impetigo or cellulitis, and invasive infections such as septicaemia, also known as “blood poisoning”. Initially, S. aureus infections were treated by using antibiotic therapy; however, S. aureus has improved its ability to resist and evade the majority of antibiotic therapies (Chambers et al., 2001), and methicillin-resistant S. aureus (MRSA) has emerged as a major problem in hospital and other healthcare environments (Chambers et al., 2009). Currently, S. aureus or MRSA infections account for ~4% of all hospital admissions in the United States, causing significantly greater mortality than any other infectious disease (Klevens et al., 2008). S. aureus produces several factors that can damage the cells using a variety of mechanisms such as epithelial barrier disruption, inhibition of opsonisation by antibodies and complement, interference with neutrophils chemotaxis, cytolysis of neutrophils, and inactivation of antimicrobial peptides (Foster et al., 2005). The common virulence factors are teichoic acid (TA) and surface proteins that promote adherence to damaged tissue such as exotoxins and haemolysins (α-, β-, and ∂-toxins) (Timothy et al., 2005). In addition, S. aureus produces enzymes such as nucleases, proteases, lipases, and collagenases that cause disruption to the epithelial barrier and improve the microbial mechanisms for invasion by causing pores in the cell membranes (Keijiet al., 2006). Mechanisms of S. aureus cell invasion start by

38 adhesion of the bacterium to the skin and disruption of the epithelial barrier components including cell-adhesion structures such as desmosomes and adherence junctions (Chavakis et al., 2002; Timothy et al., 2005).

Some studies indicate that S. aureus can produce exotoxins that attach to specific molecules on the epithelial cell surface in order to improve adhesion and microbial proliferation. For example, S. aureus produces a chemoattractant molecule, chemotaxis inhibitory protein of staphylococci (CHIP) that binds to formylated peptide receptors and the C5a receptor (C5aR) in order to reduce the ability of neutrophils from recognizing the microbe. Moreover, it inhibits the migration of neutrophils from the blood to the site of inflammation (Haas et al., 2004). S. aureus also has a specific type of protein on its surface, known as extracellular adherence protein (Eap). This is an inhibitory molecule for neutrophils chemotaxis that prevents interaction between lymphocyte and neutrophils surfaces (Chavakis et al., 2002).

Other strategy that S. aureus uses to improve its resistance to phagocytosis is expression of a protein called protein-A on its surfaces. This prevents antibody- mediated phagocytosis by blocking the Fc portion of IgG that leads to incorrect recognition by the neutrophils Fc receptor (Vuong et al.,2004). Furthermore, the majority of clinical S. aureus isolates have a thin micro-capsular layer; the polysaccharide capsule thatcan inhibit the attachment of neutrophils to the microbe. One study found that the capsule’s presence around the microbial cells could reduce the uptake of cells by neutrophils in the manifestation of normal serum opsonins; thereby demonstrating that the capsule is “anti-opsonic” (Luong et al.,2002). Furthermore, S. aureus can cause changes in immune response by producing immuno-modulator molecules, known as modulins, which are amphipathic peptides with pro-inflammatory properties. They are chemoattractant for human neutrophils and induce their activation. S. aureus only expresses modulins when the cell density is high (biofilm) in order to improve their resistance to phagocytosis (Vuong et al.,2004).

1.2.5 Wound treatment and management: Delayed healing can create problems that might impact massively on the affected individual’s quality of life. Therefore, the main aim of treating wounds is to ensure

39 the wound healing process is completed correctly in the shortest possible time. Selection of management strategies is based on the condition of the wound, as well as the patient’s circumstances. The wound healing process can be delayed or not occur at all in chronic infected wounds because of microbial infection; especially if biofilm forming organisms are present (Dowd et al., 2003). Several strategies aimed at enhancing the healing of chronic wounds are available, which are usually dependent on the factors responsible for the delayed healing. At the first stage, it should be observed whether the wound shows any clinical signs of infection. Once it occurs, the microbe should be identified and appropriate antimicrobial treatment selected. However, in complicated and more difficult cases, there are other treatment strategies such as hyperbaric oxygen, negative pressure therapy, electric stimulation and electromagnetic therapy, growth factors, bone marrow derived cells. In severe, life-threatening conditions, surgical intervention is required for tissue removal or amputation (Dowd et al., 2003).

Currently, microbiologists are faced with the challenge of developing treatments for chronic wounds that are infected with the antibiotic–resistant bacteria present in biofilms. Therefore, some studies focus on alternative therapies that might reduce infection, such as honey, essential oils and anti-microbial, moisture-retentive dressings (Okhiria et al., 2009). However, there remains an unmet need for new therapies in the treatment and prevention of wound infection. The following section discusses one emerging potential therapy “bacteriotherapy” using probiotic organisms.

1.3 Probiotics 1.3.1 Introduction: In healthy humans, certain tissues are continuously exposed to microbiota, such as skin, gut and mucous membranes. This colonisation begins during birth. The combination of organisms found at any anatomical site is the commensal microbiota. The normal microbiota of humans includes a small number of fungi and protists (Noble, 1993; Tringe et al., 2005), while the most prevalent microbes are bacteria (Noble, 1993). The normal flora and host derive benefit from each other; for instance, bacteria receive a stable supply of nutrients and protection from the environment.

From the host’s perspective, microbiota have been advocated for the maintenance of gastrointestinal health through different mechanisms of actions (Gionchetti et al., 2000 &2003). Kolida et al (2006) have reviewed that a major function of the gut microbiota is to reduce gastrointestinal diseases (Kolida et al., 2006) and prevent colonization by pathogenic microorganisms (Isolauri et al., 1999; Kolida et al., 2006). Moreover, gut microbiota play a role in human nutrition by activating the fermentation process in the gut to metabolize dietary components to end products such as short-chain fatty acids and gases (Fuller et al., 2000; Isolauriet al., 2002). This anaerobic metabolism may contribute positively to provide the colonocytes with 50% of their daily energy requirements through fermentation of carbohydrates to organic acids (Fuller et al., 2000).

Conversely, imbalances in gut microbiota growth are associated with gastrointestinal disorders. For example, a study conducted in 2005 examined the gut bacterial profiles of patients suffering inflammatory bowel disease (IBD), colitis and irritable bowel syndrome. The study found that these diseases are associated with disturbances in the natural gut bacterial population and a loss of tolerance to gut commensals (Cani et al., 2007). Since then, evidence has emerged to suggest that microbial imbalance may be related to disease and the concept that normalizing gut microbiota could contribute to a return to health has naturally appeared. Therefore, the idea that the numbers/types of organisms present in the gut could be manipulated to improve health or combat disease led to the idea of “probiotics”. Probiotics have been defined by the World Health Organization as; “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). Generally, these belong to the genera Lactobacillus or Bifidobacterium (Miettinnen et al., 1996). To date, the majority of work on probiotics has concentrated on the impact of ingestion of probiotics on intestinal health.

1.3.2 Probiotics and their beneficial effects: The concept of probiotics being beneficial to gut health and reducing gut diseases has been investigated for a number of years. Certain of the non-pathogenic Gram-positive commensal species belonging to the genera, lactobacilli and biﬁdobacteria have been identified as probiotics with beneficial health effects such as improving lymphocyte proliferation (Kirjavainen et al., 1998), enhancing innate and acquired immunity

41 responses (Gill et al., 2000), and regulating anti-inflammatory cytokine production (Pessi et al., 2000). Table 1 presents a summary for microbes from different genera being used as probiotics and their documented health beneﬁts in human clinical trials within the gastrointestinal tract diseases. In addition, L. rhamnosus GG is considered a most efficacious bacterium (Berg et al., 1996) in terms of its effectiveness in the prevention and treatment of Clostridium difficile associated diarrhoea (Makraset al., 2006), in clinical trials(Gorbach et al., 1987). There is evidence to support that probiotic Lactobacillus species play a role in the prevention of numerous gut diseases. For example, (IBD) that involves increases in cytokine production from epithelial cells with (Sartor et al., 1997) increasing apoptosis of cells (Iwamoto et al., 1996) and reduction in the intestinal microflora such as lactobacilli and bifidobacteria (Favier et al., 1997). Some studies demonstrate that Lactobacillus strains or derived materials can prevent or reduce intestinal inflammation through down regulating inflammatory cytokine production (Polk et al., 2002) that can help to reduce IBD inflammation (Polk et al., 2007). In addition, the commensal microbiota is disrupted in IBD (Favier et al., 1997), therefore, using probiotics for treatment or prevention of IBD has also been assessed in order to normalise the gut microbiota (Table 1).

Table 1: Probiotics and their recognised health benefits in human clinical trials within the gastrointestinal tract diseases Bacterial Genus and species Health benefits References Lactobacillus acidophilus La5 Reducing antibiotic associated Black et al., 1991 diarrhoea AAD Lactobacillus casei Shirota Shortening of rotavirus diarrhoea Nagao et al., 2000 and Immune modulation Sugita & Togawa 1994 Lactobacillus fermentum or Reducing colonisation by Felley et al., 2001 johnsonii Helicobacter pylor Lactobacillus paracasei F19 alleviating of irritable bowel Niedzielin et al., 2001 and plantarum 299v syndrome Lactobacillus reuteri SD2112 Shortening of rotavirus diarrhoea Shornikova et al., 1997 Lactobacillus rhamnosus GG Shortening of rotavirus diarrh oea Immune modulation and reducing Guandalini et al., 2000, Kaila of inflammatory bowel disease et al., 1992 and Gupta et al., 2000. Lactobacillus salivarius Reducing symptoms of Mattila-Sandholm et al., UCC118 inflammatory bowel disease 1999 Bifidobacterium breve or Reduced symptoms of irritable Brigidi et al.,2001 longum BB536 bowel disease Bifidobacterium lactis Bb12 Shortening of rotavirus diarrhoea Saavedra et al., 1994 and and Reducing incidence of Black et al., 1989 traveller’s diarrhoea Bifidobacteria, bacteroides Reducing levels of Clostridium Shimoyma et al., 1984 dificille 1.3.3Mechanisms of action of probiotics: Probiotics may protect against pathogenic bacteria through a variety of mechanisms. These include: I) the production of inhibitory substances such as acid or bacteriocins that can inhibit the growth of pathogen bacteria; II) modification and regulation of host innate immune responses; III) prevention of bacterial colonisation and adhesion by competitive exclusion or displacement from the cell binding sites; and IV) enhancement of epithelial barrier function.

The production of inhibitory substances from probiotics: Several studies have demonstrated the inhibitory effects of some probiotic bacteria upon pathogens; for example, Lactic acid bacteria (LAB) such as Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus caseiand Lactobacillus brevis, have antimicrobial activities against pathogens such as Escherichia coli (Sherman et al., 2005), Salmonella (Fayol-Messaoudi et al., 2005), Listeria (Touré et al., 2003), Clostridium (Kim et al., 2007), and Helicobacter pylori (Tsai et al., 2004). The antagonistic activity of LAB is mediated essentially by a number of factors secreted from LAB such as bacteriocins (Field et al., 2007).

However, the broad-spectrum antimicrobial peptide lacticin 3147 produced by lactococci is ineffective in reducing Listeria monocytogenes infection in a mouse model, even though it shows efficacy against this pathogen in vitro (Dobson et al., 2011). Therefore, further investigation is required into the influence of probiotic bacteriocins production and bacteriocins activity in order to provide greater correlation between in vitro inhibition and vivo results.

Probiotics are also able to produce anti-microbial peptides that are bactericidal, and can cause microbial cell death through formation pores in the pathogens cell wall, especially in Gram-positive bacteria (Alain et al., 2003). Moreover, Lactobacillus species have been shown to synthesise metabolites such as acetic and lactic acids that can inhibit the growth of bacterial pathogens or reduce the production of toxins (Alain et al., 2003; Asahara et al., 2004). In 2004, B. breve strain yakult protected mice against Escherichia. coli toxicity by triggering a decrease in the intestinal pH through acid production which in turn reduced the toxicity of the pathogen (Asahara et al., 2004). Another study revealed that L. rhamnosus may be able to inhibit Streptococcus pyogenes toxicity in pharyngeal epithelial cells through lactic acid production. The acidity inhibits S. pyogenes growth in addition to degrading streptococcal cytotoxic Lipoteichoic acid (LTA), which is essential in the adherence of S. pyogenes to cells (Maudsdotter et al., 2011). L. rhamnosus GG inhibited the growth of pathogenic Salmonella enterica by producing lactic acid and secreting antimicrobial molecules in one in vitro study (Marianelli et al., 2010).

Probiotics modify and regulate the host innate immune responses against pathogens: Some studies revealed that specific probiotics are able to enhance innate immune response as another strategy to reduce the pathogenic adhesion. A study conducted in 2011 investigated the protective effect of ingestion of L. plantarum against colitis. This study looked at the expression of various immuno-modulator markers such as Interleukins (IL-10, IL-12, IL-6 and IL-4) and TNF-α following the administration of probiotics in a mouse model of colitis (Raj et al., 2011). This study showed that L. plantarum might down regulate the expression of important pro-inflammatory cytokines such as TNF-α and IL-12 and could also up-regulate the production of major anti-inflammatory cytokines, such as IL-10, IL-6 and IL-4. Thus, L. plantarum strains might reduce the inflammation associated with colitis (Raj et al., 2011).

Notably, L. rhamnosus GG is reported to acts as immuno-modulator in healthy and allergic individuals that alters or regulates cytokine production from cells. For example, a one study, healthy and milk hypersensitive individuals were provided with milk that either contained or did not contain L. rhamnosus GG. Interestingly, in healthy individuals, L. rhamnosus GG stimulated the nonspecific immune response by increasing the expression of complement receptors on neutrophils as measured by flow cytometer. Whereas in allergic individuals, the L. rhamnosus GG acted as a down-regulatory factor which decreased the inflammatory response to milk (Peltoet al., 1998). Similar effects have also been found with other probiotic strains, such as, Lactobacillus acidophilus and Bifidobacterium strains which can increase phagocyte activity in vitro (Hatcher et al., 1993), and in vivo in mice (Perdigo´n et al., 1988), and in vivo (Schiffrin et al., 1997).

Probiotics prevent of bacterial colonisation and adhesion to cell binding sites: Lactobacillus species can inhibit pathogens from binding to epithelial cells by direct competition (Coconnier et al., 1993). For example, an in vitro study investigated the ability of probiotics (alone or in combinations) including L. rhamnosus NCC 4007, L. paracasei NCC 2461, to inhibit and compete with Escherichia sakazakii adherence to intestinal mucus through competitive exclusion and displacement from the binding sites (Collado et al., 2006&2008). Another study published in 2012 demonstrated that human vaginal isolates of L. crispatus and L. jensenii are able to inhibit the growth of S. aureus and block their adhesion to HeLa cells (urogenital cells) through lactic acid production and competitive exclusion from binding sites (Wang et al., 2012). Some studies suggest that probiotic strains are able to inhibit pathogen adhesion to cells by utilising adhesins factors including protein, carbohydrate, Lipoteichoic acid, or S- layer proteins (Heinemann et al., 2000, Miyoshi et al., 2006; Ruas-Madiedo et al., 2006; Chen et al., 2007). Using these mechanisms, probiotics can adhere to and prevent pathogens from residing in gut space; this is known as “colonization resistance” (Gueimonde et al., 2007; Saxelin et al., 2005; Sherman et al., 2009). For example, probiotics such as Streptococcus thermophilus ATCC 19258 and L. acidophilus ATCC 4356 have been associated with preventing the adhesion and invasion of Entero-invasive Escherichia coli in human intestinal epithelial cells in vitro (Resta-Lenert et al., 2003).Probiotic L. gasseri was evaluated for its ability to adhere to intestinal mucus, to auto-aggregate and co-aggregate with pathogens such as

Cronobacter sakazakii and Clostridium difficile (Ferreira et al., 2011); thereby demonstrating that, when combined with pathogens, L. gasseri strains bacteria have the capacity to compete with, displace and inhibit the adhesion of C. sakazakii and C. difficile in a mucus model in vitro (Ferreira et al., 2011).

In contrast, probiotics are able to modify the adhesive ability of other bacteria by producing biosurfactant. In turn, these alter the hydrophobic interactions between the bacteria and the cells (Ron and Rosenberg, 2001). Some studies suggested that probiotics, or their products biosurfactant, may be applied to patient care equipment, such as tubes or catheters or silicone rubber used in medical tools. This has the potential to decrease the colonisation of pathogens, including S. aureus and S. epidermidis ( Rodrigues et al., 2006). Stimulation of the expression of host genes such as mucin expression in intestinal epithelia suggested that L. plantarum strain 299v and L. rhamnosus GG can inhibit the adherence of Escherichia coli to intestinal epithelial cells through promoting the expression of mucins MUC2 and MUC3 that in turn reduces the pathogen adhesion to cells (Mack et al., 1999; Alain et al., 2003).

Probiotics enhance the epithelial barrier function: A number of studies have proved a relationship between utilising probiotics and the improvement of barrier function of epithelia in the gut through different mechanisms. In 2005, Parassol et al. examined whether probiotic L. casei could alter the intestinal barrier permeability in human gut epithelial cells infected with an Entero-pathogenic E. coli (EPEC). The authors found that L. casei alone did not instigate any changes in Trans-epithelial Electrical Resistance (TER), a marker of barrier function, in uninfected cells; however, when used in a co-culture with EPEC, it was apparent that L. casei could inhibit the noticeable increase in para-cellular permeability caused by EPEC (Parassol et al., 2005).

Recently, an investigation was conducted into the role of probiotic bacteria in maintaining the tight junction (TJ) proteins of gut epithelial barrier. The probiotics affect TJs either by changing the expression of their constituent proteins or by altering their distribution and localization (Madsen et al., 2012). L. plantarum MB452 has the ability to change the expression levels of Occludin, ZO-1 and ZO-2, major tight junction proteins in Caco-2 cells through increasing their protein expression resulting

46 in improved barrier function in vitro (Anderson et al., 2010; Liu et al., 2010b).Another study published in 2012 demonstrated that a mixture of probiotics (named VSL#3) or the spent culture fluid (VSL#3 CM) could modulate the TJ proteins such as claudin-2 and Occludin that was associated with an overall improvement in intestinal permeability in mice affected by ileitis. The main interesting result of the study is the ability of VSL#3 or VSL#3CM to reduce ex vivo ileal para-cellular permeability and to regulate the expression of epithelial TJ proteins through stimulation of TNF production from epithelial cells (Corridoni et al., 2012).

In 2009, a study was conducted to examine the effect of the probiotic mixture VSL#3 on epithelial permeability, TJ protein expressions and distribution, and epithelial apoptosis in a mouse model of acute colitis (Mennigen et al., 2009). The VSL#3 has been demonstrated as improving in intestinal barrier function of colitic mice through inhibiting decreases and redistributed of TJ proteins, and reducing the cells apoptosis (Mennigen et al ., 2009). Similarly, L. rhamnosus and B. lactis augmented epithelial barrier resistance with increases in phosphorylation of ZO-1 and Occludin in intestinal Caco-2 cells (Mathias et al., 2010). A gene expression study (Anderson et al., 2010) revealed that L. plantarum MB452 changed expression levels of many TJ-related genes, such as Occludin and cytoskeleton anchoring proteins and this was associated with improved intestinal barrier function (Anderson et al., 2010). Thus, probiotics, particularly lactobacilli, are considered effective through their ability to adhere to cells, exclude or displace pathogenic adherence, produce acids or bacteriocins as antagonistic to pathogen growth and support barrier properties of epithelia in many vitro studies. However, little is known about the mechanisms of lactobacilli to prevent intestinal epithelial from the pathogens in vivo; therefore, further research is required in this area.

1.3.4 Alternative uses for probiotics: Since probiotics impact positively on the gut, investigation has also been conducted into their potential effects on other body tissues, such as the urogenital tract (Reid et al., 2001) and the mouth (Haukioja et al., 2008). For example, in 2001 Reid et al. examined the impact of oral administration of Lactobacillus strains in a clinical trial of women with bacterial vaginosis (overgrowth of anaerobic bacteria in the vagina and

47 reduction in normal Lactobacillus species). The results indicated that Lactobacillus strains could inhibit and prevent the colonization of uro-epithelial cells by various anaerobic bacteria (Reid et al., 2001).

More recent reviews of the benefit effects of probiotics, mainly Lactobacillus species, on urinary tract infections have also been published. For example, two studies have demonstrated that probiotics or their spent culture fluid have the ability to inhibit the growth and reduce the adhesion of urinary tract pathogens such as Escherichia coli NCTC 9001 and Enterococcus faecalis NCTC 00775 to epithelial cells binding sites by exclusion of the pathogens in vitro (Chapman et al., 2013&2014).

Other studies have examined the potential of probiotic strains for oral health. Naseet al., in 2001, examined the impact of ingestion of milk containing L. rhamnosus GG on Streptococcus species in the mouth. The study found that L. rhamnosus GG could inhibit growth of pathogens including Streptococcus species and acidogenic lactobacilli in the mouth and could improve oral health. This yielded a significant reduction in dental caries over a period of seven months (Nase et al., 2001). Furthermore, in 2009, a probiotic lozenge, GUM® periobalance” was launched commercially. This contains a combination of L. reuteri ATCC PTA 5289 and L. reuteri ATCC 55370. This lozenge improves the natural dental flora and reduces gum inflammation (Sunstar et al., 2009; McBain et al., 2009).

Furthermore, probiotic bacteria have been used in the treatment of otitis media, middle ear infection commonly associated with Streptococcus pneumonia (Roos et al., 2001). A study conducted by Roos and colleagues demonstrated that a throat spray containing α- streptococci (non-pathogenic bacteria) could reduce significantly the reappearance rate of otitis media in children (Roos et al., 2001). In addition, probiotics have been assessed for the treatment of some chronic allergic eye diseases. For example, one study examined the effects of eye drops containing inactivated freeze dried probiotic L. acidophilus on seven patients with vernal keratoconjunctivitis (VKC) - an allergic eye disease with symptoms including itching, burning, and tearing. The authors demonstrated that the probiotic treatment for one month reduced the symptoms of the allergy without any side effects. However, controlled clinical

48 trials with a further sample of patients are required in order to prove the effects of lactobacilli on VKC patients (Iovienoet al., 2008).

1.3.5 Skin and probiotics: The application of probiotics is an intriguing idea for dermatologists as it could offer an alternative and/or prophylactic therapy to conventional antibiotics in the treatment of certain skin disorders, such as atopic dermatitis and psoriasis (Isolauri et al., 2001), or wounds (Peral et al., 2009) or infected skin (Rolfe et al., 2000; Prince et al., 2012). Moreover, probiotics can be used in the formulation of healthy skin care and anti- ageing products such as L’Oreal Youth Code Serum; therefore, consideration has certainly turned to examine the possible effects of enteric probiotics on skin health or disease.

1.3.5.1 Ingestion of probiotics and their effects on skin: Oral consumption of probiotics and their effects on skin have been the topic of many recent studies. For example, Puch et al in 2008 observed that ingestion of fermented milk containing probiotics, such as L. casei, L. bulgaricus and S. thermophiles, could improve the stratum corneum barrier function. The authors noted that in patients with dry and sensitive skin, ingestion of milk increased hydration of the stratum corneum. However, borage oil, green tea and vitamin E, as well as probiotic bacteria had been added to the milk; thereby suggesting the exact mechanism by which skin hydration improved was not investigated (Puch et al., 2008). Guéniche and colleagues (2006) studied the role of probiotics in immunoregulation associated with Ultraviolet (UV) irrradiation on skin. In general, UV radiation can cause acute or chronic skin inflammation, can induce degenerative changes in skin cells, particularly Langerhans cells by decreasing their ability to be antigen presenting cells and UV can also increase in pro-inflammatory cytokines such as IL-6 and TNF-α (Stingl et al., 1981). Guéniche’s study showed that ingested L. johnsonii NCC533 may reduce the effects of UV radiation on langerhans cell changes, and the pro-inflamatrory cytokine production observed in irradiated mice combared with untreated irradiated mice (Guéniche et al., 2006a). This result was confirmed when a 2008 study demonstrated that the ingested L. johnsonii causes acceleration in skin immune homeostasis recovery through increasing langerhans cell numbers (Peguet-Navarro et al., 2008).

Numerous studies have reported beneficial effects of ingestion of probiotics on skin inflammatory diseases, such as atopic dermatitis and psoriasis, perhaps indicating the possible role probiotics play in skin immune system (Kalliomäki et al., 2001). Atopic dermatitis is caused partly by an immune dysfunction resulting in an imbalance and dys-regulation in the formation of T lymphocytes with increasing Th2 formation. This imbalance increases the production of inflammatory cytokines, such as IL-4, IL-6 and TNF-α. However, other hypotheses suggested that atopic dermatitis is caused by the defective barrier function, which leads to the entry of antigens that result in the production of inflammatory cytokines. Both hypotheses result in increased inflammation and sensitivity of skin (Williams et al., 2009). The production of cytokines and growth factors by probiotics may play a role in reducing inflammatory reactions associated with Atopic dermatitis (Van et al., 2010). For example, a 2006 study examined the effect of oral administration of L. rhamnosus GG in mice with atopic dermatitis. The author demonstrated that L. rhamnosus GG could delay the development of atopic dermatitis through increasing IL-10 production from intestinal lymphoid organs (Sawada et al., 2006).

Another study in 1997 revealed that ingestion of L. rhamnosus GG was also shown to reduce levels of tumour necrosis factor-α (TNF-α) in patients with atopic dermatitis (Majarmaa et al., 1997). However, the exact mechanisms used by probiotics to improve Atopic dermatitis are not well understood.

All the aforementioned studies are based on the oral application of probiotic strains. Changes to skin via oral ingestion of bacteria are most likely due to immunomodulation. However, some studies indicate the beneficial effects associated with the topical application of probiotic bacteria to human skin.

1.3.5.2 Topical probiotics and the skin: A small number of studies have analysed the effects on the skin arising from the topical application of probiotics. For example, Guéniche and colleagues investigated the application of L. paracasei, combined with Bifidobacterium longum reuter extracts to treat ’sensitive skin’ such as dry/irritated skin. The study involved treatment of 33 adult females for a period of eight weeks (Guéniche et al., 2006). The authors showed that the groups receiving the probiotic products had fewer clinical signs of dryness of their legs and roughness of their face compared with those

50 receiving the placebo treatment (Guéniche et al., 2006). Another report in 2010 demonstrated that the topical application of B. longum reuter lysate, in a randomized double blind placebo trial, could increase barrier resistance and reduce skin sensitivity and dryness in patients with reactive skin. The same group found that the volunteers who used the cream containing the B. longum reuter lysate experienced a significant decrease in skin sensitivity and improvement in skin barrier function at the end of the treatment, compared with those who applied the non-probiotic cream (Gueniche et al., 2010a & 2010b). Alterations in barrier function were detected by measuring trans- epidermal water loss after barrier disruption that was caused by tape-stripping (Gueniche et al., 2010a & 2010b).

Additionally, Guéniche’s group examined the topical application of B. longum reuter lysate to ex vivo skin. Eight human skin samples were treated with or without 10% B. longum reuter lysate and cultured for 24 hours. The results showed that the signs of inflammation diminished; for example, vasodilatation, TNF-α release and oedema decreased in treated skin cultures compared to untreated cultures (Gueniche et al., 2010a). Probiotic bacteria have been assessed for their abilities to improve the skin barrier function by increasing the ceramides synthesis in normal or diseased skin. For example, Marzio and colleagues in 2003 examined the direct effects of sonicated Streptococcus thermophilus strains on those suffering from atopic dermatitis. The authors noted that the bacterial lysate improved barrier function by increasing in the levels of ceramides in the stratum corneum (Di Marzio et al., 2003).In 2008, the same group investigated S. thermophilus extracts upon the skin of either atopic dermatitis patients or ageing skin. The study suggested that bacterium could help regulate and improve ceramides production in both skin conditions by alleviating the symptoms of atopic dermatitis and improving the hydration and barrier function in ageing skin (Di Marzio et al., 2003& 2008). Miyazaki et al (2003) revealed that topical use of Bifidobacterium-fermented soymilk products could increase the production of hyaluronic acid (HA, which plays important role in water retention) from epidermal keratinocytes and dermal fibroblasts in vitro and mouse skin in vivo. This hints that the probiotic fermented the soy milk glycosides to genistein and daidzein that in turn, stimulated HA production from cells (Miyazaki et al., 2003). The same group applied the probiotic fermented soymilk product directly on mouse skin and human skin. The

51 results showed increasing in HA production and improvement in skin elasticity and hydration (Miyazaki et al., 2004). Other studies conducted in our laboratory suggested that lysates of B. longum reuter and L. rhamnosus GG enhance keratinocyte TJ function via amplification of TJ-protein expression. These data advocated that topical application of probiotics could be of importance in terms of preventing or repairing damaged TJs in skin diseases such as atopic dermatitis and psoriasis, where the TJ is known to be aberrant (Sultana et al., 2013, Watson et al, 2007). Current research is focused on understanding the concept of topical probiotics use to reduce skin infection with pathogenic bacteria (Peral et al., 2009; Prince et al., 2011), and the benefits of topical application in enhancing the wound healing process in vitro or in vivo (Peral et al., 2009).

1.3.5.3 Probiotics and skin infection: The effects of topical application of probiotics or their extracts remain speculative. For example, previous work in this laboratory has demonstrated that L. reuteri can protect keratinocytes from the pathogenic effects of S. aureus in vitro. The probiotic appears to prevent pathogen binding to the keratinocytes by competitive exclusion from binding sites in vitro. This work exemplifies the possible use of enteric probiotics as prophylactic treatment for skin infection but clearly more work is needed in this area to fully demonstrate proof of principle (Prince et al., 2011).

Probiotic bacteria have been evaluated for their inhibitory activities against skin commensals and pathogens. For example, the effects of number of different lactobacilli strains combined with glucomannanhydrolysates have been examined (Al- Ghazzewi et al., 2009) against the growth of the skin commensal, Propionibacterium acnes. This organism is associated with acnes vulgaris infections in skin (Leyden et al., 1976). The glucomannanhydrolysates were identified to promote the growth of lactobacilli (Al-Ghazzewi et al., 2009) which, subsequently, inhibited P. acnes growth in vitro and vivo studies(Al-Ghazzewi et al., 2009). Another study found that Lactococcus sp. HY 449 may be useful as anti-bacterial treatment through its ability to produce bacteriocins that was able to inhibit the growth of P. acnes and S. aureus (Oh et al., 2006). Another study examined the effects of fermentation products of P. acnes on skin infection due to S. aureus. The study’s results demonstrated that the fermentation products of P. acnes decreased significantly the growth of S. aureus

52 bothin vivo and vitro (Shu et al., 2013), suggesting the anti-S. aureus activity of the P. acnes product.

A study conducted by Kang et al (2009) highlighted that topical application of a lotion containing the spent culture fluid from probiotic Enterocococus faecalis diminished significantly, inflammatory lesions, such as papules and pustules in acne vulgaris patients following 8 weeks of treatment. This shows that the mechanism of action was by killing of P. acnes directly that led to suppression of the inflammatory response by reducing inflammatory cytokines production from either the host’s cells or the pathogens (Kang et al., 2009). Thus probiotics may have great utility in the prevention or even treatment of skin infection but clearly, more work is required for this to become reality.

1.3.5.4 Probiotics and wounds: Currently, the common pathogens isolated from wounded or burned skin that can cause infection are Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes. In general, bacterial infections identified in wounded skin are treated with antibiotics; however, most are not efficacious due to the ability of colonizing bacteria to form a biofilm, a characteristic “architecture” of micro-colonies that offers stabile structure which is resistant to a variety of antibiotics. Therefore, new therapies are urgently required. One such potential therapy is bacteriotherapy. Some studies have examined the effects of probiotic bacteria on infected wounds. For example, Ganet al in 2002 explored whether the inoculation of L. fermentum RC-14 and L. rhamnosus GR-1 and its biosurfactant product can prevent infection of silicone surgical implants in rats. The authors discovered that L. fermentum and its associated biosurfactant have the ability to reduce surgical infection with S. aureus because the tested probiotics are able to inhibit S. aureus adhesion and binding to cell surface extra-cellular matrix-binding proteins (ECMBPs) through a competitive exclusion mechanism. This study hypothesised that L. fermentum has a number of collagen- binding (Cnb) proteins that competes with S. aureus to bind to host sites (Ganet al., 2002). Another study demonstrated that a topical dressing containing nitric oxide (NO) producing L. fermentum, significantly increases wound closure and reduced the bacterial infection after 3 weeks in infected wounds in rabbit models (Jones et al.,

2012). This can be attributed to the ability of nitric oxide (NO) to kill the bacteria (Jones et al., 2012). Furthermore, a gel containing kefir (“an acid-alcoholic fermentation” usually used in Eastern Europe as milky suspensions due its potential health benefits, strains found in kefir are Lactobacillus, Leuconostoc, Kluyveromyces and Acetobacter genus) were applied directly to infected burn wounds in rat models; thereby demonstrating stimulation in wound re-epithelialisation and collagen formation in a short period, compared with silver sulfadiazine (a common wound treatment) treated controls (Huseini et al., 2012).

A study conducted by Valdez in 2005 revealed that L. plantarum inhibited the infection-causing capacity of Pseudomonas aeruginosa. The mechanism appeared to be through inhibiting and blocking the production of quorum sensing molecules (acyl- homoserine-lactones AHLs) and elastase from P. aeruginosa. Subsequently, this affects the ability of the organism to form biofilms. The author also examined the ability of L. plantarum to inhibit P. aeruginosa infection in vivo in a burned mouse model by injecting the probiotics into the infected burned areas. The author highlighted that L. plantarum inhibited P. aeruginosa by producing a very different pattern of cytokines and chemokines that interacted with pathogenic bacteria cytokine production (Valdéz et al., 2005).

In addition, a study in 2009 tested the ability of topically applied L. plantarum as a treatment for burns infected with P. aeruginosa. The authors examined 80 patients who had suffered burn injuries and divided them into three groups: infected second- degree burns; infected third-degree burns; and non-infected third-degree burns. The authors found that L. plantarum reduced the bacterial numbers in the infected wound area. L. plantarum also promoted tissue granulation and healing, and protected the wounds against infection. These results could be due to the ability of L. plantarum to induce anti-inflammatory cytokines from host inflammatory cells, which can oppose cytokines induced by pathogens (Peral et al., 2009); however, other mechanisms may also be involved. Another study was conducted by the same group in 2009 to investigate the effects of L. plantarum on P. aeruginosa infected chronic venous ulcers. The topical application of L. plantarum caused a reduction in the pathogenic numbers in the injury area and decreased IL-8 production induced by P. aeruginosa. L. plantarum induced increases in the IL-12 and tumour necrosis factor-α (TNF- α)

54 production from peripheral blood mononuclear cell (PBMC) that activates cytotoxicity against pathogens (Peral et al., 2009).

Additionally, one study examined the effects of probiotic spent culture fluid on the wound healing process; in particular, the proliferation and migration of the human keratinocytes in vitro. The study hypothesised that the L. plantarum (spent culture fluid) contains a specific substance, Plantaricin A, which stimulates keratinocyte migration and proliferation in a scratch assay (Daniela et al., 2011). The author explored the mechanism underlying the improved migration and proliferation by measuring the level of growth factors, such as TGF-ˇ1, VEGF and FGF-7. The results revealed that Plantaricin A increased the expression of the growth factors promoting the wound healing process (Daniela et al., 2011). However, further work is required in order to elucidate completely whether topical probiotics or their extractions can be used as treatment or prophylaxis for wound infections, and the possible effects.

1.4 Aims and Hypothesis Numerous studies have demonstrated the importance of probiotics and their potential therapeutic effects within the gut. In contrast, few studies have analysed the use of topical probiotics. The evidence suggests that enteric probiotics could contain crucial therapeutic value in terms of the prevention of infection and promotion of wound healing. However, to date, comparatively few probiotic strains have been investigated. Additionally, very little research has been conducted into the mechanisms underpinning the effects of probiotics on skin. The main aims of this project are to investigate the potential of topically-applied enteric probiotics to modify pathogenic effects and wound healing in a model of skin, and to understand some of the basic mechanisms underlying these effects. In particular, the aims of this research are:  To screen a range of probiotic strains for their ability to inhibit keratinocyte infection caused by skin pathogen, S. aureus  To determine the effects of probiotic-derived materials in reducing S. aureus infection in vitro.  To fractionate L. rhamnosus GG lysate and determine the effective molecule(s) in terms of its ability to reduce S. aureus infection in vitro.

 To screen a range of probiotic lysates for their effect on keratinocyte re- epithelialisation in a wound model to determine whether the “topical” application of probiotic lysates could enhance the wound healing process via activation of cytokines or growth factors.

Hypothesis Based on the positive influence of probiotics on gut health we hypothesise that: probiotic lactobacilli or their extracts will inhibit the toxic effects of S. aureus on keratinocytes. The “topical” application of probiotic lysates will positively augment wound re-epithelialisation in vitro and ex vivo. All the above will be mediated by specific molecules within the probiotic bacteria.

Chapter Two Methods and Materials

2.0 Reagents and materials All bacteriological media were purchased from Oxoid, Basingstoke, UK. Tissue culture media and reagents were purchased from Promocell, Heidelberg, Germany.

Tissue culture plates were obtained from Nunc™ or Fisher Scientific Ltd. (Loughborough, UK). Bio-Rad protein assay lits, TEMED, SDS molecular weight broad range standard and 10x TGS buffer (Tris-Glycine-SDS) were purchased from Bio-Rad laboratories (Hemel Hempsted, Herts). Protease inhibitor (set III) was purchased from Calbiochem (Beeston, Nottingham).

All liquid Chromatography columns were obtained from Phenomenex, Huddersfield, UK. Protein extraction reagents and buffers were purchased from Thermo Scientific, Rockford,USA.The primary antibodies used in this study were purchased from R&D Systems, Minneapolis, USA.

Reverse Transcription Polymerase Chain Reaction (QPCR) reagents and kits such as TaqMan® Micro-RNA Reverse Transcription Kit, TaqMan® Gene Expression Master Mix and TaqMan® Array Human Chemokines were supplied by Invitrogen, Life Technologies Ltd, Paisley, UK. All other reagents unless otherwise stated were purchased from Sigma-Aldrich, Dorset, UK.

2.1 Bacterial cell culture Lactobacillus organisms (Lactobacillus reuteri, Lactobacillus rhamnosus GG, Lactobacillus fermentum, Lactobacillus salivariusand Lactobacillus plantarum) were obtained from LGC Standard Limited (ATCC, Middlesex, UK). Staphylococcus aureus was donated kindly by Dr. Andrew McBain, University of Manchester and was originally isolated from a chronic wound. All bacteria that used have been listed in Table 2. Bacteria were grown on Mannitol Salt agar –MSA or Man-Rogosa Sharpe agar (MRS) for S. aureus or lactobacilli respectively. The plates containing lactobacilli

57 were incubated overnight at 37°C in an anaerobic incubator (Don Whitley Scientific, Shipley, UK), whereas, S. aureus plates were incubated overnight at 37°C in an aerobic incubator. As described by Cowan (2003), Gram stains were performed for the basic characterisation of organisms. Where broth cultures were required, a single colony of plated bacteria was inoculated into 10ml of Wilkins-Chalgren Broth (WCB) and incubated aerobically for S. aureus and anaerobically for lactobacilli overnight. Thereafter, larger volume broth cultures were prepared (Section 2.1.3) and used for further experiments.

Table 2: Organisms used in this study Probiotic organisms Lactobacillus reuteri ATCC-55730 Lactobacillus salivarius UCC-118 Lactobacillus rhamnosus GG ATCC-53103 Lactobacillus fermentum ATCC-14932 Lactobacillus plantarum ATCC-10241 Pathogenic organism Staphylococcus aureus subsp. aureus

2.1.1 Growth curves Overnight broth cultures of the organisms were diluted, 10µl culture in 90µl of WCB on a 96 well plate, and incubated in triplicate in a Powerwave XS plate reader (Biotek, Bedfordshire, UK) for 24h at 37oC in order to determine the growth curve for each organism. Growth curves of overnight cultures for all bacteria were constructed and analysed using the Gen5 Software program (Biotek, Bedfordshire, UK).

2.1.2 Viable counts of bacterial cultures A 1ml aliquot from 10ml overnight culture of all bacteria was used to perform serial dilutions, as described by Aneja et al., (2003). Briefly, a 1ml of bacterial culture was mixed with 9 ml of sterile WCB to create a 1/10 dilution. A 1ml of this dilution was added to a further 9 ml of WCB in a separate tube to create a dilution of 10-2. This was repeated until dilution up to 10-9 was produced. A 50µl of dilution (10-1 – 10-9) was added to agar plates that were labelled with the corresponding dilution (10-1 – 10-9). A sterile plastic spreader was wiped gently over the surface of the agar plate to spread the

58 dilution. Dilutions were platted on Mannitol Salt agar for S. aureus. The experiment was performed three times with triplicate samples within each individual experiment. Following overnight incubation at 37oC, the number of colonies on each plate was counted. Plates with greater than 200 colonies or fewer than 35 were not counted. The number of bacteria was calculated using the formula as described by Anejaet al., 2003: Number of bacteria CFU/plate = Number of colonies/ amount of platted x dilution factor

2.1.3 Preparation of bacteria for experiments using mammalian cell culture A 10ml of 106CFU/ml of S. aureus and a 10ml of 108CFU/ml of probiotics (listed in Table 2) were centrifuged at 15,000x g for 10 minutes and were then washed twice in phosphate buffered Saline (PBS), and re-suspended in 10ml of keratinocyte medium (Section 2.4). Then, a 100μl of this bacterial suspension was added directly to 5 x 105 of confluent keratinocytes.

For some experiments, bacterial cells were pelleted, in a centrifuge at 1500x g for 5 minutes. The spent culture fluid (CM) was collected and filtered using a 0.22µm pore filter (Millipore, Billerica, USA). 50μl of the CM was transferred to agar plates to confirm that there were no residual viable bacteria. In other experiments utilising heat killed L. rhamnosus GG, bacteria were re-suspended in 10ml of keratinocyte medium (Section 2.4) and heat-inactivated by placing the vessel of L. rhamnosus GG in a water bath for 45 minutes at 85°C. Samples were gram stained to ensure no lysis of bacterial cells had taken place. A 50μl of the samples were then transferred to agar plates to confirm they were heat-inactivated.

2.1.4 Preparation of bacterial lysates Overnight cultures of 100ml of probiotic bacteria were harvested using 10 minutes of centrifugation at 12000xg. Once harvested, the cells were washed with phosphate buffered Saline (0.01M PBS, pH=7.4), and the pelleted cells were re-suspended in 25ml of PBS. This solution was sonicated for 3 minutes using a MSE Soniprep 150 at 16kHz set to 240 volts as outlined by Villaverdeet al. (1997). The sonicated solution was kept in ice in order to preserve the sample. Following sonication, the samples were passed through 0.22um Millex-GV syringe filters that were obtained from Millipore (Bedford, MA, USA). Then, 50μl of thesolution was plated on agar plates to confirm

59 the complete removal of all viable bacteria. Approximately 100µl of this lysate was used to treat approximately 5x 105 keratinocytes. In separate experiments investigating the effects of protease on the activity of the lysate, 1ml ofL. rhamnosus GG lysate was centrifuged and re-suspended in 1ml of Trypsin (0.4%) for 1h at 37°C. After this, 1ml of Trypsin Neutraliser solution (0.05%) was added; SDS-page was performed as described in Section 2.4.5 to check whether the lysate protein was affected by protease treatment. In other experiments investigating the effects of heat, lysate was placed in a bath of boiling water for 5 minutes. Moreover, SDS-page was conducted to assess the effect of heat on lysate protein, as outlined in Section 2.3.5.

2.1.5 S. aureus dose and time response assays Keratinocytes were grown to 80% confluence in 24 wells plate and exposed to105, 106, 107 or 108 CFU/ml of S. aureus. Samples were set up in triplicateand incubated for 24h at 37°C. Keratinocytes were subsequently washed twice in phosphate buffered saline (0.01M PBS, pH=7.4). A trypan blue exclusion assay (Section 2.2.1) was then conducted to determine the viability of the keratinocytes in the presence of different concentrations of pathogen. In other experiments, a time course was conducted using 106 CFU/mlS. aureus. Keratinocytes were infected with 106 CFU/ml S. aureus for 6, 8, 12 and 24h prior to assessment of the keratinocyte viability using trypan blue exclusion (Section 2.4.1). The experiment was performed three times with triplicate samples within each individual experiment.

2.1.6 L. rhamnosus GG dose response assay To determine the ratio of L. rhamnosus GG to S. aureus for use in experiments, confluent keratinocytes growing in 24 well plates were exposed to a range of L. rhamnosus GG numbers (104,105,106, 107, and 108 CFU/ml) plus 106 CFU/ml S. aureus simultaneously, in triplicate for 24h incubation and keratinocyte viability was assessed using trypan blue exclusion assays (Section 2.4.1).

2.2 Screening of inhibitory activity of L. rhamnosus GG 2.2.1 Determination of bacterial antagonism (Spot on Lawn Assays) In the protocol adopted, usually 10μl of 106 CFU/ml cultures of the S. aureus was inoculated in 7ml of the appropriate soft-agar media (0.7% agars). This mixture (top

60 agar) was transferred to plates pre-poured with agar base. 50μl of organism or extracts of bacteria were spotted onto this lawn and plates were incubated overnight at 37oC in an upright position. The inhibition zone was evaluated following overnight incubation. This was achieved by using a ruler to measure in mm the diameter of the zone. Additionally, Universal pH indicator was added to visualise the pH change on some plates. During these experiments, the pH of combination cultures and lysates from two probiotics was measured. The experiment was performed three times and results were taken as the average of triplicates.

2.2.2 Measurement of S. aureus viability in cell culture For the purpose of determining whether L. reuteri, L. rhamnosus GG lysates or spent culture fluid (CM) were able to inhibit the growth of S. aureus in keratinocyte cultures, keratinocytes were grown to confluence in a 24 well plate. Confluent keratinocytes were exposed to either 106 cfu/ml S. aureusor S. aureus plus 100µl lysates or 100µl spent culture fluid. After 24h exposure, the medium was removed and the keratinocytes were trypsinised. 500µl of 0.25% v/v Triton-X-100 in PBS was then added for approximately 30 minutes in order to lyse the cells. The well contents were used for serial dilution plate counts to determine the total number of viable staphylococci as described in Section 2.1.2.

In separate experiments, cells were exposed to L. rhamnosus GG lysates 2, 4, 6, 8 and 12h once the S. aureus infection had commenced. After 24h exposure, the total number of viable staphylococci was counted as described above. The experiment was performed three times with triplicate samples within each individual experiment.

2.2.3 Live/Dead staining At 24h, BacLite ™ Live/Dead stains were performed for 1ml of S. aureus plus 100µl lysates or 100µl spent culture fluid,according to the manufacturer’s instructions (Invitrogen, Life Technologies Ltd, Paisley, UK). Bacteria were counted under the microscope for three different areas and processed using ImageJ 64 software program (obtained from http://imagej.nih.gov). Non-viable bacteria were identified by their red colour, while viable bacteria were identified by their green colour at these randomly picked fields and the average calculated for three individual experiments with triplicate samples within each individual experiment.

2.2.4 Measurement for bacterial adhesion to keratinocytes Confluent keratinocytes were exposed to either 108 cfu/ml probiotic either at the same time as, or 2h before, or up to 12 h after the addition of 106 cfu/ml S. aureus for 1h. After incubation, cells were washed three times in PBS to remove non-adherent bacteria. The cells were trypsinised and serial dilution plate counts performed to assess the number of adherent bacteria present (2.1.2). MSA agar was used for growth of staphylococci and MRS agar was used for growth of probiotics. In some experiments instead of using live probiotic, either 100µl of the lysate or the spent culture fluid was used.

2.3 Fractionation of the L. rhamnosus GG lysate by Reverse Phase Liquid Chromatography 2.3.1 Reverse Phase Liquid Chromatography  Fractionation of L. rhamnosus GG lysate using Strata-XL A 60ml Strata XL column (pore size 100 µm) was washed using 99.99% (v/v) methanol in water. 30ml of lysate prepared as described in 2.1.3 was pH adjusted to 5.8 using 0.1% (v/v) trifluoroacetic acid (TFA) in water and then was applied to the column and allowed to flow through. The flow through was collected into a 50 universal tube. The column was washed with 45ml of 50% methanol pH 2 to remove any unbound proteins. After that, all proteins bound to the column were eluted gradually from the column in 60ml 90% methanol at pH 2. The eluted proteins were collected into two 25ml universal tube. Then the eluted proteinswere evaporated to remove alcohol for 3h in the centrifugal evaporation system (Biotek, Bedfordshire, UK). The fractions were maintained at 4oC for further analysis.

 Fractionation of L. rhamnosus GG lysate by Sep-Pak C18 A 5 ml Sep-Pak C18 cartridge (pore size 37-55µm) was washed with 5ml of 100% acetonitrile (Fisher scientific, Loughborough, UK), and then washed with 5ml of 0.1% (v/v)TFA in water (Fisher scientific, Loughborough, UK). Then, the column was loaded with 5ml of the sample previously obtained from the Strata-XL column; the resulting flow through fraction was collected in 10ml universal tube and labelled as flow through. The sample was eluted using increasing concentrations of 10-70% (v/v) acetonitrile containing 0.1% (v/v) TFA solution. 5ml of each fraction was collected in

10ml universal tube separately according to acetonitrile concentrations. Then the eluted fractions were evaporated to remove the acetonitrile for 3h in centrifugal evaporation system (Biotek, Bedfordshire, UK). 1ml of each fraction was subjected to SDS-page analysis and stained with Instant Blue (Harston, Cambridgeshire, UK) for overnight to visualise the protein bands detailed in Section 2.3.5. The fractions were maintained at 4oC for further analysis.

2.3.2 Reversed Phase High-performance Liquid Chromatography (RP-HPLC) The HPLC column packed with Silica, C12, 90µm pore size in 10mm inner diameter × 250 mm (Catalogue Number = 00G-4396-N0) was used for a third round of purification for fractions previously obtained from the Sep-Pak C18 cartridge. The HPLC column was washed with 0.1% (v/v) TFA in water (pH 2) at flow rate 5ml/minute. Then, 2ml of sample were injected onto the column and the proteins were eluted with increasing concentrations of 10-99% (v/v) acetonitrile containing 0.1% (v/v) TFA solution that was forced through the column by high pressure in the range 6000-9000 psi distributed by a pump at flow rate 2ml/minute for 50 minutes. A UV detector was used to measure the protein amount at 215nm. The output yielded from this detector is referred to as a “chromatogram peak”. Eluted fractions were then collected separately in 2ml Eppendorf tubes. High peak fractions (Figure 5) were selected and evaporated over 3h in a centrifugal evaporation system (Biotek, Bedfordshire, UK); meanwhile, the 1ml volume of fractions was subjected to SDS- page analysis, as described in Section 2.3.5. The activities of fractions were assayed for inhibitory activity against the S. aureus, using the spot on-lawn assay and adhesion assay. Some fractions were donated kindly by Dr. Rawshan Reza Choudhury (University of Manchester, Center of Dermatology) for the purpose of examining their activities. Selective fractions containing functional proteins were kept at 4oC for MS/MS analysis.

Figure 5: Chromatographic peak of the eluted compound. Chromatographic represents peaks for each separated compound and the peak height was measured from the baseline to determine the amount of proteins.

2.3.3 Preparation of Protein from keratinocytes Confluent keratinocytes were grown in 24 well plates and the proteins were extracted from one well using 100µl HEPES cell extraction buffer (120mM NaCl, 25mM

HEPES, pH 7.5), 1%Triton X100, 2mM EDTA, 25mM NaF, 1mM NaVO4, 100µM PMSF, 1% SDS, 10% Glycerol and Protease cocktail Inhibitor. Cells were scraped and gathered into 1.5ml Eppendorf tubes. The tubes were incubated on ice for 30 minutes, with frequent flicking at 5-minute intervals to re-suspend pellets. The tubes were centrifuged using an Eppendorf MiniSpin (Eppendorf, UK Limited, Stevenage, UK) for 5 minutes at 1340x g with the aim of sedimenting the cellular debris. The supernatants were collected into new Eppendorf tubes and frozen at -20°C. The protein concentrations of the samples were determined using the Pierce®Bicinchoninic Acid Assay kit (BCA, Thermo Scientific, Rockford, USA) according to the manufacturer’s instructions (Section 2.4.4). The sample protein bands were visualised using SDS-page (2.3.5).

2.3.4Protein concentrations Protein concentrations of the proteins extracted from keratinocytes (2.3.3) or lysate or fractions (2.4) were determined using the Pierce® Bicinchoninic Acid Assay kit (BSA, Thermo Scientific, Rockford, USA), in accordance with the manufacturer’s instructions. A standard curve was constructed using Bovine serum albumin (BSA, 2mg/ml) as the standard (Table 3). Samples were diluted 1 in 10 with distilled water. In

64 each well of a 96 well plate (Corning, Amsterdam, The Netherlands) 25µl of the diluted sample was added to 200µl of the BSA working reagent. Three replicates were made for each sample and the plate incubated at room temperature for 30-45 minutes. The results were then delivered via a microtitre plate reader (540-590nm) and the protein concentrations of samples determined using the standard curve (Figure 6).

Table 3: Standard protein solutions used to make a standard protein curve

Protein (mg/ml) BSA (2mg/ml) Distilled water 0.0 - 100µl 0.25 12.5µl 87.5µl 0.50 25.0µl 75.0µl 0.75 37.5µl 62.5µl 1.00 50.0µl 50.0µl 1.50 75µl 25.0µl 2.00 100µl -

Figure 6: A standard protein curve.The protein concentrations of the samples were determined using the Bovine serum albumin (BSA, 2mg/ml)as standard protein solutions.

2.3.5 SDS-PAGE and Western blotting The 25μl of probiotic lysates or fractionswere combined with 2X laemmli buffer (0.125M Tris-Cl, 4%w/v SDS 20% w/v glycerol, 10% w/v 2-mercaptoethanol, pH6.8) The mixture was boiled for 3 minutes in order to denature the proteins, and then loaded in equal volumes (35μl) into each of the wells of into a 4-12% Bis-Tris gel from Invitrogen (Life Technologies Ltd, Paisley, UK) along with a broad range molecular weight marker. The proteins were separated electrophoretically for 1h at 150V with

10xrunning buffer (0.25M Tris, 1.92M Glycine, 1%w/v SDS) until the bromophenol had reached the bottom of the gel. Gels were stained with Silver or Instant Blue according to manufacturer’s instructions (Life Technologies Ltd, Paisley, UK). Silver staining was performed as described in 2.3.4.1.

 Western blotting Post SDS-PAGE, proteins (from unstained gels) were transferred electrophoretically onto a polyvinylidene fluoride (PVDF) membrane (Hybond ECL, Amersham Biosciences, USA) for 1 hour at 30mv with Transfer buffer (Table 2.3). Following transfer, membranes were blocked in a 5%skimmed milk solution (10ml TBS of 10% w/v TBS + 5g milk powder) for 1h at room temperature, and then incubated with the primary antibodies CXCR2/IL-8 RB (2.5µg in1ml of PBS) or Human FGF-7 antibody (5.0 µg in 1ml of PBS)overnight at 4Co. All Western blot buffer recipes are detailed in Table 4. Membranes were washed for 3x 5minutes with TBS/Tween containing 5% skimmed milk solution and then incubated with secondary antibody (HRP-conjugated goat anti- mouse IgG) at 1:5000 dilutions for 1h at room temperature. The membrane was washed 3 more times in skimmed milk, and 3 times in TBS. Membranes were visualised using enhanced chemiluminescence (ECL; Amersham, UK), according to the manufacturer’s instructions. Blots were quantified using densitometry, as described by Mclaughlin et al. (2004). The experiment was performed three times with triplicate samples within each individual experiment.

Table 4: Western blot buffer recipes

10% SDS (Sodium dodecyl sulfate) 50g SDS in 500ml H2O Tank Buffer (10X)(Running buffer) (0.25M Tris, 1.92M Glycine, 1% SDS) 12g Tris base, 57.6g Glycine, 40ml SDS in 400ml H2O 2X Loading buffer (0.125M Tris-Cl pH6.8, 4% SDS, 20% glycerol, 10% 2- mercaptoethanol): 2.5g Tris base, 4.0ml SDS, 2.0ml Glycerol, 1.0ml 2-mercaptoethanol, 0.5mg bromophenol blue in 10ml H2O. Transfer Buffer (25mM Tris, 192mM glycine, 15% methanol): 3.05g Tris base, 14.4g Glycine, 150ml Methanol to 1000ml H20. Tris Buffer Saline (TBS) (10x): 22.1g Tris base, 40g NaCl to 500ml (pH to 7.6) H2 TBS + 0.5%Tween: 100ml TBS (10x), 5.0ml Tween 20 to 1000ml H2O TBS containing 5% (w/v) non-fat dried milk 10ml TBS (10% ) + 5g milk powder + 90ml water. powder Primary (1°) antibody made up in 2.5g milk powder + 5ml TBS/Tween + 45ml water + 10µl 1° TBS/Tween containing 5% (w/v) non-fat Ab. dried milk powder. Secondary (2°) antibody made up in 2.5g milk powder + 5ml TBS/Tween + 45ml water + 10µl 2° TBS/Tween containing 5% (w/v) non-fat Ab. dried milk powder.

2.3.4.1 Silver staining Following the separation of the proteins by SDS-page, gels were fixed in 30% v/v Ethanol in water and 10%v/v Acetic acid in water at room temperature for 3h. The gel

was sensitized for 1 minute in 0.6g Sodium thiosulphate (Na2S2O3), 0.3g Potassium

Ferro-cyanide (KFe (Cn6)) and 0.1g Sodium carbonate (Na2CO3) in 200ml of deionised water. The gels were then washed in deionised water and exposed to Silver nitrate

0.1% w/v AgNO3 in water, for 30 minutes. The gels were developed by 2.5% w/v

Na2CO3, 0.02% w/v formaldehyde in water until desired staining was achieved. Staining was terminated in 1% acetic acid for 5 minutes and the gel was preserved at 4 ºC in 2% w/v glycerol, 30% v/v acetic acid in water.

2.3.6 Tandem mass spectrophotometric analysis of proteins Tandem Mass spectrophotometric (MS/MS) analysis of proteins was conducted using the ‘gel top’ method. Prior to the MS/MS analysis the proteins were separated electrophoretically for 10 minutes at 150V by SDS-PAGE as described in Section 2.4.4; Gels were stained with Instant Blue (Harston, Cambridgeshire, UK) to visualise protein bands. Tandem mass spectrophotometric analysis of proteins was conducted entirely in the faculty of Life Sciences, Biomolecular Analysis Unit (University of Manchester) by Dr David Knight according to their established protocols. Protein identification was performed using the MASCOT database search engine (Matrix Science,http://www.matrixscience.com). Searches were performed and analysed by a Biomolecular Analysis specialist; Dr David Knight). MS/MS determined the most

67 abundant proteins present in gels within the molecular weight range approximate 5- 500kDa. MS/MS data for some fractions was provided kindly from Dr. Rawshan Reza Choudhury (University of Manchester, Centre for Dermatology).

2.4 Mammalian cell culture Normal human epidermal keratinocytes (NHEK, Promocell, Heidelberg, Germany) were maintained in keratinocyte medium containing a supplement mix: bovine pituitary extract (0.004mg/ml), epidermal growth factor (recombinant human) 0.125ng/ml, insulin (recombinant human 5µg/ml), hydrocortisone (0.33µg/m), epinephrine (0.39µg/ml), transferrin, (holo human 10µg/ml) and 0.06mM CaCl2. Medium was changed twice weekly and cells were cultured at 37oC in a humid atmosphere of 5% CO2 in T-75 culture flasks. Once the cells had grown to 80% confluency, they were washed with 100µl Phosphate Buffer Saline (PBS) per cm2 of the vessel’s surface. Cells were then detached using 100µl of 0.3% Trypsin (0.4% / EDTA) per cm2 of vessel surface until they had become detached. This was followed by adding the same volume of 0.1% Trypsin Neutraliser solution (0.05% / BSA. Cells were then sedimented by subjecting them to 3 minutes of centrifugation at 220xg. The supernatant was discarded and the cells were re-suspended in keratinocyte medium. A viable count was performed on this suspension using trypan blue exclusion assays (2.4.1). The cells were then re-seeded into cell culture vessels at approximately 5 x 105 cells/cm2.

2.4.1 Trypan blue exclusion assay Culture medium was removed from keratinocytes growing in 24 well plates and keratinocytes were washed twice in PBS. The cells were then detached, as described in Section 2.2. 20µl of 0.4% (w/v) Trypan Blue solution (Invitrogen, Life Technologies Ltd, Paisley, UK) (filtered to remove particulates) was added for 30 seconds. Cells were counted under the microscope in four 1 x 1mm squares of one chamber of a hemocytometer. Non-viable cells were identified by their blue colour, while viable cells remained unstained. Percentage viability of keratinocytes was calculated using the following equation: Percentage viability = Viable count / Total count x 100/1.

All experiments were conducted a minimum of three times with triplicate samples within each individual experiment.

2.5 Wound healing study in vitro and ex vivo models 2.5.1 Scratch wound healing assay Keratinocytes were seeded in 24 well plates at a density of 5×105cells/well in growth medium. Cells were grown to a confluence of about 80%. A scratch was then made through each monolayer using a sterile 100μl pipette tip. Cells were washed twice with 0.01M PBS (pH=7.4) and fresh medium added to the cells. Scratches were documented at 6, 12, 18 and 24 hours post scratching by staining cells with 200µl of filtered 0.1% Crystal Violet solution (Invitrogen™, UK) for 30 seconds. Scratches were documented under the Keyence microscope (x5 magnification, 50µm scale bar, Keyence, Osaka, Japan) after the staining procedure. The images were analysed using ImageJ 64 software program (obtained from http://imagej.nih.gov). This enabled determination of the percentage of scratch area (gap) with respect to the percentage of starting scratch area at time zero (Figure 7). For each image, the area between one side of scratch and the other was measured. Quantification of scratch closure was obtained by comparing the area of the scratch at time zero with that at a specific time point using the following equation: Percentage of re-epithelialisation= {area of the scratch (µm) at t=0h - sample area (µm) at t=t} / {area of the scratch (µm) at t=0h} x 100 Where t=t is a specific time point post scratching.

In some experiments, scratches were treated with 100µl bacterial lysates (0.93mg/ml)or 0.5mg/ml of Mitomicin C from Streptomyces caespitosusand lysate. In other experiments scratches were pre-treated with blocking antibodies for 45min at 8oC before the addition of lysate. The antibodies were Human CXCR2/IL-8 RB Allophycocyanin MAb (Clone 48311) (2.5µg in 1ml PBS) and Human FGFR-2 (alpha isoform) antibody (Clone 98706) (5.0µg in 1ml PBS) used to block the specific receptors. A negative control antibody Human IgG1 (Clone 97924) (500µg)was used in this assay.

Figure 7: Schematic representation of the measurement scales for scratch wound healing. Quantification of scratch closure was obtained by comparing the area of the scratch at time zero with that at a specific time point.

2.5.2 Keratinocyte migration assay Sub-confluent keratinocytes were harvested with trypsin, as described in Section 2.1, and re-suspended in keratinocyte culture medium. 100µl aliquots of the cell suspension containing 2.5x105 cells per well were plated in the upper wells of a 24-transwell chamber (Invitrogen™, UK). 100μl of probiotic lysates were placed in the lower chamber and an 8μm pore-size permeable polycarbonate membrane separated the compartments. Trans-wells were incubated at 37 ◦C in a humidified atmosphere, and cells were allowed to migrate for 2, 4, 6 and 8h toward probiotic lysates present in the lower well of the chamber. After incubation, the membrane was treated with 100% methanol for 20 minutes and then stained with 50μl of 0.1% Crystal Violet solution for a further 20 minutes. Non-migrating cells in the upper well were removed with a cotton swab, whereas cells adhering to the lower surface of the membrane were considered as migrated. The chamber was then mounted on a glass slide and the number of migrated cells was determined by counting them using a Keyence microscope (Keyence, Osaka, Japan). Images from three different areas of the membrane were acquired and processed using ImageJ 64 software program. Using this software, cells were counted at these randomly picked fields and the average calculated for three individual experiments and thensubtracted from the initial number of cells seeded i.e. 2.5×105. The results were taken three times with triplicate samples within each individual experiment.

2.5.3 Keratinocyte proliferation assay The proliferation assay (CellTiter 96® AQueous) was performed in line with the manufacturer’s instructions (Promega, Madison, USA). Cells were seeded at an initial density of 2.5×105 cells per well in 96-well plates. After 24h incubation, cells were

70 exposed to 100μl of probiotic lysates. Plates were then incubated at 37oC, for 6, 12, 18 and 24h. Once each time point was reached, the medium was aspirated and replaced with 100μl per well of the CellTiter 96® AQueous. After 3h of incubation, the absorbance of the solutions was measured at 490nm in a Titertek micro-plate reader (Flow Laboratories Ltd.UK). The absorbance of cells in the lysate-treated wells was compared with that in their untreated counterparts.The control absorbance was normalized to 2.5×105 cells absorbance (original seeding number), while that of treated cells was normalized to control. This was because absorbance is directly proportional to the number of living cells in a culture. The experiment was conducted three times with triplicate samples within each individual experiment.

2.5.4 Ex-vivo wounded model (human skin) Once informed consent and ethical approval were granted (University of Manchester, UK), human skin was obtained from three participants who had undergone elective cosmetic surgery (one 47 year old female, and two males aged 45 and 47 respectively). The “punch-in-a-punch” design of Moll et al. (1998) was utilized. First, 6 mm incisions were made in the skin using a biopsy punch (PFM, Köln, Germany). Smaller (2 mm) biopsy punches were then used to create “punch-in-a-punch” skin wounds. These were positioned individually, with the epidermis facing up, in 12-well plates and cultured in serum-free Williams E medium (minimal medium), supplemented with (10 μg/mL insulin, 10ng/mL hydrocortisone, 2mM L-glutamine and 100IU/mL penicillin / 0 10μg/mL streptomycin) in 37 C, 5% CO2 incubator. The skin was cultured in the absence or presence of 500μl of probiotic lysates with medium changes every 48h. Biopsies were harvested at days 0, 1, 3 and 7, then were mixed in a cryopreservation medium such as optimal cutting temperature (OCT, Thermo Fisher Scientific,

Waltham, MA, U.S.A.) compound before freezing in liquid N2.

2.5.5.1 Slide preparation

Frozen tissue were taken out of the liquid N2 and sited on specialized metal grids (Bright Co Ltd, Cambridgeshire, UK) that fit onto the cryostat OTF5000 (Bright Co Ltd, Cambridgeshire, UK). The tissue was cut 5μm thick in the cryostat chamber at −20°Cwithin 1 min of cutting a tissue section; the section was transferred to a microscope slide by touching the slide to the tissue. Then the slides were stored at - 80oC in a slide container.

2.5.5 Histology staining for Ex-vivo wounded model Frozen sections (5μm thick), were taken out of freezer (- 80 °C) and air dried at 37oC for 10 minutes. After that the sections were fixed in 4% w/v Paraformaldehyde in 0.1M Phosphate buffer pH=7.4 at room temperature before the staining. In some protocols, the sections were fixed in 100% Acetone or Ethanol with Acetic acid (2:1) solution for 5 minutes at room temperature. Histology staining was performed by colouring the fixed samples with Mayer’s Haematoxylin (Merck, Darmstadt, Germany) and 0.1% Eosin E (Sigma-Aldrich, St Louis, MO, U.S.A.) as described in Table 5. Images were captured using a Keyence Biozero-8000 Microscope (Keyence Corporation, Osaka, Japan) and analysed using ImageJ 64 software program (obtained from http://imagej.nih.gov). This enables determination of the wound area and quantification of re-epithelialisation by measuring the overall length of newly formed inner epithelial tongue (Figure 8). In separate experiments, the stained sections were used to measure the number of keratinocytes and the thickness of epidermis in 50µm areas. Images were captured at x40 magnification; bars=100µm for three randomly selected fields (Figure 8). The experiment was performed three times with triplicate samples within each individual experiment.

Table 5: Protocol for histological staining with haematoxylin and Eosin Reagents Time Haematoxylin 6 mins exactly

Running tap water 2 mins exactly

0.2% v/vAcid (HCl)/water 20 secs exactly

Running tap water 1 min

37mM ammonia (or blueing reagent) 2 mins All reagents from Running tap water 1 min Sigma-Aldrich, St 70%v/v methanol/water 1 min Louis, MO, U.S.A 0.1%Eosin 3 mins exactly 70% v/v methanol/water 20 secs exactly 100% methanol 15 secs exactly 50:50 100%Xylene/100% methanol 30 secs exactly 100%Xylene 1 min 100%Xylene 1 min 100%Xylene 1 min and mount Mount using Pertex

Thickness of epidermis in 3 selected points by measuring from the top of the stratum corneum to the basement membrane Number of cells in 3 selected areas (H&E staining, Ki-67 and TUNEL staining )

Quantitation of the Pixel Intensity of Immuno-histology Staining (Anti-Cortactin, CK10 and Involucrin) Measuring area 50µm Length of inner tongue /µm

Figure 8: Schematic representation of the measurement scales for full-thickness wound biopsies.Measuring the length of new tongue, number of cells, thickness of epidermis and the Immuno-staining intensity that were all quantified in wounded skin.

2.5.6 Immuno-staining for Ex-vivo wounded skin 2.5.6.1 Ki67/TUNEKL staining Human skin sections (5um) were housed on slides prepared as in section 2.5.2.1. Staining for the proliferation marker Ki67 and the apoptosis marker TUNEL was carried out using the kit exactly as described by the manufacturer (Apoptag ® fluorescein detection kit; Millipore, Watford, UK). Ki67 staining was performed by using Anti-Ki67 primary antibody [SP6] (ab16667, Abcam, Cambridge, UK) at 1:100 dilution. Detection of Ki-67 was performed with Goat anti-mouse-IgG-FITC/- Rhodamine (JIR, Westgrove, PA; USA; 1:200). Images were captured using a Keyence Biozero-8000 Microscope (Keyence Corporation, Osaka, Japan) and analysed with ImageJ 64 software program (obtained from http://imagej.nih.gov). This defined the proliferating and apoptotic cells in wound areas and new epithelial tongue by calculating the number of coloured cells. Images were captured at x60 magnification; bars=100µm and the measurement scale is illustrated in Figure 8.

2.5.6.2 Cortactin staining To assess migration, immuno-staining analysis of migration marker 16-228/Human Anti-Cortactin (p80/85) Antibody (Clone 4F11, Alexa Fluor® 488, Millipore, CA,

USA) was performed on fixed cryo-sections (2.5.6.1). Following overnight incubation with 1:100 dilution of the primary antibodies, immuno-fluorescence was detected using Goat anti-mouse IgG Alexa Fluor® 488 1:1000 dilution (JIR, Westgrove, PA; USA). Images were captured using a Keyence Biozero-8000 Microscope (Keyence Corporation, Osaka, Japan) and analysed using ImageJ 64 software program (obtained from http://imagej.nih.gov). This facilitated the determination of the pixel intensity of Phosphorylation cortactin (migration marker) in wound area and new tongue. Images were captured at x60 magnification; bars=100µm (Figure 8).

2.5.6.3 Cytokeratin 10 andInvolucrin staining To assess cell differentiation, Immuno-staining analysis of differentiation markers, Human Anti-Cytokeratin 10 antibody (ab7797) and Human Anti-Involucrin antibody (ab68) (abcam, Cambridge, UK), was performed on fixed cryo-sections (Table 5). Following overnight incubation with the 1:100 dilution of the primary antibody, immuno-fluorescence was detected using Goat anti-mouse IgG Alexa Fluor® 488 1:1000 dilution (JIR, Westgrove, PA; USA). Images were captured and analysed using a Keyence Biozero-8000 Microscope (Keyence Corporation, Osaka, Japan) and ImageJ 64 software program (obtained from http://imagej.nih.gov) respectively. Images were captured at x60 magnification; bars=100µm (Figure 8). Measuring the pixel intensity was adjacent to the selected wound areas; the following formula was used to calculate the average intensity of the pixels/corrected total cell fluorescence (CTCF). CTCF = Area of selected cell (µm) X Mean fluorescence of background readings. These were used to identify the pixel intensity of Cytokeratin 10 and -Involucrin in the wound area, expressed as the mean ratio of positive layers divided by the total number of epidermal cell layers (mean of three measurements). The experiment was performed three times with triplicate samples within each individual experiment. Table 6shows the general protocol for all previous immuno-staining that were used in this study.

Table 6: A general protocol for immuno-staining of ex vivo-wounded skin The fixed section were pre-incubated with 10% v/v Goat normal For 20 minutes serum in PBS The sections were incubated with the primary antibody Overnight Washing in PBS 3x10 minutes The sections were incubated with secondary antibody For 45minutes Washing in PBS 3x10 minutes For each section, the cell nuclei were visualized with 1 μg of 4,6- For 1 minute diamino-2-phenylindole (DAPI) in 1ml PBS Washing in PBS 3x10 minutes The slides were covered with Fluoromount (Southern Overnight Biotechnologies, Birmingham, UK)

2.6 Microarray study 2.6.1 RNA extraction and concentration Confluent keratinocytes were scratched and exposed to bacterial lysate for 12h at 37°C in 5% CO2. Cells were washed twice in PBS and the total RNA was extracted using 1ml Trizol per 10cm2 of the culture dish (Invitrogen, Life Technologies Ltd, Paisley, UK). Lysing of the cells was performed directly in the culture dish by pipetting them up and down several times. RNA was purified using an Ambion® PureLink® RNA Mini Kit according to the manufacturer’s instructions (Invitrogen, Life Technologies Ltd, Paisley). The RNA concentrations of the samples were determined using the Nanodrop instrument (Thermo scientific, Wilmington, USA).

2.6.2 Quantitative Reverse Transcription Polymerase Chain Reaction (Q-PCR) and Gene-expression microarray assay Following RNA extraction (Section 2.6.1), single-stranded cDNAs were synthesized from total RNA usingTaqMan® Micro-RNA Reverse Transcription Kit according to the manufacturer’s instructions. cDNAwas synthesized by PCR (2720 Thermal cycler, Applied Bio-system, Life Technologies Ltd, Paisley, UK) using 45 cycles of: 30 minutes at 16oC, 30 minutes annealing at 42oC and 30 minutes extension at 85oC, with 10 minutes extra extension for the final cycle. This procedure was conducted usingTaqMan® Gene Expression Master Mix in accordance with the manufacturer’s instructions. TaqMan® Array Human Chemokines is a gene signature plate with TaqMan probes and PCR primer sets for specific Chemokines dried-down in 96-well format (Table 7). The 10

µL of the cDNA for each sample and 10 µL master mix solution were added to each well of the plate and the plate was covered using MicroAmp® Optical Adhesive Film. The plate was centrifuged to mix the solution of the wells at 100xg for 1 min. After that, The reaction was run in thermocycler (Chromo4™ System, Applied Bio-system, Life Technologies Ltd, Paisley, UK) for 40 cycles of 10-minute at 95oC; denaturing occurred for 15 seconds at 95oC and 1minute annealing/extension at 60oC. An additional extension of 10 minutes was allowed for the final cycle. The standard plate format provides real-time PCR results in 30–45 min. The experiment was performed three times with triplicate samples within each individual experiment.

Table 7: Gene signature plates 1 2 3 4 5 6 7 8 9 10 11 12 18S GAPDH HPRT1 GUSB AGTRL1 CXCR5 C5 CCL1 CCL11 CCL13 CCL16 CCL17 CCL18 CCL19 CCL2 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28 CCL3 CCL4 CCL5 CCL7 CCL8 CCR10 CXCR2 CCR3 CCR4 CCRL1 CCRL2 CKLF CLCF1 CMTM1 CSF2 CSF3 CX3CL1 CXCL1 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL16 CXCL2 CXCL3 CXCL5 CXCL6 CXCL9 CXCR4 DARC TYMP GDF5 GPR81 GRB2 HIF1A IFNG IFNGR1 IFNK IL10 IL12A IL12B IL13 IL16 IL18 IL1A IL1B IL1RAP IL2 IL4 IL5 IL6 IL8 IL9 JAK2 MYD88 NFKB1 PF4 PPBP SCYE1 SLIT2 SOCS5 STAT3 STAT4 TLR2 TLR4 TNF TNFRSF1A TNFSF10 TNFSF14 XCL1 XCL2

 Gene-expression microarrays assay In another experiment, the total RNA was extracted from scratched keratinocytes after 12h incubation with or without lysate, and then purified as detailed previously (Section 2.6.1). The samples gathered from three individual set experiments, were passed to a Bioinformatics specialist (Dr Leo Zeef, University of Manchester) in order for him to perform Gene-expression microarrays and data analysis. RNA quality was checked using the RNA 6000 Nano Assay, and analysed on an Agilent 2100 Bioanalyser (Agilent Technologies). RNA was quantified using a Nanodrop ultra-low-volume spectrophotometer (Nanodrop Technologies). Human Genome U133 plus 2.0 AffymetrixGeneChips were run according to manufacturer’s instructions. Bioinformatics analysis was performed with dChip (V2005) (www.dchip.org, Cheng Li and Wing Hung Wong, 2001). The model-based analysis of oligonucleotide arrays and gene expressions analysis were carried out using a Bioconductor as described by Bolstadet al., (2003). Filtering for probe sets with a P≤ 0.05 created a gene list of differentially expressed genes. Furthermore, the expression of a target gene in all treated samples is expressed as either an increase or decrease

76 fold change relative to control. Data was analysed using the Ingenuity Knowledge Base program (IPA, http://www.ingenuity.com/products/ipa) with the aim of identifying the genes related to the wound-healing process.

2.7 Enzyme Linked Immunosorbent Assays (ELISA) Confluent keratinocytes were grown in 24 well plates, in which scratches were made using a sterile 100ul pipette tip, and exposed to 100μl of bacterial lysate for 12h and 18h. This was done to evaluate secreted CXCL2 production by keratinocytes in response to lysate. Following exposure, the spent cell culture fluid yielded from the plates was removed. CXCL2 levels were determined using CXCL2 ELISA development kits (Uscn Life Science, USA), in adherence to the manufacturer’s instructions. By using the same kits, CXCL2 levels were also determined in the spent cell culture fluid from ex-vivo wounded skin culture after 1, 3 and 7 days incubation with lysate or without.

In other experiments, scratched keratinocytes or ex-vivo wounded skin were exposed to bacterial lysate in order to evaluate the production of Fibroblast Growth Factor (FGF 7) from cells. This was in response to lysate after 12h and 18h in cell cultures, or after days 1, 3 and 7 post-woundingin ex-vivo culture. FGF7 level was determined using Human FGF-7 ELISA development kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions.The optical density of wells was determined using a Labtek LT400 microplate reader (Labtech International Ltd, Ringmer, UK) set at 450nm. The experiment was carried out three times with triplicate samples within each individual experiment.

2.8 Statistical analysis All experiments were performed three times with triplicate samples within each individual experiment. For experiments comparing two or more treatments, a two-way ANOVA with post hoc Tukey test were utilised to analyse the main effects of, and interactions between, multiple factors. Dose response and competition assays were analysed using linear regression and two-way ANOVA respectively. Results were considered significant if P≤ 0.05, and all analysis were performed using the SPSS (IBM SPSS Statistics version 16.0) program.

Chapter Three An in vitro investigation into the effects of species of lactobacilli on toxicity of S. aureus to primary human keratinocytes Probiotics have been suggested to prevent or treat a variety of gastrointestinal diseases in adults and children (Rembacken et al., 1999; Szajewska et al., 2001). In particular, the ability of these organisms to help the gut combat infection is proposed by their positive influence on certain conditions, such as “travellers” diarrhoea (Collado et al., 2008). The mechanisms by which probiotics are thought to limit colonisation of the gut by pathogens include:  Increasing epithelial barrier function (Parassol et al., 2005).  Modulation of the immune response, particularly innate immune responses, such as defensin production and cytokine reactions (Borruel et al., 2003; Wehkamp et al., 2004).  Inhibition of pathogen adherence and down regulation of virulence factors (Backhed et al., 2005).  Modifying the pH of the immediate environment through the production of organic acids (Karska-Wysocki et al., 2010).

The potential effects of probiotics on body tissues other than the gut, such as the urogenital tract (Reid et al., 2001) and the mouth (Haukioja et al., 2008), have also been investigated. For example, some studies evaluate the role of lactobacilli in the prevention of urinary tract infections and in encouraging normal vaginal flora to colonise; thereby preventing/reducing bacterial vaginosis (BV) (Reid et al., 2001, Barrons et al., 2008). Other studies have highlighted the impact of lactobacillion oral cavity infections and dental caries, through their ability to produce bacteriocins that can reduce colonisation of, for example, S. mutans (Çaglar et al., 2006; Nase et al., 2001). More recently, the topical application of probiotics has been explored in a number of studies in relation to the potential of probiotic organisms to inhibit pathogenesis (Karska-Wysocki et al., 2010; Hasslöf et al., 2010; Prince et al., 2012).

This chapter examines the possibility of using “topically applied” probiotic organisms, or their extracts, to inhibit a common skin pathogen. S. aureus was selected as the model organism because normally it is a “transient” coloniser of the skin; however, its production of microbial virulence factors can damage and disrupt the epithelial barrier, resulting in diseases, ranging from impetigo to septicaemia (Zanger et al., 2010).

In the current study, monolayers of normal human epidermal keratinocyte (NHEK) were utilised as a model system to screen for possible protective effects of different probiotic species. The aims of this investigation were to:  Demonstrate the growth pattern of each test bacterium used in this study  Investigate the effects of S. aureus on keratinocyte viability  Screen a range of probiotics for their ability to protect human keratinocytes from the effects of S. aureus by measuring cell viability.  Investigate the mechanism involved in any observed protective effects.

3.0 Bacterial growth curves Growth curves for L. reuteri. L. rhamnosus GG, L. plantarum and L. fermentum were generated following inoculation of the bacteria into Wilkins-Chalgren Broth (WCB). This was done to determine the time point at which each organism entered the stationary phase of growth. This refers to the time at which the total cell absorbance (as measured by optical density) reaches maximum value and apparent net growth ceases. This is important because some probiotics may produce bacteriocins during the stationary phases of growth, which may affect the pathogen toxicity (Ogunbanwo et al., 2003). For S. aureus, the time at which this organism entered the log phase was determined because it is the growth phase in which staphylococcimay produce toxic and adhesion proteins, which may affect the cells (Bowler et al., 2001) (Figure 9).Overnight cultures of the organisms in use were diluted 1:100 in WCB in a 96-well plate. The absorbance of each well was measured automatically every 1h at 660nm for a 24h period; the experiment was repeated three times (Section 2.0). The graphs depicted in Figure 9 were created using the mean values of the absorbance. These demonstrate that the probiotic species began the stationary phases at approximately 10h post-inoculation; whereas S. aureusbegan the log phase at approximately 4h post-inoculation.

A) L. reuteri B) L. rhamnosus GG 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0

0 4 8 12 16 20 24 0 4 8 12 16 20 24

Absorbance 660nm Absorbance Absorbance 660nm Absorbance Time (Hours) Time (Hours)

C) L. plantarum D) L. fermentum 1 1.4 0.8 1.2 1 0.6 0.8 0.4 0.6 0.4 0.2

0.2 Absorbance 660nm Absorbance

0 660nm Absorbance 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 Time (Hours) Time (Hours)

E) S. aureus 1.4 1.2 1 0.8 0.6 0.4 0.2

Absorbance 660nm Absorbance 0 0 4 8 12 16 18 24 Time(Hours)

Figure 9: Growth curves for probiotic species and S. aureus in batch culture Th growth curve of the probiotic species A) L. reuteri, B) L. rhamnosus GG, C) L. plantarum, D) L. fermentum and pathogen E) S. aureus. The stationary phase of probiotic species began at approximately 10h post inoculation for A-C (n=3), whereas, log phase of S. aureus began at approximately 4h post inoculation (n=3).A) L. reuteri (Max SE=0.1), B) L. rhamnosus GG (Max SE=0.2), C) L. plantarum (Max SE=0.2), D) L. fermentum (Max SE=0.1) and E) S. aureus (Max SE=0.01). Error bars (SE) have been shown only at selected time points to allow greater clarity.

3.1 The effect of S. aureus on the viability of keratinocyte monolayers Keratinocytes were exposed to different concentrations of bacterial cells for 24h to determine the effects of the S. aureus on keratinocyte viability. This was monitored using a trypan blue exclusion assay (Section 2.1.1). Trypan blue is a membrane- impermeable dye that penetrates cells with a damaged membrane, allowing differentiation between viable and dead cells. When control (uninfected) keratinocytes were incubated for 24h, approximately 93%±0.1 of the cells remained viable; whereas, the percentage of keratinocytes that remained viable following 24h infection with S. aureus was significantly lower (P=0.02, n=3). At number 105 cfu/ml of S. aureus, approximately 41%±0.3 of the keratinocytes remained viable following 24h exposure to the pathogen. Using higher concentrations of pathogen led to lower keratinocyte viability, e.g. at 108 S. aureus cfu/ml, only ~ 5%±0.4 of the keratinocyte remained viable. At 106cfu/ml of S. aureus 25%±0.3 of keratinocyte were viable after 24h incubation with the pathogen (Figure 10 A). The number 106 cfu/ml is relevant physiologically as this number of organisms has been observed in infected non-healing skin wounds (Bowler et al., 2001; Edwards et al., 2004). Therefore, a time course of keratinocyte viability in the presence of 106cfu/ml S. aureus was also conducted. This number induced significant cell death between 8h and 24h incubation with S. aureus (P=0.04, Figure 10 B). 106S. aureus cfu/ml was chosen for use in further experiments.

A ) 1 0 0 *

8 0

i l

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4 0 %

2 0

0 C o n tr o l 1 0 ^ 5 1 0 ^ 6 1 0 ^ 7 1 0 ^ 8 S .a u r e u s L o g c fu /m l

B ) 1 0 0 * N .S N .S N .S

8 0

t i

l 6 0

i V

4 0 %

2 0

0 U n tr e a te d 2 4 6 8 1 2 1 8 2 4 T im e (H o u r s)

Figure 10: The effect of S. aureus numbers on keratinocyte viability A) After 24h incubation, 93%of untreated cells were viable. The viability of keratinocyte was reduced significantly (Overall P=0.02, n=3) as the number of S. aureus used to infect them increased. B) Time course of keratinocyte viability in the presence of 106 CFU/ml S. aureus.At 106 CFU/ml of S. aureus induced keratinocyte death between 8h and 24h of infection (P=0.04, n=3). Results are expressed as the mean ± SEM,*P<0.05. NS= Non-Significant.

3.2 The effect of probiotic species on the viability of keratinocytes The viability of keratinocytes in the presence of probiotics was assessed. The species selected were L. reuteri, L. rhamnosus GG, L. plantarum or L. fermentum; all used at a concentration of 108 cfu/ml. As highlighted in Section 3.1, 106 cfu/ml of S.aureus

82 induced significant cell death in keratinocytes (P=0.04). Conversely, general exposure of keratinocyte to specific lactobacilli did not result in any significant cell death, compared with untreated cells (P=0.07). However, monolayers of cells incubated with L. fermentum exhibited a reduction in cell viability and only ~45%±0.5 of cells were still live after 24h incubation, compared with 95% in control monolayers (P=0.008, n=3, Figure 11).

1 0 0 *

8 0

y t

i 6 0

4 0 %

2 0

0 C o n tr o l L G G L R L P L F T r e a tm e n ts

Figure 11: The effect of specific lactobacilli species of keratinocyte viability L. reuteri (LR), L. rhamnosus GG (LGG) and L. plantarum (LP) did not cause cell death with 87%±0.4, 88%±0.2, and 81%±0.3 respectively of cells remaining viable following a 24h incubation (P=0.07). However, L. fermentum (LF) reduced keratinocyte monolayer viability significantly to 45%±0.1(P=0.008, n=3) compared to 95%±0.2 in control monolayers. Results are expressed as the mean ± SEM, *P<0.05.

3.3 Specific species of lactobacilli protect keratinocyte from the pathogenic effects of S. aureus An investigation was carried out into the ability of species of lactobacilli to protect keratinocytes from the effects of S. aureus. Keratinocytes were exposed simultaneously to a combination of 106 cfu/ml of S.aureus and 108 cfu/ml of each probiotic, L. reuteri, L. rhamnosus GG, L. plantarum or L. fermentum. Previously, a study in this group demonstrated that L. reuteri protected keratinocytes from S. aureus toxicity; therefore, L. reuteriwas tested as a comparator. Monolayer of keratinocytes incubated with pathogen and either L. reuteri or L. rhamnosus GG were found to have significantly higher percentage viabilities (55%±0.4 and 60%±0.3, P=0.01 and P=0.001 respectively, n=3) than monolayers infected with pathogen alone (Figure 12A & B). However, no significant difference was noted in the percentage of viable keratinocytes in monolayers infected with S. aureus and L. plantarum (39%±0.2, P=0.07, n=3) compared to keratinocytes infectedwith S. aureus alone. A combination of S. aureus and L. fermentum significantly diminished keratinocyte viability (27%±0.4, P=0.01, Figure 12A &B).

B ) * 1 0 0 *

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t i

l 6 0

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4 0 %

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0 C o n tr o l S A L G G + S A L R + S A L P + S A L F + S A T r e a tm e n ts

Figure 12: Effect of lactobacilli on the viability of keratinocyte monolayers infected with S. aureus A)Representative images of infected cells stained with trypan blue (magnification x60) which were used to assess viability. B) Uninfected cells had a mean viability 90% whereas the mean viability of S. aureus infected cells was 25%. Keratinocytes infected with a combination of S. aureus and L. rhamnosus GG (LGG+SA) had a mean viability of 60% (P=0.001). The viability of S.aureus and L. reuteri (LR+SA) infected monolayers was 55% (P=0.01), and the protection afforded by L. rhamnosus was significantly higher than thatafforded by L. reuteri (P=0.04). A combination of S. aureus and L. plantarum (LP+SA) resulted in 39% viability (P=0.07) and a combination of S. aureus and L. fermentum (LF+SA) resulted in 27% viability (P=0.01, n=3). Results are expressed as the mean ± SEM, *P<0.05.

3.4 The effect of L. rhamnosus GG lysates and spent culture fluid on keratinocyte monolayers infected with S. aureus. Of all the lactobacilli screened, L. rhamnosus GG was the most efficacious species with respect to its ability to protect keratinocytes from pathogens. Next, the effects of bacterial lysates (Section 2.0.3) and spent bacterial culture fluid (Section 2.0.2) were examined to test whether a live organism is required in order for the effects to be observed. Bacterial lysates or spent culture fluid did not impact significantly on the viability of keratinocytes (P=0.08). However, 100μl of L. rhamnosus GG lysate or spent culture fluid (extracted from 108cfu/ml of L. rhamnosus GG) reduced the toxicity of S. aureus to the extent thatthe viability of infected keratinocytes was 65%±0.2 and 57%±0.3 respectively, compared with 25%±0.4 in those infected solely with S. aureus (P=0.006 and P=0.01 respectively, n=3, Figure 13 A & B). Formerly, a study in this group established that L. reuteri cells or lysate protected keratinocytes from S. aureus toxicity (Prince et al., 2012); however, the spent culture fluid was not tested. Therefore, L. reuteri lysate and spent culture medium was also tested as a comparator. Keratinocytes exposed to a lysate (extracted from 108 cfu/ml of L. reuteri) had a 53%±0.1 significant percentage of viable keratinocytes (P=0.02, n=3) compared with keratinocytes infected with S. aureus alone.Interestingly, L. rhamnosus GG lysate had a more significant protective effect from S. aureus toxicity on keratinocytes than L. reuteri lysate (P=0.03, n=3). However, the spent culture fluid from L. reuteri had no protective effect on keratinocyte monolayers.

B ) 1 0 0

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y t

i 6 0

4 0 %

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0 C o n tr o l S A S A + S A + L G G L G G L G G C M L G G L Y S C M L Y S

T r e a tm e n ts

C ) 1 0 0

8 0

y * t

i 6 0

4 0 %

2 0

0 C o n tr o l S A S A + L R S A + L R L R L R C M L Y S C M L Y S T r e a tm e n ts Figure 13: Lysate or spent culture fluids from L. rhamnosus GG protect keratinocyte from the effects of S. aureus A)Illustrative images of infected cells stained with trypan blue (magnification x60). B) Keratinocyte exposed to a lysate (LGGLYS) or spent culture fluid (LGGCM) had significantly higher percentage viabilities (P=0.006 and 0.01 respectively, n=3) than keratinocyte exposed to S. aureus alone. C) Keratinocyte exposed to a lysate of L. reuteri (LRLYS) had a significant percentage of viable keratinocytes (P=0.02, n=3) but the same effect was not found with the spent culture fluid of L. reuteri (LRCM). Results are expressed as the mean ± SEM,* P<0.05.

3.5 The protective effect of L. rhamnosus GG or lysate is not time-dependent The next aspect of investigation focused on the timing of the protective effect of L. rhamnosus GG. This was achieved by adding the live bacteria, the lysate or the spent culture fluid either pre or post-infection with S. aureus. The percentage of viable keratinocytes was significantly greater in monolayers exposed to L. rhamnosus GG for 2h prior to being infected with S. aureus (58%±0.2, P=0.006), than in those infected solely with S. aureus (25%±0.1). Both the lysate and spent culture fluid displayed similar levels of protection; the percentages of viable keratinocytes were 57% ±0.4 and 55%± 0.4respectively (P=0.005, 0.004 respectively, n=3, Figure 14). During the ‘postexposure’ experiment, keratinocytes were exposed to S. aureus for 2, 4, 6, 8 and 12h before adding the live L. rhamnosus GG, lysate or spent culture fluid. The viability of the monolayer was then measured at 24h post-infection with S. aureus. The data presented in Figure 15 (A&B) illustrate that both the live probiotic and its lysate could

88 protect the keratinocytes when added following infection with S. aureus. Even at 12h post-S. aureus infection, L. rhamnosus GG or lysate continued to protect the keratinocytes; that are, 58%±0.3 and 55%±0.4 of cells remained viable respectively, compared with 25%±0.2 exposed to S. aureus alone (P=0.003, 0.01 respectively, n=3). However, the spent culture fluid from L. rhamnosus GG had no protective effect on keratinocytes when added after S. aureus (Figure 15 C).

1 0 0

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Figure 14: L. rhamnosus GG or lysate and spent culture fluids protect keratinocytes from S.aureus Keratinocytes were treated with either live L. rhamnosus GG (LGG+SA) or a lysate (LGG LYS) or a spent culture fluid (LGGCM) for two hours prior to the addition of S. aureus. In all cases, the percentage of keratinocytes remaining viable following 24h incubation with S. auresus was significantly higher than in cells treated with S. aureus alone (P=0.006, 0.005, 0.004 respectively, n=3). Results are expressed as the mean ± SEM,* P<0.05.

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0 Control SA LGG+ 2h 4h 6h 8h 12h LGG+SA SA Post-exposed Pre-exposed

Treatments B)

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0 Control SA SA+ 2h 4h 6h 8h 12h LGGCM LGG CM + Post-exposed SA Pre-exposed Treatments

Figure 15: L. rhamnosus GG or lysate, but not its spent culture fluid, rescue keratinocytes from S. aureus mediated toxicity A) The viability of uninfected keratinocyte incubated overnight was approximately 90%. The viability of keratinocyte infected with S. aureus (SA) was significantly (P=0.003) higher in cells exposed to live L. rhamnosus GG (LGG) after 12h from infection had begun. B) There was a significant difference between the viability of cells (P=0.01) treated with L. rhamnosus GG lysate (LGGLYS) after 12h from infection had begun, whereas the viability of keratinocyte infected with S. aureus (SA) alone was 25%. C) Cells post-exposed to L. rhamnosus GG spent culture fluid (CM) did not have significant protection (P=0.15) after 12h whereas, the co-exposed cells had a significant percentage of keratinocytes (around 56%) still alive (P=0.01, n=3) compared to 25% of live cells with S. aureus (SA) alone. Results are expressed as the mean ± SEM,* P<0.05.NS= Non-Significant.

3.6 Mechanisms of protection by L. rhamnosus GG 3.6.1 Direct effect on S. aureus growth It has been reported that certain probiotics produce substances that can inhibit the growth of other organisms (Karska et al., 2010). Therefore, the ability of probiotic lysate or spent culture fluid to impede directly the growth of S. aureus was assessed using competition assays. S. aureus cultures were diluted 1:100 in WCB in a 96-well plate before being treated with 100µl of probiotic lysate or spent culture fluid (Sections

2.0.2 & 2.0.3). The absorbance of each well was measured hourly at 660nm over a 24h period (Section 2.2.1). Competition assays demonstrated a significant reduction in S. aureus growth when the pathogen was incubated with 100µl of L. rhamnosus GG lysate (Figure 16A). L. rhamnosus GG lysate was heated by placing it in a water bath for 10 minutes at 100°C, as described in section 2.0.3; after which, 100µl of heated lysate was added to the S. aureus culture. Heating of the lysate resulted in loss of the inhibitory effect on S. aureus growth (P=0.07, n=3, Figure 16 B).

Combinations of S. aureus with either 100µl L. reuteri lysate or the spent culture fluid from L. rhamnosus GG had no effect on the growth of S. aureus, in comparison with untreated cultures (P=0.06, n=3, Figure 16C& D). Moreover, the total number of viable Staphylococcus was assessed in the presence of 100µl ofprobiotic lysate or spent culture fluid following an incubation period of 24h (Section 2.2.3). The number of viable

Staphylococci decreased significantly to 5 log10 cfu/ml±0.4 in the presence of the L. rhamnosus GG lysate, compared with 8 log10 cfu/ml±0.5 for S. aureus grownin isolation(P=0.02, Figure 3.9A) but without spent culture fluid. As a comparison, the effect of L. reuteri lysate on the viability of S. aureus was also investigated. However, no reduction was observed in the number of viable staphylococci present in the L. reuteri lysate. Furthermore, the total number of viable staphylococci in the presence of keratinocytes diminished over time by the L. rhamnosus GG lysate in post-exposed cultures,even after 12h incubation (P=0.05, n=3, Figure 17A). A live-dead count of the culture at 24h also demonstrated a significant reduction in S. aureus cell viability (Figure 17B).

A) 1.60 SA SA+LGG LYS B) 1.60 SA SA+ heated LGG LYS

1.20 1.20 660nm

0.80 0.80

0.40 0.40 Absorbance Absorbance Absorbance 660nm Absorbance 0.00 0.00 0 1 2 4 6 18 20 22 24 0 1 2 4 6 18 20 22 24 Time (Hours) Time (Hours)

C) 1.60 SA SA+LGG CM D) 1.60 SA SA+LRLYS

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660nm 660nm 0.80 0.80

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0.00 0.00 Absorbance Absorbance 0 1 2 4 6 18 20 22 24 Absorbance 0 1 2 4 6 18 20 22 24 Time (Hours) Time (Hours)

Figure 16: The effect of L. rhamnosus GG or L. reuteri lysates and L. rhamnosus GG spent culture fluid on S. aureus growth in competition assay The optical densities of cultures of S. aureus (SA, Max SE=0.1) growing in the presence of (A) L. rhamnosus GG lysate (LGG LYS, Max SE=0.01) or (B) heated L. rhamnosus GG lysate (heated LGG LYS, Max SE=0.02) or (C) spent culture fluid (LGG CM, Max SE=0.01) or (D) L. reuteri lysate (LR LYS, Max SE=0.01) were determined every hour to monitor the growth pattern of the bacteria. The growth of S. aureus in the presence of the L. rhamnosus GG lysates was significantly lower than in S. aureus cultures alone (P=0.02, n=3), whereas the heated L. rhamnosus GG lysate or spent culture fluid and L. reuteri lysate had no significant effect (P=0.06, n=3). Error bars have been presented only for selected time points to allow clarity. The maximum standard error has been specified for each graph.

A )

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Figure 17: The effect of L. rhamnosus GG lysate and spent culture fluid on Staphylococcal viable count A) The number of viable S. aureus (SA) treated with L. rhamnosus GG lysate was 5log10 cfu/ml, compared to 8log10 cfu/ml for S. aureus grownalone in co-exposed cultures.Spent culture fluid (LGG CM) or L. reuteri lysate (LR LYS) did not produce this effect. Additionally, the total number of viable staphylococci in keratinocyte culture was reduced by the L. rhamnosusGG lysate in a post-exposed assay (2-4-6-8 and 12 hours), even after 12h incubation (P=0.05, n=3) compared to S. aureus alone. B) Live- dead staining of S. aureus following 24h exposure to L. rhamnosus GG lysate also demonstrated a significant reduction in bacterial cell viability.Results are expressed as the mean ± SEM,* P<0.05.

3.6.2 Studying the inhibitory effect of L. rhamnosus GG or lysate on S. aureus The anti-microbial properties of L. rhamnosus GG and lysate were evaluated using a spot-on-lawn assay (Section 2.2.2). This highlighted the significant inhibition of S. aureus growth. Zones of inhibition were observed when S. aureus was treated with anaerobic cultures or lysates of the probiotic grown anaerobically. However, no such zone was yielded by organisms when grown aerobically. Zones of inhibition were visible for the live bacterium or lysates, as presented in Table 8 (An example is given in Figure 18).A live L. reuterior lysate was included as a comparator control, but these did not inhibit S. aureus growth under either of the aforementioned conditions.

Figure 18: Zone of inhibition (mm) produced by L. rhamnosus GG bacteria or lysate on pathogenic bacteria using a spot on lawn assay A representative image of the experiment demonstrating that S. aureus (SA) growth was inhibited by either L. rhamnosus GG (LGG) or lysate (LGG LYS) under anaerobic condition, but not under aerobic condition after 24h incubation (Black arrows indicate the spots). However, neither live L. reuteri (LR) nor lysate (LR LYS) inhibited S. aureus growth under either condition.

Table 8: Diameters of inhibition zone (ZOI) for spot-on lawn assays (n=3)

Treatments Zone of inhibition mm Zone of inhibition mm (Aerobic) (Anaerobic) SA+LGG No inhibition 11 1.3

SA+LGG LYSATE No inhibition 18 0.7

SA+LR No inhibition No inhibition

SA+LR LYSATE No inhibition No inhibition

3.6.3 Acid production from L. rhamnosus GG In order to eliminate the possibility that acid production by L. rhamnosus GG was responsible for the inhibition of growth of S. aureus, the pH of spent culture fluid of the S. aureus culture was measured. The pH of the fluid collected from live L. rhamnosus GG, lysate or spent culture fluid with S. aureus and L. reuteri lysate with S. aureus was measured after 24h incubation with keratinocytes (Figure 19A). The mean pH value of culture medium of S. aureus was 7. By contrast, the mean pH value of culture fluid from live L. rhamnosus GG or S. aureus cultures combined with 108log CFU/ml L. rhamnosus GG was 5.1 (P=0.03, n=3). However, the mean pH value of S. aureus cultures combined with probiotic extracts was 7 (Figure 19 A). In addition, a Universal Indicator Stain was added to plates with live L. rhamnosus GG or lysate, an obvious change in the pH was observed in the zones of inhibition between plates incubated with live bacteria, where the acid was produced, and lysate (Figure 19B).

A ) 1 0 * * 8

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Figure 19: Mean pH values of S. aureus cultures after treating with L. rhamnosus GG or its derived material A) Mean pH of S. aureus cultures treated with L. rhamnosus GG lysate (SA+LGG LYS) was 7.51. S. aureus cultures treated with L. rhamnosus GG spent culture fluid (SA+LGG CM) or L. reuteri lysate (SA+LRLYS) was 7.46. However, mean pH of S. aureus cultures treated with108cfu/ml L. rhamnosus GG (SA+LGG) was 5.1 (P=0.03, n=3), *P<0.05. B) Universal Indicator stained spot-on-the-lawn assay for S. aureus with L. rhamnosus GG or lysate. The mean pH of S. aureus agar with L. rhamnosus GG was approximately 4-5 (orange colour) while the pH of S. aureus agar with lysate was approximately 7 (light green colour) in the zones of inhibition (n=3).

3.6.4 L. rhamnosus GG inhibits adhesion of S. aureus to keratinocytes Another mechanism by which live bacteria, lysate or spent culture fluid of L. rhamnosus GG may provide protection to the keratinocytes is through the inhibition of pathogenic adhesion. A previous study in this field proved that agents that inhibit pathogen

97 adhesion to keratinocytes may reduce S. aureus toxicity (Prince et al., 2012). Therefore, a hypothesis was proposed that L. rhamnosus GG protects the keratinocytes through the inhibition of adhesion.

Adhesion assays were performed to determine whether inhibition could be attributed to competition, exclusion or displacement of pathogens from binding sites on keratinocytes (Section 2.2.4). The data presented in Figure 20A highlight that both S. aureus and L. rhamnosus GG adhered to keratinocytes within 1h of cell exposure to bacteria (Figure 20A). Next, an investigation was performed to determine whether L. rhamnosus GG or lysate or spent culture fluid could inhibit S. aureus adhesion to keratinocytes as a result of competing with the pathogen for binding sites on keratinocytes. Figures 20

A&Billustrate that the number of adherent S. aureus was reduced from 7.9 log10 cfu/ml±0.4, to 4.5 log10cfu/ml±0.1, 5 log10cfu/ml±0.3 and 6.2 log10cfu/ml±0.4, respectively (P=0.04 and P=0.003, P=0.03 respectively) when cells were co-exposed to live L. rhamnosus GG or lysate or spent culture fluid alongside S. aureus. In comparison, the lysate, but not the spent culture fluid of L. reuteri also reduced the numbers of S. aureus bound to keratinocytes (Figure 20B). L. salivarius, which does not protect keratinocytes, did not reduce pathogen adhesion either as a lysate or spent culture fluid, which concurs with the findings of Prince et al (2012).

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T r e a tm e n ts Figure 20: Live L. rhamnosus GG lysate or spent culture fluids inhibit S. aureus from adhering to keratinocytes A) 108cfu/ml of L. rhamnosus GG (LGG) or 106cfu/ml of S. aureus (SA) bind to keratinocytes after 1h incubation. Cells co-exposed to S. aureus plus L. rhamnosus GG (LGG+SA) had significantly fewer staphylococci adhering to keratinocytes compared to cells infected with S. aureus alone (P=0.04, n=3). B) Cells co-exposed to S. aureus plus L. rhamnosus GG lysate (LGGLYS+SA) or spent culture fluid (LGG CM+SA) had significantly fewer staphylococci adhering to keratinocytes compared to cells infected with S. aureus alone (P=0.003, 0.03 respectively, n=3). L. reuteri lysate (LR+SA) also reduced S. aureus adhesion to keratinocytes (P=0.04, n=3). L. rhamnosus GG lysate

99 showed significant anti-adheison activty compared to L. reuteri lysate (SA+LR LYS) or L. rhamnosus GG spent culture fluid (SA+LGG CM) (P=0.01, n=3). However, keratinocytes exposed to L. salivarius lysate (SA+LSLYS) or spent culture fluid (SA+LSCM) did not affect S. aureus adhesion to keratinocytes (P=0.07, n=3).Results are expressed as the mean ± SEM, * P<0.05.

Next, the ability of L. rhamnosus GG or its extracts to reduce the adhesion of S. aureus by exclusion was investigated. The data shown in Figure 21 demonstrate that L. rhamnosus GG, lysate or spent culture fluid were able to reduce the pathogen binding if cells were pre-exposed to live L. rhamnosus GG, lysate or spent culture fluid for 2h before adding the S. aureus. The number of adherent S. aureus was 5.73log10 cfu/ml±0.4, 5.9 log10 cfu/ml±0.2 and 6.4 log10 cfu/ml±0.1 with L. rhamnosus GG, lysate and spent culture fluid respectively, compared with 7.9 log10 cfu/ml±0.3 of adhering S. aureus with cells alone (P=0.04, n=3).

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L G G S A L G G + L G G L Y S + L G G S A S A C M + S A T r e a tm e n ts Figure 21: L. rhamnosus GG lysate or spent culture fluid inhibit S. aureus from adhering to keratinocytes by competitive exclusion Pre-exposed cells with live L. rhamnosus GG (LGG+SA), lysate (LGGLYS+SA) or spent culture fluid (LGG CM+SA) had significantly fewer staphylococci adhering to them compared to cells infected with S. aureus (SA) alone (P=0.04, n=3). * P<0.05.

Furthermore, Figure 22 shows that if live L. rhamnosus GG, or lysate was applied to cells 12h after the initiation of S. aureus infection, a significant reduction was observed

100 in S. aureus adhesion to keratinocytes. Approximately 5.5 log10cfu/ml, and 5.4 log10cfu/ml of S. aureus adhered to cells treated with live L. rhamnosus GG and lysate respectively (P=0.01, 0.02 respectively, n=3).However, addition of the probiotic spent culture fluid subsequent to pathogen infection did not decrease the number of adherent staphylococci to keratinocytes (P=0.06, n=3, Figure 22).

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S A L G G -1 2 h L G G L Y S L G G C M - 1 2 h -1 2 h T r e a tm e n ts Figure 22: L. rhamnosus GG inhibited S. aureus from adhering to keratinocytes by competitive displacement Post-exposed cells with L. rhamnosus GG (LGG-12h) or lysate (LGGLYS-12h) had significantly fewer staphylococci adhering to them after 12h compared to cells infected with S. aureus (SA) alone (P=0.01, 0.02 respectively, n=3). However, the post exposed cells with L. rhamnosus GG spent culture fluid (LGG CM-12h) did not reduce the number of adherent S. aureus (P=0.06, n=3). Results are expressed as the mean ± SEM, * P<0.05.

3.7 Discussion This study examined whether enteric probiotics, particularly Lactobacillus species, could protect keratinocytes from Staphylococcus aureus toxicity. Human keratinocyte cell cultures were used as a model. The staphylococci colonise epithelial surfaces and cause a variety of cutaneous and systemic infections as a result of microbial virulence factors. These disrupt the epithelial barrier by inducing cell death in keratinocytes (Keiji et al., 2006). Therefore, initial experiments determined the effect of S. aureus on keratinocyte viability. The

101 staphylococci reduced significantly the viability of keratinocyte monolayers when cells were incubated with the pathogen for 24h. This occurred in a dose and time-dependent manner (Figure 10 A&B), which is in accordance with another report produced on the toxic effects of S. aureus on keratinocytes (Prince et al., 2012).

In contrast to S. aureus, L. reuteri, L. rhamnosus GG and L. plantarum, exhibited no significant effects on the viability of keratinocyte monolayers following 24h incubation (Figure 11). However, L. fermentum may have a toxic effect on keratinocytes as it reduced the viability of keratinocytes to 45% (Figure 11). This suggests that care must be taken when selecting species for use on skin, and appropriate experiments should be conducted to assess potential toxicity.

Incubation of cells with S. aureus and L. reuteri, or with S. aureus plusL. rhamnosus GG, yielded a protective effect. This could be noted in the substantial increase in the number of viable keratinocytes, in comparison with keratinocytes infected solely with S. aureus (Figure 11 A&B). This protective effect was species-specific, as demonstrated by L. plantarum, which afforded less protection than L. reuteri or L. rhamnosus GG. Conversely, L. fermentum was unable to protect keratinocytes from S. aureus-induced cell death; rather, it caused more cell death (Figure 11). Thus, the protective effect depends entirely on the specific species of bacterium. Even within particular genera, the species is critical. This is evidenced by the fact that not all lactobacilli tested were able to confer protection to keratinocytes in the presence of pathogens.

L. rhamnosus GG was the most efficacious species with respect to the ability to protect keratinocytes from pathogen; therefore, it was subject to further examination. For this organism, the protective effect did not require viable bacteria because a lysate or the spent culture fluid also afforded protection from S. aureus to keratinocytes (Figure 12). This could be a vital observation, as it suggests the use of lactobacilli on skin will not be limited necessarily by the need to keep them viable; thereby simplifying the formulation process. Moreover, it proposes the negation of potential safety concerns by applying live bacteria to skin. This will be especially important for conditions where the skin barrier is breached and where live lactobacilli could enter the blood stream. In comparisons of two materials derived from L. rhamnosus GG, the lysate afforded higher protection against S. aureusthenspent culture fluid. The timing of the application of L.

102 rhamnosus GG or lysate did not affect the degree of protection conferred by the probiotic lysate against S. aureus induced cell death; keratinocytes co-exposed, preexposed or post-exposed to L. rhamnosus GG lysate were afforded equal protection(Figures 13&14). However, the spent culture fluid only protected keratinocytes when added either before or at the same time as the pathogen. These data suggests that two possible separate mechanisms are involved in the protective effects of L. rhamnosus GG.

One mechanism of protecting keratinocytes may be to reduce S. aureus growth, which is a feature of the lysate but not the spent culture fluid. This was demonstrated through competition assays and viable counts of staphylococci. In all these tests, the numbers of viable staphylococci were reduced by the presence of the lysate (but not the spent culture fluid). This direct effect of L. rhamnosus GG on pathogenic growth appeared to be species-specific because the lysate from L. reuteri, made following an identical method had no effect on the growth of S. aureus (Figure 15 D). Furthermore, the effects of the L. rhamnosus GG lysate on the growth of S. aureus could be negated by heating the lysate to 100oC for 10min; thus suggesting the existence of a heat labile molecule, mediating the effects. It is possible that this molecule(s) may be synthesized but not secreted because the lack of impact of L. rhamnosus GG spent culture fluid had no impact on the viability of S. aureus (Figure 16&17). On the other hand, it is also possible that the molecule is secreted but that the effective concentration in the spent culture medium is insufficiently high for the protective effects to be observed. If L. rhamnosus GG contains bacteriostatic substances, then this may also explain at least in part, the protective effect of the probiotic in keratinocyte survival assays.

Probiotics, particularly lactobacilli, have been shown previously to exert a strong inhibitory effect on S. aureus viability. Certain species or strains of Lactobacillus have been reported to be highly antagonistic to bio-film-forming S. aureus (Ammoret al., 2006a; Charlieret al., 2008; Hernandez et al., 2005). Other studies have reported that probiotics can improve gut health by inhibiting microorganism’s growth through production of several anti-microbial compounds, for example, bacteriocins that impede the growth of the other bacteria (Brassart and Shiffrin, 1997; Rolfe et al., 2000; Servin and Coconnier, 2003). Moreover, probiotics have been demonstrated to exert protective effects against pathogen-induced cell death in other epithelia. For example, a study

103 examined the impact of oral administration of Lactobacillus strains in a clinical trial of women with Bacterial vaginosis (BV). The results indicated that Lactobacillus strains could inhibit and prevent the colonisation of uro-epithelial cells by pathogens (Reid et al., 2001). Another study investigated the potential of probiotic strains for oral health. The impact of ingestion of milk containing L. rhamnosus GG on Streptococcus species in the mouth was examined. The study demonstrated that L. rhamnosus GG could inhibit pathogen growth; thereby resulting in a significant reduction in dental caries over a 7-month period (Naseet al., 2001).

Acid production by probiotics is a common method of protection against pathogens in the gut (Asahara et al., 2004) and uro-genital tract (Marianelliet al., 2010). However, although live L. rhamnosus GG did produce acid, it is not a likely mechanism to explain the effects of L. rhamnosus GG lysate. The pH of cell cultures exposed to S. aureus with or without L. rhamnosus GG lysate did not differ from the pH of media containing no bacteria. This hints towards the existence of another mechanism that is responsible for this inhibition of growth of S. aureus. In addition, the lysate was prepared in a buffered medium (pH 7.0); thus confirming that the acidity is it not a likely mechanism. Furthermore, a lysate of L. reuteri did not affect the growth of S. aureus, despite this bacterium being known to produce acid (Flynn et al., 2002). Hence, although this does not rule out the possibility of acid production by L. rhamnosus GG, it does suggest that it is not a main mechanism by which the lysate inhibits S. aureus growth.

Another mechanism by which live bacterium or lysate, or spent culture fluid of L. rhamnosus GG, may protect keratinocytes is by inhibition of pathogenic adhesion. Data gathered from the present study highlight a reduction in the adhesion of S. aureus to keratinocytes when the bacterium or lysate or spent culture fluid were added before or at the same time as the pathogen (Figures 18&19). This suggests a mechanism of competitive exclusion, as observed previously for L. reuteri (Prince et al., 2012). However, interestingly, live L. rhamnosus GG or lysate (but not spent culture fluid) can also inhibit adhesion of S. aureus when added to existing infections. These data suggest that another mechanism of protection mediated by L. rhamnosus GG such as competitive displacement of the pathogen (Figure 19). Species-specific effects were also observed because L. salivarius lysate or spent culture fluid did not affect S. aureusadhesion to keratinocytes (Figure 19B). Live L. salivarius also did not protect

104 keratinocytes from pathogenesis,as was observed previously in Prince et al. (2012). All these observations confirm a link between adhesion and the protection against S. aureus toxicity.

As mentioned previously, the presence of multiple mechanisms may hint at the involvement of separate molecules in triggering these effects. However, at present, it is not possible to rule out the chance that the displacement of pathogens may be related to the inhibition of pathogen growth. L. rhamnosus GG lysate reduced the population of pathogenic bacteria by 5 log10 cfu/ml, a level that was less than that found in the control. Since the spent culture fluid from L. rhamnosus GG did not displace, but only excluded, the pathogen, the ability of L. rhamnosus GG to exclude and displace pathogens from keratinocytes may be based on different molecules.

The results of this study concur with other reports evaluating the ability of different probiotics to reduce pathogenic adhesion. For example, Lactobacillus species can inhibit Enterobacter sakazakii adhesion to intestinal mucus by competitive exclusion (Collado et al., 2008). Another study has demonstrated that some probiotics, such as L. plantarum strain 299v and L. rhamnosus GG, inhibit the adherence of Escherichia coli to intestinal epithelial cells through promoting the expression of mucin MUC2 (Mack et al., 1999). Furthermore, work in vitro has suggested that probiotics use various mechanisms to inhibit pathogens, including direct competition or displacement for binding sites on epithelial cells. For example, Satu and colleagues (2006) found that certain lactic acid bacteria, mainly L. rhamnosus GG, were able to protect the intestinal cells by reducing the adhesion and viability of S. aureus. The authors found that the number of adherent S. aureus in human intestinal mucus was reduced by 39–44% after treated with L. rhamnosus GG (Satu et al., 2006).

In summary, the data presented in this chapter suggest strongly that the protective effects observed from L. rhamnosus GG against S. aureus rely on two mechanisms: first, the direct effect of probiotic lysate on the pathogen growth (bacteriostatic effects); second, the inhibition of the binding of S. aureus to keratinocyte by competitive exclusion or competitive displacement to binding sites (anti-adhesion effects). Since L. rhamnosus GG lysate is also extremely effective against S. aureus, the utility of L. rhamnosus GG on skin will not be limited by whether it can grow and survive on skin

105 because a lysate of the organisms is just as efficacious as live bacteria. Furthermore, the lysate could be useful as prophylaxis, e.g. in hand washes, and potentially as an adjunct or even an alternative to antibiotics in existing infection. Nevertheless, further research is required to distinguish between the protective mechanisms of L. rhamnosusGG lysate against S. aureus infectionand determine the exact molecule(s) that response for this protective effects. For this reason, the purification L. rhamnosus GG lysate and activity of eachpartially purified against S. aureus were investigated. This is explored further in Chapter 4.

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6 Chapter Four

Fractionation of Lactobacillus rhamnosus GG lysate

The investigations conducted in Chapter 3 demonstrated the ability of L. rhamnosus GG lysates to protect human keratinocytes from the toxic effects of S. aureus. This chapter will examine the constituents of the lysates following fractionation. This was done for two reasons: i) in an attempt to identify the molecules within the lysate that may be efficacious in preventing pathogen-induced cell death and ii) fractionation of the lysate may help provide clarification on whether the inhibition of S. aureus growth by the probiotic is linked to the ability of the Lactobacillus to displace pathogens from keratinocyte binding sites. Thus, this approach may determine whether the same molecules involved in both activities. In this study, a series of Reverse Phase Liquid Chromatography (RP-LC) methods were adopted to partially purify the lysate in combination with functional assays to eludicate in which fractions the efficacious molecules were contained. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to assess the number of proteins in the fractions. Finally, Mass Spectrometry-based protein sequencing was used to identify putative proteins in the fractions. In summary, the aims of this study were to:  Fractionate L. rhamnosus GG lysate  Determine which fractions of the lysate have the ability to inhibit the growth or adhesion of S. aureus  Clarify the link (if any) between the displacement and the inhibition by the probiotic lysate of pathogen growth.

4.0 Heat-killed or protease-treated lysate does not protect keratinocytes from S. aureus. Initially, the possibility that proteins are the main effective molecules involved in the effects of the L. rhamnosus GG lysate against S. aureus was investigated by using heat-killing the bacteria, or by implementing protease treatment of the bacterial lysate. A sample containing 108cfu/ml of L. rhamnosus GG was heated

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by placing it in a water bath for 45 minutes at 85°C, as described in section 2.1.3, before the lysate was prepared (Section 2.1.4). When this was tested in a keratinocyte viability assay (trypan blue exclusion assay, as described in Section 2.4.1), the data yielded that the treated lysates had lost the ability to protect keratinocytes from the pathogen thereby suggesting the involvement of a heat- labile molecule, such as a protein (P=0.08, n=3, Figure 23).In order further to establish whether proteins are the main effector molecules in the L. rhamnosus GG lysate, a trypan blue exclusion assay was performed using a lysate treated with the protease, trypsin, as outlined in Section 2.1.4. Trypsin treatment removed the ability of the L. rhamnosus GG lysate to protect keratinocytes from the pathogen; the results demonstrated no significant protection of infected keratinocytes against S. aureus (P=0.07, n=3, Figure 23).

1 0 0

8 0 *

t i

l 6 0

i V

4 0 %

2 0

0 C o n tr o l S A S A + S A + S A + L G G L Y S H T -L G G P R - L G G L Y S T r e a tm e n ts Figure 23: Heat or protease treatment of the L. rhamnosus GG lysate removed its ability to protect keratinocytes from the effects of S. aureus The viability of S. aureus-infected keratinocytes treated with L. rhamnosus GG lysate (LGGLYS) was 65%±0.2 compared to 25%±0.1 in keratinocytes infected with S. aureus alone (P=0.006). In contrast, heat-killed L. rhamnosus GG lysate (HT- LGG) did not protect infected keratinocytes (P=0.08). Protease treatment (PR- LGGLYS) also resulted in the loss of the ability of the lysate to protect keratinocytes, was not significantly different to that of keratinocytes infected with S. aureus alone (P=0.07). Results are expressed as the mean ± SEM,*P<0.05.

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4.1 Separation of L. rhamnosus GG lysate proteins by Reverse Phase Liquid Chromatography (RP-LC) and analysis by SDS-PAGE The term “chromatography” is defined as the separation of a solute between a stationary (sorbent) and a mobile phase (solvent). In liquid chromatography, the mobile phase is a liquid and the stationary phase is a solid (liquid solid chromatography). Reversed Phase Chromatography is based on the adsorption of hydrophobic molecules (sample/solute) onto a hydrophobic, solid (stationary phase) support in a polar mobile phase. Decreasing the mobile phase polarity by adding more organic solvent, such as methanol or acetonitrile plus 0.1% of trifluoroacetic acid (TFA), reduces the hydrophobic interaction between the solute and the solid support; consequently, desorption occurs. On application to the column, the sample (in this case the L. rhamnosus GG lysate) distributes between the two phases and some molecules will remain fixed in the stationary phase, while others will proceed to the mobile phase. For example, highly polar analytes require low concentrations of organics and a short time in which to elute them from the column; whereas non-polar compounds need higher concentrations and more time. As detailed in Section 2.0.3, a 30ml aliquot of L. rhamnosus GG lysate was fractionated using a Strata-XL column. Separation of protein using this column is based on hydrophobic interaction between the protein and the column matrix. Proteins were eluted using 90% methanol at pH 2, based on the hydrophobic interaction between the lysate proteins and the stationary phase (see above description). The eluted fractions from this step were combined and evaporated for 3h in a centrifugal evaporation system to remove the alcohol. The resulting volume (10ml) was kept for further separation. The aim of this process was not only the removal of unwanted compounds, but also to increase the concentration of the desired proteins into a smaller volume. The proteins eluted after this step, were analysed using SDS-PAGE, and the gel featured in Figure 24A illustrates minimal loss of total proteins during this step.

The second separation method involves a Sep-Pak C18 cartridge containing a hydrophobic silica-based matrix, which also separated the proteins based on their hydrophopicity. A gradient of acetonitrile (10-70% in 0.1%TFA) was used as a mobile phase to elute proteins. Seven fractions, each of 5ml volume, were collected separately according to acetonitrile concentrations and evaporated to remove

109 acetonitrilefor 3h using a centrifugal evaporation system. The remaining volume (1ml) was then analysed by SDS-PAGE (Figure 24).The gel in Figure 24B demonstrates that most of the proteins eluted in 30-60% acetonitrile, with some small molecular weight proteins eluting at lower concentrations. Eluted fractions containing proteins were then tested against S. aureus using a spot-on-lawn assay, trypan blue assay or adhesion assay with keratinocytes. The entire experimental set- up is presented in the flow diagram depicted in Figure 25.

Figure 24: SDS-PAGE separation for L. rhamnosus GG lysate A)Proteins present before (2) and after (3) separation by Strata-XL column; lane (1) shows molecular weight marker (250-10kDa). B) Proteins eluting after separation by Sep-Pak C18 cartridge; elution was performed using a gradient of increasing concentrations of 10–70% acetonitrile with 0.1% TFA. Gels were stained with Instant Blue Staining to visualise protein bands. Lanes (1) show

110 molecular weight marker (250-10kDa) and (2) L. rhamnosus GG lysate protein. Lanes (3) show fractions in 10% acetonitrile, (4) 20% acetonitrile (5) 30% acetonitrile, (6) 40% acetonitrile, (7) 50% acetonitrile, (8) 60% acetonitrile and (9), 70% acetonitrile.

100ml of 108 CFU/ml of L.rhamnosus GG Sonicated for 3minutes 30 mL of L.rhamnosus GG lysate

Filitered using a 0.45µm Exteraction mixture

Strat-XL 60ml column (hydrophobic interaction) To concentrate and clean the lysate Fraction eluted with 90% Methanol pH2 Evaporated for 3h SDS-page assay Collected fraction amounts≈ 5mL of Fraction 10 ml injected Set-pack C18 , 5ml cartridge(hydrophobic interaction)

Seven fractions eluted with increasing concentration of 10-70% acetonitrile with 0.1% TFA Evaporated for 3h Fration collected based on the gradiant Protein concentration assay increase of acetonitrile SDS-page assay

Fractions activities against S.aureus Trypan blue Spot on lawn assay assay Adhesion assay

Figure 25: Extraction and fraction scheme of the L. rhamnosus GG lysate The entire fractionated set up is shown in the flow diagram.

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4.2 Specific L. rhamnosus GG lysate fractions protected keratinocyte from S. aureus infection The fractionated lysate was tested in the trypan blue assay to assess which fractions contained the proteins required for protection of keratinocytes against S. aureus. The results demonstrated that the protective effect was contained within fractions eluting in specific concentrations of acetonitrile; that is, 30%, 40% 50% and 60% acetonitrile. These fractions all reduced S. aureus toxicityto the extent thatthe viability of infected keratinocyte monolayers was 60%±0.2, 48%±0.3, 65%±0.1and 62%±0.3respectively, compared with 25%±0.2 in monolayers infected solely with S. aureus (P=0.04, P=0.01, P=0.04 and P=0.03 respectively, n=3). In contrast, none of the other fractions provided any significant protective effect against S. aureus (overall P=0.07, n=3, Figure 26).

1 0 0

9 0

8 0 * * 7 0 *

* y

t 6 0

i b

i 5 0

% 4 0

3 0 N .S N .S

2 0

1 0

0 C o n tr o l S A L G G 2 0 % 3 0 % 4 0 % 5 0 % 6 0 % 7 0 % L Y S T r e a tm e n ts Figure 26: The protective effect of the lysate is contained within specific fractions of the L. rhamnosus GG lysate The viability of infected keratinocyte treated with whole L. rhamnosus GG lysate (LGGLYS) or fractions of 10-70% acetonitrile eluted from the Sep-Pak C18 column. Fractions were tested in three independent experiments. Asterisk denotes significant protection of keratinocytes from the effects of S. aureus (SA).NS= Non-Significant.

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4.3 Specific fractions of of L. rhamnosus GG lysate inhibit the growth of S. aureus The spot-on-lawn assay was used to investigate the inhibitory activity against growth of S. aureus of the fractions eluted from the Sep-Pak C18 cartridge. The diameter of the resultant zones of inhibition indicated that specific fractions possess inhibitory action against S. aureus growth. Whole lysate, 30% acetonitrile fraction and 60% acetonitrile fractionproduced large zones of growth inhibition when tested against S. aureus, while other fractions did not have any effect against S. aureus; namely, 20%, 40%, 50% and 70% acetonitrile fractions (Table 9). In this experiment, 99.9% acetonitrile plus 0.1% TFA was used as a control to ensure the elution buffer did not have any inhibitory effects (Figure 27).

Table 9: Diameters of inhibition zones forL. rhamnosus GG lysate and its fractions by spot-on lawn assays (n=3)

Figure 27: Specific fractions of the L. rhamnosus GG lysate inhibit the growth of S. aureus in a spot-on lawn assay The fractions eluting in 60%or 30% acetonitrile inhibited the growth of S. aureus, while neither the negative control nor the 20%, or 40% or 50% acetonitrile fractions inhibited S. aureus growth.

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4.4 Specific fractions of of L. rhamnosus GG lysate have anti-adhesive action against S. aureus The mechanism by which L. rhamnosus GG lysate prevents pathogen-induced cell death appears to be due, at least in part, to the inhibition of S. aureus adhesion, as outlined in Chapter 3. Therefore, the ability of the fractions eluted from the Sep-Pak C18 cartridge to inhibit S. aureus adhesion to keratinocytes was tested. The data demonstrated that if infected cells were co-exposed to L. rhamnosus GG lysate or specific fractions (30%, 40%,50%and 60% acetonitrile fractions) together with S. aureus, the binding of S. aureus to cells decreased significantly (P=0.01, P=0.016 P=0.012, P=0.015 respectively, n=3) compared with S. aureus adhesion to cells alone (Figure 28A). However, the fractions eluting in 20% acetonitrile did not hinder the adhesion of S. aureus to cells.

Similar results were observed in cells exposed to S. aureus for 2h before the addition of lysate fractions as a measure of the displacement activity of the fractions. The graph in Figure 28B depicts a reduction in adhesion of S. aureus to keratinocytes in post-exposed cultures. This is due to the proteins eluting in the 30%, 40% and 60% acetonitrile fractions. The numbers of adherent pathogens treated with these fractions were 5.6 log10cfu/ml, 6 log10cfu/ml and 5.9 log10cfu/ml respectively, compared with

8 log10cfu/ml of S. aureus in the absence of any fractions (P=0.034,P=0.035, P=0.033 n=3). Interestingly, the 50% acetonitrile fraction from L. rhamnosus GG lysate was significantly more effective at inhibiting S. aureus adhesion to keratinocytes in post-exposed cultures than in other fractions (P= 0.02, n=3, Figure 28B).

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A ) 1 0 * * 9 * * N .S 8

7 L

6 m

/ * * u

f 5

o 4 L 3

0 S A L G G L Y S 2 0 % 3 0 % 4 0 % 5 0 % 6 0 % 7 0 %

T r e a tm e n ts

B ) * 1 0 * * * 8

L 6

f c

* * g

o 4 L

0 S A L G G L Y S 2 0 % 3 0 % 4 0 % 5 0 % 6 0 % T r e a tm e n ts Figure 28: Specific fractions from L. rhamnosus GG lysate inhibit S. aureus adhesion to keratinocytes A)Cells co-infected with L. rhamnosus GG lysate (LGGLYS) had significantly fewer adherent staphylococci compared to cells infected with S. aureus (SA) alone. The adhesion of the pathogen to keratinocytes was significantly lower in cultures treated with fractions eluting in 30%, 40%, 50% and 60% acetonitrile (P=0.01, P=0.016 P=0.012, P=0.015 respectively, n=3). B) In post-exposed cultures, specific lysate fractions (30%, 40%, 50% and 60% acetonitrile fractions) inhibited S. aureus adhesion to keratinocytes by competitive displacement (P=0.034,P=0.035, P=0.01, P=0.033 respectively, n=3). There was no difference between the numbers of staphylococci adherent to cells exposed to 20% acetonitrile fractions in either

115 assay(P=0.06, n=3). However, there was a significant difference in the number of adherent staphylococci adherent to cells exposed to 50% acetonitrile fraction compared with other fractions in both assays(**P=0.02). Results are expressed as the mean ± SEM,*P<0.05. NS= Non-Significant.

4.5 Anti-adhesive activity of the 50% acetonitrile fraction from L. rhamnosus GG lysate Since the protective effect conferred by the 50% acetonitrile fraction was the greatest, relatively speaking, this fraction was selected to examine its ability to displace S. aureus from keratinocytes following a prolonged period of exposure; for example, 12h from the initial infection. The results presented in Figure 29 show that the fraction eluting in 50% acetonitrile was able to displace significant numbers of S. aureus from keratinocytes; therefore, only 5.4 log10cfu/ml±0.2 of treated S. aureus remained adherent (post-exposed, P=0.002, n=3), compared with 8 log10cfu/ml±0.1 in untreated cultures (Figure 29).

1 0

9 *

7 L

/ u

f 5

o 4 L 3

0 S A C O -e x p o s e d P o s t-e x p o s e d

T r e a tm e n ts Figure 29: The 50% acetonitrile fraction from L. rhamnosus GG lysate reduces S. aureus adhesion to keratinocytes by competitive displacement A 50% acetonitrile fraction caused significant reduction in staphylococci adhesion to cells compared to cells infected with S. aureus (SA) alone in either co-exposed (at the same time, P=0.01) or post-exposed (after 12h from infection has commenced, P=0.002) cultures. Results are expressed as the mean ± SEM,*P<0.05.

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4.6 Further purification of the 50% and 60% acetonitrile fractions from L. rhamnosus GG lysate using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) The investigation described in Sections 4.4 & 4.5 demonstrated that the 50% acetonitrile fractionfrom the lysate was the most effective anti-adhesive fraction; however, it did not inhibit the growth of S. aureus in a spot-on-lawn assay. Conversely, the 60% fraction impeded both adhesion and growth of S. aureus, suggesting that each function is performed by a different protein(s). Further exploration was performed for 50% and 60% acetonitrile fractions by initiating a third round of purification using RP-HPLC coupled with Mass-Spectrophotometer (MS/MS) protein sequencing. The aim of this process was to identify possible candidate proteins and separate those contained in the active fractions. Moreover, it was performed to narrow down the single protein involved in inhibitory assays and identify individual proteins present in specific lysate fractions. The specific fractions (50% and 60%) were applied initially to the column in water containing 0.1% TFA, while the solutes were eluted by the addition of organic solvent to the mobile phase (99.9% acetonitrile combined with 0.1% TFA) as described in Section 4.1. Elution was carried out by a gradient elution, where the amount of acetonitrile is increased over a period of time. In order to make a quantitative measurement of the protein, its peak height was measured based on the UV absorption at 215nm because in HPLC peaks are indicative of eluted proteins; whereas, in pervious purification methods the protein content of fractions could not be determined until all eluted fractions were analysed by SDS-PSGE or used in inhibitory assays.

 Further purification of the 50% acetonitrile fraction using RP-HPLC: The 50% sample was fractionated using a HPLC- C18 reverse phase column adopting a gradient of 0 to 100% acetonitrile. On this occasion, the gradient was applied over a 50-minute period and fractions were collected separately based on the UV absorption at 215nm. The UV maximum plot for fractions is given in Figure 30 and highlights the presence of 4 peaks (F1, F2, F3, F4) eluting from the column at 30-40 minutes (Figure 30A). This hints towards the presence of protein in these fractions, as the increase in peak height in the UV chromatogram is proportional to increase in protein concentration. Furthermore, the HPLC fractions of 50% acetonitrile were analysed using

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SDS- PAGE to confirm that the fractions F1, F2, F3, and F4 contained proteins. Each fraction contained 4-6 abundant protein bands except F4 that has 7 abundant protein bands (Figure 30B). The proteins present in these fractions concentrated into sharp bands that have molecular weights ranging from 50 to 10kDa. However, F4 also contains a band with a higher molecular weight of 100kDa (Figure 30B).

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A) mAU

1200

1000

800 F3

600

F4 F2 F1

400

200

-200

0.0 10.0 20.0 30.0 40.0 50.0

Time (Minutes)

Figure 30: The UV chromatograph and SDS-PAGE of 50% acetonitrile fraction from L. rhamnosus GG lysate A)The UV maximum plot for maxium peak height was observed for four fractions, F1, F2, F3 and F4 between retention times of 30 to 40 minutes. B) SDS-PAGE indicates the bands of fractions that may contain the proteins. MW= molecular weight marker (250-10kDa).

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The effects of four fractions, F1, F2, F3 and F4, on S. aureus were investigated using a trypan blue assay. The viability of keratinocytes infected with S. aureus and treated with 50% acetonitrile fraction was 59%±0.1 or 60%±0.2 with the F4 HPLC fraction, compared with 25%±0.1 for keratinocytes infected with S. aureus alone (P=0.04 and P**=0.003 respectively, n=3). Conversely, the remaining fractions displayed less protection of infected keratinocytes than fraction F4. The viability of keratinocytes infected with S. aureus and treated with fractions F1 was 39%±0.1, F2 was 38.5%±0.3and F3 was 41%±0.2 (Figure 31).

1 0 0

9 0

8 0

7 0 * y

t 6 0 * * *

i b

i 5 0

4 0 %

3 0

2 0

1 0

0 C o n tr o l S A S A + S A + S A + S A + S A + 5 0 % F 1 F 2 F 3 F 4

T r e a tm e n ts Figure 31: The protective effect of the 50% acetonitrile fractions is contained within specific HPLC fractions The viability of keratinocytesinfected with S. aureus and treated either with 50% acetonitrile fraction (SA+50%) or with the F4 HPLC fraction (SA+F4) significantly increased compared to keratinocytes infected with S. aureus alone (P=0.04 and P**=0.003 respectively, n=3). However, the other fractions (SA+F1, SA+F2, SA+F3) afforded less protection to keratinocytes than cells treated with F4 (P=0.02,n=3). Results are expressed as the mean ± SEM,*P<0.05.

Section 4.5 emphasised the anti-adhesive activity as the main mechanism utilised by 50% acetonitrile fraction against S. aureus. Based on this, the effect of fraction F4 on S. aureus adhesion was examined using an adhesion assay. The data indicated that fraction F4 causes a significant reduction in S. aureus adhesion to keratinocytes (P=0.002, n=3), even when added 12h after the addition of the pathogen. There were

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5.5log10cfu/ml±0.2 of adherent S. aureus in treated cultures compared with 8log10 cfu/ml±0.2 of adherent S. aureus to keratinocytes in untreated cultures (Figure 32).

1 0

8 L

m 6

o 4 L

0 S A 5 0 % F 4

T r e a tm e n ts Figure 32: Specific HPLC fraction from 50% acetonitrile fractions reduced S. aureus adhesion to keratinocytes Cells exposed to S. aureus (SA) for 12h and then treated with either 50% acetonitrile fraction(50%) or HPLC fraction F4 (F4) have significantly fewer staphylococci adhering to the cells than cells infected with S. aureus (SA) alone. Results are expressed as the mean ± SEM,*P<0.05.

 Reverse Phase purification of the 60% acetonitrile fraction of the lysate The investigation described in Sections 4.3 established that the fraction eluting in 60% acetonitrile inhibited S. aureus growth and created a significant zone of inhibition in a spot-on-lawn assay. Therefore, the 60% acetonitrile fraction was further fractionated using the C18 reverse phase column (Section 4.6). The UV maximum plot for the fraction elution is illustrated in Figure 33. Protein was detected after 20 and 35 minutes in the UV chromatogram and a general increase in the peak height at these retention times for the compounds was observed in two fractions, R1 and R2 (Figure 33A). The 60% acetonitrile fractions was also analysed by SDS- PAGE as described in Section 4.1 to confirm that the fractions R1 and R2 have proteins (Figure 33B). The gel in Figure 33B showed that most of the abundant proteins eluted in fraction R2 about 2 bands with molecular weight 50-37 KDa; whereas no clear protein bands in R1. The effects of these two fractions, R1and R2, against S. aureus infection were investigated by the using a spot-on-lawn assay (Section 2.2.2).

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The data presented in Figure 34 illustrates that fraction R2 produced zones of growth inhibition when tested against S. aureus, while the R1 fraction did not have any effect against S. aureus (Figure 34). However, the zone produced by the R2 fraction was smaller than that produced by the original 60% fraction. A summary flow diagram for L. rhamnosus GG lysate fractions activities against S. aureus can be seen in Figure 35.

A) mAU

1200

1000

800 R2 R1

600

400

200

-200 0.0 10.0 20.0 30.0 40.0 50.0 Time (Minutes)

Figure 33: The UV chromatograph and SDS-PAGE of 60% acetonitrile fraction from L. rhamnosus GG lysate A)The UV maximum plot for maxium peak height was observed for two fractions, R1 and R2, between retention times 20 and 35 minutes. B) The SDS-PAGE assay indicated the bands of fractions that may contain the proteins. MW=molecular weight marker (250-10kDa).

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Figure 34: Spot-on Lawn assay A) Spot-on-lawn assay showing zones of inhibition produced by HPLC fractions from the 60% acetonitrile fraction against S. aureus. R2 produced a zone of inhibition, whereas R1 did not inhibit S . aureus growth. B) The table presents the diameters of inhibition zones for HPLC fractions R1 & R2 (n=3).

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Figure 35: Scheme of activities of L. rhamnosus GG lysate fractions against S. aureus Summary flow diagram for L. rhamnosus GG lysate shows fractions activities against S. aureus.

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4.7 Identification of proteins from the 50% and 60% acetonitrile fractions using Tandem Mass spectroscopy (MS/MS) Identification of proteins contained within the lysate fractions was done by isolating the proteins directly from SDS-PAGE gels (as described in Section 2.3.6), and by analysing them by Tandem MS/MS. This is currently the most widely-used technique for high through protein identification. The approach used was “Gel digestion” (Section 2.3.4), whereby the protein bands were digested usingtrypsin to produce peptides. These were then identified according to their mass, by Tandem MS/MS. The masses of the peptides were then compared with a database containing known protein sequences using the program “Mascot” (http://www.matrixscience.com).Gel digestion-based methods are limited, as proteins below 10kDa diffuse frequently from the gel, and proteins of around 500kDa do not always enter. However, MS/MS is a very sensitive technique to determine the most abundant proteins present in samples within a molecular weight range of approximately 5-500kDa.

 Further analysis of the 50% fraction In order to obtain more information about the protein profile of the 50% acetonitrile fractions and fraction F4 from HPLC, further analysis was conducted using Tandem MS/MS. The scrutiny of the 50% acetonitrile fraction indicated that the fractions contained a complex mixture of proteins. The list of identified proteins is provided in Table 10A; from which, a small number of proteins have been associated previously with anti-adhesive activity for probiotics. For example, elongation factor thermo unstable (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, and triosephosphateisomerase (TPI) are regarded as multifunctional surface-bound proteins with anti-adhesive activity (Bernet et al., 1994; Pancholi et al., 1998). In addition, the same proteins have been identified in fraction F4 from HPLC by Tandem MS/MS (Table 10B). Tables 10 A&B demonstrate that the greater the number of matches with a protein’s unique number in the database, the more certain the identification: i.e., 1 match is a possible identification; 2-3 matches indicate a probable identification; and 4 or more signifies an almost certain identification.

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Table 10: Proteins were identified from L. rhamnosus GG lysate fraction 50% acetonitrile and HPLC fraction F4 by mass spectroscopy (MS) Mass spectroscopy was determined the most abundant proteins present in the (A) 50% acetonitrile fraction and (B) the F4 fraction. The red colour highlighted the proteins that may be related to L. rhamnosus GG lysate functions, particularity are related to anti-adhesion effect against S .aureus that have been published before. A) Identified Proteins Molecular Weight 50% UDP-glucose 4-epimerase OS=Lactobacillus rhamnosus 90 kDa 3 Beta-galactosidase, chain D Beta-galactosidase, chain D 33 kDa 6 30S ribosomal protein S7 OS=Lactobacillus rhamnosus 95 kDa 5 Acyl carrier protein OS=Lactobacillus rhamnosus 9 kDa 5 Glyceraldehyde-3-phosphate dehydrogenase 36 kDa 11 Elongation factor Tu OS=Lactobacillus rhamnosus 43 kDa 10 Transcription elongation factor greA OS=Lactobacillus rhamnosus 23 kDa 4 Phosphopentomutase OS=Lactobacillus rhamnosus 43 kDa 8 Elongation factor Tu OS=Lactobacillus rhamnosus 44 kDa 11 Triosephosphate isomerase OS=Lactobacillus rhamnosus 27 kDa 8 50S ribosomal protein L11 OS=Lactobacillus rhamnosus 15 kDa 5 Dihydroxyacetone kinase OS=Lactobacillus rhamnosus 21 kDa 7 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 6 Asparaginyl-tRNA synthetase OS=Lactobacillus rhamnosus 50 kDa 6 Enolase OS=Lactobacillus rhamnosus 47 kDa 15 GMP synthase [glutamine-hydrolyzing] OS=Lactobacillus rhamnosus58 kDa 4 UPF0342 protein LRH_01568 OS=Lactobacillus rhamnosus 13 kDa 3 30S ribosomal protein S5 OS=Lactobacillus rhamnosus 32 kDa 3 Glucose-1-phosphate thymidylyltransferase OS=Lactobacillus rhamnosus75 kDa 4 Beta-galactosidase, chain D Beta-galactosidase, chain D 33 kDa 6 DNA-directed RNA polymerase subunit alpha OS=Lactobacillus rhamnosus38 kDa 5 Zinc OS=Bacillus pumilus ATCC 7061 GN 23 kDa 5 Phosphoribosylpyrophosphate synthetase OS=Lactobacillus rhamnosus25 kDa 4 Phosphoglycerate mutase family protein OS=Lactobacillus rhamnosus46 kDa 2 Aspartyl-tRNA synthetase OS=Lactobacillus rhamnosus 64 kDa 2 M29 family aminopeptidase OS=Lactobacillus rhamnosus 21 kDa 3 Glycine cleavage system H protein OS=Lactobacillus rhamnosus 11 kDa 2 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 6 UPF0342 protein LRH_01568 OS=Lactobacillus rhamnosus 13 kDa 3 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 6 UPF0342 protein LRH_01568 OS=Lactobacillus rhamnosus 13 kDa 3 B) Identified Proteins Molecular Weight F4 Acyl carrier protein OS=Lactobacillus rhamnosus 9 kDa 1 Glyceraldehyde-3-phosphate dehydrogenase 36 kDa 4 Elongation factor Tu OS=Lactobacillus rhamnosus 43 kDa 5 Transcription elongation factor greA OS=Lactobacillus rhamnosus 23 kDa 3 Phosphopentomutase OS=Lactobacillus rhamnosus 43 kDa 1 Elongation factor Tu OS=Lactobacillus rhamnosus 44 kDa 5 Triosephosphate isomerase OS=Lactobacillus rhamnosus 27 kDa 3 50S ribosomal protein L11 OS=Lactobacillus rhamnosus 15 kDa 1 Dihydroxyacetone kinase OS=Lactobacillus rhamnosus 21 kDa 1 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 1 Asparaginyl-tRNA synthetase OS=Lactobacillus rhamnosus 50 kDa 1 Enolase OS=Lactobacillus rhamnosus 47 kDa 3 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 2 UPF0342 protein LRH_01568 OS=Lactobacillus rhamnosus 13 kDa 2 50S ribosomal protein L22 OS=Lactobacillus rhamnosus 13 kDa 2

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Furthermore, a SDS-PAGE assay (Section 2.5.4) was used to determine the most abundant proteins present in 50% acetonitrile fraction and F4 fraction within the molecular weight range of approximately10-250 kDa (Figure 36). Four dominant protein bands were noted that had molecular weights corresponding to the published molecular weights of enolase, EF-Tu protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphateisomerase (TPI) (Figure 36) (Granato et al., 2004; Ramiah et al., 200) with some small molecular weight proteins (11-13kDa) that are ribosomal proteins.

Figure 36: SDS-PAGE for protein profile of 50% acetonitrile fraction and its HPLC fraction F4 Major abundant protein bands correspond to proteins of molecular weights 64KDa, 47KDa, 44kDa, 36kDa, 27kDa and 13kDa in the 50% acetonitrile fraction and the F4 fraction according to the molecular sizes. Pattern: Molecular Weight (MW) marker, Lane 50% acetonitrile fraction protein bands and Lane F4 fraction protein bands.

 Mass spectroscopy of the 60% acetonitrile fraction of the lysate In order to obtain more information on the 60% acetonitrile fraction protein profile, further analysis was conducted using Tandem MS/MS. This process is described in detail above. Proteins identified from the 60% acetonitrile fraction are listed in Table 11. In fact, the most identified proteins contained in this table are enzymes that contribute to stimulate metabolic pathway, such as carbohydrate catabolism, and have roles to play in the bacteria’s physiological activities (Gorbach et al., 1987; Conway et al., 1987).Based on the list of

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protein identification (Table 11), no conclusive results could be obtained that can be associated with the antibacterial effect of this fraction against S. aureus.

Table 11: Proteins were identified from L. rhamnosus GG lysate fraction 60% acetonitrile by tandem mass spectroscopy Tandem mass spectroscopy determined the most abundant proteins present in the 60% acetonitrile fraction.

Identified Proteins Molecular Weight 60% L-lactate dehydrogenase 2 OS=Lactobacillus rhamnosus 36 kDa 17 Phosphoglycerate kinase OS=Lactobacillus rhamnosus 42 kDa 6 D-fructose-6-phosphate amidotransferase OS=Lactobacillus rhamnosus 66 kDa 12 60 kDa chaperonin OS=Lactobacillus rhamnosus 57 kDa 5 Adenylate kinase OS=Lactobacillus rhamnosus 24 kDa 4 Pyruvate kinase OS=Lactobacillus rhamnosus 63 kDa 4 Uracil phosphoribosyltransferase OS=Lactobacillus rhamnosus 23 kDa 4 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 1 OS 26 kDa 7 Phosphomannomutase OS=Lactobacillus rhamnosus 64 kDa 4 Lactate dehydrogenase OS=Lactobacillus rhamnosus 37 kDa 4 Universal stress protein UspA related nucleotide-binding protein 17 kDa 4 Aldo/keto reductase OS=Lactobacillus rhamnosus 36 kDa 2 50S ribosomal protein L23 OS=Lactobacillus rhamnosus 117 kDa 3 50S ribosomal protein L9 OS=Lactobacillus rhamnosus 49 kDa 4 Glucose-6-phosphate isomerase OS=Lactobacillus rhamnosus 22 kDa 3

4.8 Discussion The data presented in Chapter 3 demonstrated that L. rhamnosus GG lysate has the potential to protect keratinocytes from the toxic effects of the skin pathogen, S. aureus. Three potential mechanisms were identified: inhibition of pathogen growth; competitive exclusion; and displacement of the pathogen from keratinocyte binding sites. In this chapter, the putative molecules eliciting these effects have been investigated.

First, the possibility was considered that the main effective molecules in the lysate were proteins. In order to further explore this, the activity of lysates comprising heat- killed probiotic, or protease-treated lysates, was explored. The ability of these lysates to protect keratinocytes against S. aureus appeared to decrease (Figure 23); thereby proposing that heat labile molecules are responsible for the protective effect, and that proteins are most likely the main effective molecules in the L. rhamnosus GG lysate.

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The proteins were purified in part from the lysate by a series of Reverse Phase liquid chromatography methods based on hydrophopicity. Consequently, the partially- purified proteins were examined for their anti-microbial activities against S. aureus infection. Trypan blue assays were performed and the resulting data demonstrated that specific fractions eluted in 30%, 40%, 50% and 60% acetonitrile protect keratinocytes. However, other fractions eluted in 10%, 20% and 70% acetonitrile did not protect keratinocytes from the pathogen (Figure 24). An adhesion assay also was performed to detect the anti-adhesion activity for lysate fractions, as there is a clear link between adhesion and toxicity of S. aureus (Chapter 3; Prince et al., 2012). The adhesion assay indicated that the 30%, 40%, 50% and 60% acetonitrile reduced the binding of the pathogen to the keratinocytes (Figure 25). Taken together, the data suggest that under the experimental conditions used in the present study, the 30%, 40%, 50% and 60% acetonitrile fractions from L. rhamnosus GG lysate protected keratinocytes from S. aureus infection probably by inhibition of adhesion of the pathogen. A spot-on-lawn assay showed that 30% and 60% acetonitrile fractions had the ability to inhibit the pathogen growth, while other fractions did not (Figure 26). These data suggest that the proteins required to inhibit adhesion may be different from those required to inhibit growth. On the basis of the above findings, and in order to determine the protein profile of L. rhamnosus GG lysate, HPLC coupled with mass spectrometry protein sequencing were used on the fractionated proteins and identified the active proteins in the probiotic extractions, particularly 50% and 60% acetonitrile fractions. These two fractions are the most effective protein fractions in terms of their abilities to work against S. aureus toxicity as anti-adhesion fractions (Figures 26 & 27). The activity of the fractionated proteins was determined by performing adhesion assays for HPLC fractions from 50% acetonitrile. The anti-adhesion activity for F4(HPLC fraction) from 50% acetonitrile was able to compete with the S. aureus adhesion to keratinocyte binding sites and displace S. aureus (Figure 28). MS/MS data for 50% acetonitrile fraction and HPLC F4 fraction indicated the presence of some cell surface proteins of lactobacilli such as the elongation factor Tu, glyceraldehyde- 3-phosphate dehydrogenase, enolase and triosephosphateisomerase (TPI), which were described previously as anti-adhesion proteins (Beck et al., 2009; Izquierdo et al., 2009). These proteins are likely to be the major constituents of fraction F4 because the most abundant proteins in this fraction (as judged by SDS-PAGE) corresponded to the known molecular weights of these adhesion molecules. In theory, this work is

129 incomplete and further investigation is required, such as applying these isolated adhesion proteins (possibly in recombinant proteins form) directly to infected keratinocytes with S. aureus in orderto confirm their ability to reduce adhesion. While it is likely that this proteomic profile (protein purification and function) represents the proteins displayed most abundantly in 50% acetonitrile and HPLC fraction F4 that fractionated from L. rhamnosus GG lysate, identifying the precise proteins would necessitate an additional period of study.

In agreement with the present work, the ability of lactobacilli to adhere to epithelia and prevent colonisation of other bacteria through production or secretion of adhesion and cell wall proteins, “moonlighting proteins” has been previously demonstrated (Morita et al., 2009; Ohashi&Ushida et al., 2009). Moonlighting proteins, categorised by their numerous “autonomous” functions, have been identified in various bacteria. Remarkably, many of these proteins are present in the cytoplasm and contribute to the stimulation of the metabolic pathway and bacterial response. In addition, moonlighting proteins may also be localised to the bacterial surface where they have additional activities, which have been postulated to involve in bacterial pathogenicity or bacterial benefit, such as bacterial interference between different types of bacteria (Pancholi et al., 2003; Henderson et al., 2011). The main recognised functions of moonlighting proteins include adhesion to host epithelia or host components, such as extracellular matrices (ECMs) and plasminogen, in addition to the modification of host immune responses (Pancholi et al., 1992; Seifert et al., 2003; Carneiro et al., 2004; Burnham et al., 2005; Blau et al., 2007; Xolalpa et al., 2007). Surprisingly, many of these proteins, namely, enolase, elongation factor Tu, GAPDH and TPI are present in the L. rhamnosus GG lysate (Tables 10A&B). Hence, these proteins produced from L. rhamnosus GG could be the molecules preventing S. aureus adhesion. Elongation factors are group of cell surface proteins that have been shown to play a potential part in L. rhamnosus GG adhesion. Generally, elongation factors are cytoplasmic enzymes that play an essential role in protein synthesis; however, some studies have suggested that EF-Tu are found on the cell surface of probiotic lactobacilli and bifidobacteria that bind to intestinal epithelial cells, inducing a very different pattern of cytokines and chemokines in comparison with pathogens (Beck et al., 2009; Izquierdo et al., 2009; Ramiah et al., 2008; Ruiz et al., 2009a; Beck et al., 2009; Kelly et al., 2005; Ruiz et al., 2009a; Ruiz et al., 2009a). Furthermore, GAPDH

130 has been suggested as being involved in lactobacilli surface adhesion to fibronectin (Sanchez et al., 2009c) and human colonic mucin (Kinoshita et al., 2008). Enolase from L. crispatus can bind to laminin and collagen I, which reduce the S. aureus adhesion to epithelial cell lines through these binding sites (Antikainen et al., 2007a). Enolase from L. plantarum has been reported as binding to fibronectin and competing with S. aureus adhesion to epithelial cell lines through fibronectin (Castaldo et al., 2009).

Other moonlighting proteins that contribute to bacterial adhesionhave been found in lactobacilli. For example, triosephosphateisomerase (TPI) from L. plantarum plays a role in the adhesion of lactobacilli to Caco-2 cells, and has the ability to compete with pathogens such as Clostridium sporogenes and Enterococcus faecalis by excluding and displacing them from the cell-binding sites (Ramiah et al., 2008; Siciliano et al., 2008; Kainulainen et al., 2012). Interestingly, these probiotic proteins were identified in the current study’s MS/MS data for L. rhamnosus GG lysate (Table 10 A&B), which could elucidate the ability of lysate in the competitive exclusion and displacement of S. aureus from keratinocyte binding sites.

Other types of protein have been reported for their ability to reduce pathogen adhesion. For example, SpaCBA pili on the cell surface of L. rhamnosus GG, mediates its adhesion to the mucus layer of the intestinal tract (Ossowski et al., 2010). In addition, probiotic bacterial adherence is associated frequently with the interference of pathogenic bacteria adhesion (Ohashi &Ushida et al., 2009). Many studies have identified a variety of Gram-positive pathogens or probiotics interfere with each other’s adhesion to the GI tract (Beck et al., 2009; Izquierdo et al., 2009; Ramiah et al., 2008; Ruiz et al., 2009a; Sanchez et al., 2009a). For example, probiotic L. rhamnosus GG cell wall-bound pili (SpaCBA pili) prevents pathogenic bacterial adhesion by competitive exclusion (Kankainen et al., 2009).

Taken together, it can be speculated that L. rhamnosus GG lysate utilises different mechanisms to protect keratinocytes from S. aureus toxicity. The present study indicates that the proteinaceous substances are involved in anti-adhesion activity. Moreover, the study provides evidence of their capacity for interfering with the binding of S. aureus to the keratinocyte binding sites in vitro. This is achieved by

131 displacing the pathogen and preventing the severity of pathogen infection, while other partially purified proteins from L. rhamnosus GG lysate could inhibit pathogen growth and protect keratinocytes from its toxicity. This study postulates that the proteins involved in inhibiting adhesion may differ from those required impeding growth. Further studies are in progress to elucidate the precise anti-adhesion proteins and anti-microbial proteins of L. rhamnosus GG lysate against S. aureus in human keratinocyte models.

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7 Chapter Five

An in vitro study to investigate the potential use of probiotic lysates as a therapy for wound healing A number of studies suggest that enteric probiotics, particularly L. rhamnosus GG, or their extracts may be able to promote ulcer healing in the gut (Lam et al., 2007). This suggests the attractive hypothesis that probiotics may also have the potential to promote cutaneous wound healing. Indeed, some limited data suggest the possibility that specific, live probiotics or their spent culture fluid may be efficacious in the treatment of wounds. However, in general, the mechanisms underlying the effects have not been explored (Peral et al., 2009; Remus et al., 2011). Although probiotic bacteria have GRAS (Generally Recognized as Safe) status in terms of their use in foods, there is no evidence currently that their topical use as live micro-organisms will be safe. Indeed, a probiotic bacterium in a wound site may have the ability to access the circulation, where it is potentially, a hazardous pathogen. For this reason, the use of lysates is considered a safer alternative. In this study, the lysate made from L. rhamnosus GG was tested for its capacity to promote re-epithelialisation using an vitro. Furthermore, the mechanisms underlying this effect were investigated. The specific aims of the study were:  To test the ability of the L. rhamnosus GG lysate in an vitro model of wounding, i.e. keratinocyte scratch assay  To compare the effects of L. rhamnosus GG with those of other lactic acid bacteria on scratched keratinocytes  To determine the effects of L. rhamnosus GG on gene expression on scratched keratinocytes using microarray technology.  Determine the effects of L. rhamnosus GG lysate on protein expression for any genes of interest (identified above).

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5.0 Probiotic lysates modulate the re-epithelialisation of keratinocyte monolayers in a species-dependent manner The scratch assay is a commonly used method of investigating the effects of different treatments on re-epithelialisation. The foundation of the assay is that a “wound” is created in a cell monolayer by making a scratch that removes keratinocytes from the monolayer. “Healing” of this scratch is then observed over time. This assay facilitates the study of an important component of wound healing; re-epithelialisation. Generally, this occurs due to keratinocyte migration, proliferation or a combination of both, resulting in closure of the gap. Factors altering the migration or proliferation of the cells may be able to increase or decrease the rate of “healing” (Lampugnani et al., 1999; Yarrow et al., 2004). A scratch was made in a keratinocyte monolayer using a sterile 100µl pipette tip. Scratched monolayers were treated either with 100µl bacteria lysates (Section 2.0.3), or untreated so as to be used as controls. Scratches were measured at 6, 12, 18 and 24 hours post-scratch by staining cells with crystal violet solution and taking microscope images. These were analysed using Image-J 64 software to determine the scratch area in relation to that taken at time zero (Section 2.3.1,). The following equation was used to obtain quantification of scratch closure by comparing the area of the scratch at time zero with a specific time point: Percentage of re-epithelialisation= {area of the scratch (µm) at t=0h - sample area (µm) at t=t h} / {area of the scratch (µm) at t=0h} x 100 Where t=t is a specific time point post scratching.

The scratch assay was used to determine the effect of L. rhamnosus GG, L. plantarum,L. fermentumand L. reuteri lysates on keratinocyte re-epithelialisation. Compared with the control, 100µl of L. rhamnosus GG lysate accelerated significantly the re-epithelialisation of the monolayer to the extent that by 18h, 95%±0.55 (P=0.03, n=3) of the scratch area was re-epitheliased compared with 72%±0.65 in the control monolayer (Figures 37 A&B). Similar results were found when scratched cells were subjected to treatment by100µl of L. reuteri lysate; whereby 85%±0.55 (P=0.05, n=3) of the scratch area was closed at 18h (Figure 37 A&B). Conversely, L. fermentum lysates caused a significant reduction in the monolayer re-epithelialisation in comparison with the control (31%±0.48, P=0.01, Figure 37 A&B). However, scratches treated with L. plantarum lysate did not heal any differently from the

134 control, untreated monolayers (70%±0.45, P=0.06, n=3, Figures 37 A&B). Keratinocyte Growth Factor (KGF) was utilised as a positive control due to its known role in the wound-healing process (Tsuboi et al., 1993; Sato et al., 1995; Finch et al., 1989). KGF increased significantly the percentage of the re-epithelialisation compared with untreated control (P=0.04, n=3, Figure 37 A&B). Interestingly, both L. reuteri and L. rhamnosus GG lysates created equal promotion in the monolayer re- epithelialisation as KGF.

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Figure 37: Specific probiotic lysates stimulated keratinocytes re-epithelialisation in vitro A) Representative images of monolayer re-epithelialisation in the presence/absence of different treatments. B) The table shows the percentage of scratch re-epithelialisation in cultures treated with/without probiotic lysates at different time points. The red colour highlights significant data. C) Shows the same data graphically.Results are expressed as the mean ± SEM, *P<0.05.

5.1Effect of L. rhamnosus GG and L. reuteri lysates on the rate of keratinocyte migration Since re-epithelialisation could be attributed to keratinocyte migration and/or proliferation, the effect of L. rhamnosus GG and L. reuteri lysateson these keratinocyte activities was tested. A keratinocyte migration assay was performed by plating the cells in the upper chamber of trans-wells and then placing 100μl of probiotic lysate in the lower chamber. The chambers were separated by an 8μm pore- size permeable membrane that enabled the keratinocytes to move down towards potentially chemo-attracting probiotic lysates. At 2, 4, 6 and 8h post-inoculation, the membrane was stained with crystal violet in order to visualise the cells. Non- migrating cells in the upper surface were removed, whereas cells adhering to the lower surface of the membrane were considered to have migrated. The number of migrated cells was determined by calculating the value (mean ± standard error) of the

136 migrated cells from at least three wells for each experimental group in three high- powered fields that subtracted from the number of seeding cells (2.5x105 cells). Images were captured at 40x magnification; Section 2.3.2. There were 48x105±0.32 and 24x105±0.42 migrated cells after 4h in cultures treated respectively with L. rhamnosus GG lysate and L. reuteri lysate (P = 0.001, P= 0.004, respectively, n=3), compared with 7x105±0.22 cells in control cultures (Figures 38 A&B). For comparison purposes, the effect of KGF was also tested as a positive control as a result of its well-documented role in promoting cell migration or proliferation (Tsuboi et al., 1993; Sato et al., 1995; Finch et al., 1989). In this assay, KGF stimulated the migration of 34x105± 0.33 cells. Importantly, a significant difference was observed between two lysates (P=0.002, n=3), with L. rhamnosus GG lysate being the most efficacious in this scenario (Figures 38 A&B). However, L. rhamnosus GG lysate acted similarly to KGF in terms of stimulating keratinocyte migration, while the L. reuteri lysate afforded less stimulation than KGF (Figure 38 A&B).

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Figure 38: L. rhamnosus GG lysate and L. reuteri lysate increased the rate of keratinocyte migration A)Representative images ofmigrated keratinocytes in serum-free keratinocyte medium alone (control) and medium containing L. rhamnosus GG lysate (LGGLYS) or L. reuteri lysate (LRLYS)determined by trans-well chamber assay The black arrows indicate migrated cells (dark purple) at 40x magnification; bars=100µm that were counted from 3 randomly picked fields in membranes. B) Either L. rhamnosus GG lysate or L. reuteri lysate significantly increased the number of migrated cells (P=0.001, P=0.004, respectively n=3). However, L. rhamnosus GG lysate was more efficacious than L. reuteri lysate (P**=0.002, n=3) and had similar effect to KGF. Results are expressed as the mean ± SEM, *P<0.05.

5.2 Effect of L. rhamnosus GG lysate and L. reuteri lysate on the rate of keratinocyte proliferation The second possible contributor to re-epithelialisation (e.g. proliferation) was studied using MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay. This is a colorimetric technique based on quantifying the cell production of Formazan (Tim et al., 1983). The absorbance of Formazan is proportional directly to the number of living cells in culture at different time points of incubation (Section 2.3.3). Incubation of keratinocytes with 100µl of L. rhamnosus GG lysate or L. reuteri

138 lysateresulted in significant proliferation in treated cultures, as double the number of cells was found in these cultures relative to control cultures (P=0.02, P=0.03, respectively, n=3) at 12h post-inoculation (Figure 39). In this experiment, the KGF was used also as a positive control. KGF caused a two-fold increase in the number of cells, compared with the untreated control (Figure 39). Interestingly, both lysates afford equal stimulation of proliferation as KGF to keratinocyte proliferation.

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Figure 39: L. rhamnosus GG lysate and L. reuteri lysate increased keratinocyte proliferation L. rhamnosusGGlysate and L. reuteri lysate increased significantly the cell proliferation by doubling the number of cells compared with control cultures (P=0.02, P=0.03, respectively, n=3) after 12h incubation. KGF increased cell proliferation twice that of the control at 12h. However, no difference was observed between the effects of the three treatments. Results are expressed as the mean ± SEM, *P<0.05.

5.3 Effect of the L. rhamnosus GG lysate and L. reuteri lysate on keratinocyte re- epithelialisation in the presence of Mitomycin C Since the probiotic lysates increased the rates of keratinocyte proliferation and migration, a scratch assay was performed in the presence of the proliferation inhibitor, Mitomycin C (0.5 mg/ml), in order to differentiate between the two possible effects of the lysate. The data presented in Figure 40A illustrate that cells treated with L.

139 rhamnosusGGlysate and Mitomycin C resulted in 94.5%±0.10 re-epithelialisation after 18h incubation (P=0.03,n=3), compared with 57%±0.30 in control with Mitomycin C alone. However, in the presence of Mitomycin C, the L. reuteri lysate- treated monolayer was only 52%±0.32 re-epithelialised, in comparison with 90%±0.13 in cells treated solely with L. reuteri lysate (P=0.01, n=3,Figure 40B).

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0 6 1 2 1 8 2 4 T im e (H o u r s) Figure 40: Efect of L. rhamnosus GG lysate and L. reuteri lysate on keratinocytes treated with Mitomycin C A) L. rhamnosus GG lysate accelerated significantly the cell re-epithelialisation of scratches treated with Mitomycin C by 18h (P= 0.03, n=3). B) In cells treated with L. reuteri lysate and Mitomycin C, only 52% of the scratch area was closed, compared with 90% in L. reuteri lysate alone (P=0.01, n=3). However, the cell re- epithelialisation was 57% in cells treated solely with Mitomycin C, compared with untreated control cultures. Results are expressed as the mean ± SEM, *P<0.05.

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5.4 Genome-wide Affymetrix microarray study of the effects of the L. rhamnosus GG lysate on keratinocyte gene expression and quantitative RT-PCR studies So far, the data presented suggest that the L. rhamnosus GG lysate increases re- epithelialisation by enhancing keratinocyte migration and proliferation. Therefore, the pathways underlying these effects were explored in the following set of experiments. Techniques, such as microarray analysis (gene expression profiling) permit the measurement of thousands of genes in a single RNA sample. Thus, microarray analysis of scratched keratinocytes treated vs untreated with L. rhamnosus GG lysate was chosen as a starting point from which to understand the mechanisms involved. Keratinocyte monolayers were scratched and treated with L. rhamnosus GG lysate for 12h and RNA was extracted and processed using an RNA extraction assay kit (as described in Section 2.5.1). The quantity of extracted RNA was established using a Nano-drop, while triplicate samples were analysed using Affymetrix gene-array chips (Section 2.5.2). A full list of genes whose expression was changed by treatment with the lysate is available in Appendix 2. Further assessment using the ‘Ingenuity Pathway Analysis’ program (IPA-Ingenuity®Systems, www.ingenuity.com) was performed to identify those genes whose regulation altered more than two-fold in the L. rhamnosus GG lysate treated cultures vs untreated controls, and where the P values were <0.05.The entire analysis is outlined in the flow diagram in Figure 41.

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Keratinocyte monolayers were scratched and treated with L. rhamnosus GG lysate

After 12h incubation

RNA was extracted from treated and untreated cells that was performed for three individual experiments

Analysed RNA by using Affymetrix gene array chips to identfiy the gene expressions

List of over 6000 genes whose expression was changed by treatment with the lysate

List of 3700 genes have been selected according to the p values that are <0.05 and the fold changes that are > 2 from 6000 genes(Appendix 2)

Genes were then grouped according to cellular functions by IPA program

Cellular functions related to wound healing process were selected based on the literature reviews (Table 12)

Cell migration and prolifertaion were selected as main mechansims for wound healing process based on the literature reviews

over 1760 genes involved in proliferation and migration altered in response to the L. rhamnosus GG lysate were idenitfied (Appendix 3)

The top10 genes for each mechansim have been selected that have the highest fold changes compared to other genes (Tabls 13 & 14)

Figure 41: Microarray analysis of scratched keratinocytes treated vs untreated with L. rhamnosus GG lysate The flow diagram outlines the entire analysis set up for microarray data (n=3).

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The genes identified in the microarray were grouped using IPA which groups genes according to cellular function. Table 12 considers only those functions relevant to the wound-healing process. This was based on a literature review (Florin et al., 2005; Devalaraja et al., 2005; Miyamasu et al., 1998). From Table 12, it can be observed that more than 1760 genes involved in proliferation and migration, are altered in response to the L. rhamnosus GG lysate with significant p-value <0.05. Interestingly the group that had the most significance was cell movement, but all the genes involved in this were common to the group called ‘cell migration’. The top 10 genes from this common group are presented in Table 13 for cell migration. Table 14 shows the top 10 genes for proliferation which was also demonstrated to be a highly significant group from the micro array analysis. Interestingly, CXCL2 also appears in this group. The complete gene list for these two mechanisms is available in Appendix 3.

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Table 12: Bioinformatic analysi of L. rhamnosus GG lysate effects on cell activities The table summarises the cellular biological processes that related to wound healing process, corrected P-values and the number of associated molecules affected by L. rhamnosus GG lysate. Gene values treated with lysate are considered significant when the overall P-value was <0.05,compared with the control. The data represent three individual experiments.

In Table 13, it can be observed that the expression of two genes in particular was highly regulated by the L. rhamnosus GG lysate. The first gene is CXCL2 and the second is a corresponding receptor, CXCR2 (Table 13). It has been demonstrated previously that keratinocytes express CXCR2, the receptor for IL-8 and CXCL2, and the interaction of keratinocyte CXCR2 with its ligands(s) have been exhibited in both

145 murine and human models as playing an essential role in wound repair by stimulating cell migration (Devalaraja et al., 2000). CXCl2 is a chemokine that stimulates cell movement and chemotaxis was also a group noted in the IPA analysis in Table 12. In addition, the IPA program was used to generate a network interaction of the genes associated with cell migration following exposure to L. rhamnosus GG lysate (Figure 42).

Table 13: Uniprot function of some genes following L. rhamnosus GG lysate treatment The IPA knowledge -base was cross-referenced with the data set for known genes interacting with cell migration. This table lists the top 10 of 100 genes that interact specifically with cell migration and their corresponding P-values, fold changes and their functions according to Uniprot. The highest fold change genes are highlighted in red, such as CXCL2 and its receptor CXCR2. Gene values treated with lysate are considered significant when P-value is <0.05and the fold change is greater than two– fold, compared with control (n=3).

Gene name Fold change p-values Functions CXCL2 19.899 9.12E-10 Chemotaxis, stimulation cytokines and growth factors CXCR2 10.18 1.48E-04 Receptor for IL-8 and CXCL1, stimulate cells migration CCL20 4.505 0.0012 Activate cell-cell signaling, chemoattractant EDN2 5.698 0.001 Stimulate cells invasion and movement TNFRSF18 5.80 0.001 Positive regulation of CXCL2 IGFBP3 4.550 0.002 Regulate protein phosphorylation and cells movement TNF 10.05 0.004 Positive regulation of CXCL2, cytokines activity CXCL10 4.427 0.006 Chemotaxis, cAMP- dependent protein kinase regulator, Cortactin 3.00 0.01 Stimuli to promote and rearrangement of the actin cytoskeleton MAP3K13 3.193 0.001 Positive regulation for cell migration. Activated MAPK cascade

Figure 42: Network of genes associated with cell migration IPA was used to generate a network of the genes (Table 13) associated with cell migration following exposure to L. rhamnosus GG lysate. Solid lines indicate a relationship of expression that increased the cell migration. Red indicates that expression of two top genes is up-regulated, whilst gray indicates up-regulation of other gene expressions. Circles highlight the two main genes investigated in present study; CXCR2 and CXCL2.

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Table 14 highlights the over-expression of candidate genes involved in proliferation, such as FGF7 (Fibroblast growth factor 7) and its receptor FGFR2, which was up regulated respectively 10.89-fold and 9.84-fold greater than the control (Table 14). A number of studies provide evidence of the important role played by FGF7 and its receptor in cutaneous wound repair by stimulating the cell proliferation, either in vitro or in vivo models (Semenova et al., 2008;Turner et al., 2010). However, CXCL2 appears in both proliferation and migration mechanisms as over-expression in response to L. rhamnosus GG lysate. Moreover, the IPA program was used to create a network interaction of the genes associated with cell proliferation following exposure to L. rhamnosus GG lysate (Figure 43).

Table 14: Uniprot function of some genes following L. rhamnosus GG lysate treatment The IPA knowledge-base was cross-referenced with the data set for known genes interacting with cells proliferation. This table lists the genes that interact specifically with cell proliferation and their corresponding P-values, fold changes and their functions according to Uniprot. The highest fold change genes are highlighted in red, such as FGF7 and FGFR2. Gene values treated with lysate are considered significant when P-value is <0.05and the fold change is more than two-fold, compared with the control (n=3).

Gene name Fold change p values Functions CXCL2 19.899 9.12E-10 Activate two pathways MYD88 and NFkB pathways FGF7 10.898 7.59E-03 Mitogenic , activates growth factors signaling FGFR2 9.842 1.48E-04 G-protein coupled receptor binding to FGF7 LEP 9.567 1.06E-03 Activates AMPK signaling, cytokines activity BMP2 9.415 5.08E-03 Activate cells phosphorylation and formation CDKN1B 4.62 5.01E-03 Cyclin-dependent protein kinas inhibitor CXCL1 4.871 5.03E-03 Binds to CXCR2 , stimulates growth factors CXCL10 4.427 5.06E-03 Regulates cAMP- dependent protein kinase CXCL5 3.409 5.09E-03 Binds to CXCR2 to recruit cells, chemoattractant IGFBP3 3.418 1.06E-02 Regulate protein phosphorylation and cells movement

Figure 43: Network of genes associated with cell proliferation IPA was used to generate a network of the genes associated with cell proliferation following exposure to L. rhamnosus GG lysate. Solid lines indicate a relationship of

147 expression that increased the cells proliferation. Red indicates that expression of three top genes is up-regulated, whilst gray indicates up-regulation of other gene expressions. Circles highlight the two main genes investigated in the present study; FGF7 and FGFR2.

Additionally, in order to validate gene expression data of CXCL2 and FGF7, quantitative RT-PCR was performed (Section 2.5.2). Quantitative RT-PCR confirmed that treatment of keratinocytes with L. rhamnosus GG lysate resulted inup-regulation of the expression of these genes. This was given as a fold change in the gene expression related to control after 12h incubation (data not shown).

5.5 Direct measurement of CXCL2 and FGF7 secretion by keratinocyte cultures L. rhamnosus GG lysate up-regulated the gene expression of CXCL2 and its receptor CXCR2; moreover, it stimulated the gene expression of FGF7 and its receptor FGFR2. This suggests that L. rhamnosus GG lysate may accelerate the re- epithelialisation process in keratinocytes by stimulating both the chemokine or growth-factor receptors and their ligands. In order to confirm this hypothesis, the secretion of CXCL2 and FGF7 by scratched keratinocytes in response to L. rhamnosus GG lysate after 12hincubation was measured using ELISA assays (Section 2.6). The results reveal that CXCL2 was secreted at a higher level by L. rhamnosus GG lysate-treated keratinocytes, with 6.30µg/ml±0.4, compared with 3.10µg/ml±0.7 in untreated cells (P=0.002, n=3, Figure 44A). Furthermore, a significant increase was observed in FGF7 production, relative to the control following incubation with L. rhamnosus GG lysate (10.10µg/ml±0.44, P=0.003, n=3); whereas, control cells secreted 5.30µg/ml±0.32 (Figure 44B).

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5.6L. rhamnosus GG lysate alters the expression of CXCR2 and FGFR2 protein receptors in keratinocyte cultures Sections 5.4 and 5.5 highlight that L. rhamnosus GG lysate induced up-regulation in the expression of the mRNA for CXCR2 and FGFR2, in addition to stimulating the production of the corresponding ligands in keratinocytes. Therefore, Western blot analysis was performed to quantify the changes in the levels of CXCR2 and FGFR2 protein receptors post-treatment with L. rhamnosus GG lysate for 12h. Western blot analysis of extracts from cells that had been exposed to L. rhamnosus GG lysate for 12h demonstrated that L. rhamnosus GG lysate caused an up-regulation of CXCR2 protein levels. This was four times greater than the control, and the FGFR2 protein level, which was double that of the control(P=0.0009 and P=0.04 respectively, n=3, Figure 45A & B).

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T r e a tm e n ts Figure 45: Western blot analysis confirmed altrations in the level of CXCR2 and FGFR2 protein receptors post-treatment with L. rhamnosus GG lysate A) Representative images of immuno-blots for CXCR2 and FGFR2. B) Densitometry analysis of the western blots was performed to quantify the change in protein levels for treated cell cultures vs. untreated controls. Changes were normalized against β- actin. L. rhamnosusGG lysate increased CXCR2 protein level and FGFR2 protein level compared to untreated control (P=0.0009 and P=0.04 respectively). Data are representative of three individual experiments and are expressed as the mean ± SEM, *P<0.05.

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5.7 Effect of CXCR2& FGFR2 neutralising antibodies on the healing of scratched keratinocyte cultures Neutralising CXCR2 and FGFR2 proteins with antibodies can provide valuable information pertaining to whether L. rhamnosus GG lysate may mediate acceleration of the re-epithelialisation of scratched keratinocytes through these protein receptors. CXCR2 and FGFR2 receptors in scratched keratinocytes were blocked separately with 2.5μg/ml CXCR2 and 5μg/ml FGFR2 antibodies, and then incubated for 18h and 24h with or without L. rhamnosus GG lysate (described in Section 2.3.1).The re- epithelialisation of these cultures decreased to 45%±0.33 in the presence of the CXCR2 antibody combined with L. rhamnosus GG lysate (P=0.0003). This is compared with a 95%±0.56 re-epithelialisation with cultures treated alone with L. rhamnosusGG lysateat 18h (Figures 46A &47A). Similar results were yielded when scratched cells were subjected to treatment by L. rhamnosus GG lysate with the FGFR2 antibody, as 63%±0.12 (P=0.04) of the scratch area was closed at 18h (Figures 46B & 47B). However, a minor but significant decrease in keratinocyte re- epithelialisation was observed with CXCR2 and FGFR2 antibodies in control cultures. This amounted to 37%±0.12 and 28%±0.16 respectively, compared with 73%±1.19 re-epithelialisation in control cultures alone (P=0.004). A negative control antibody (IgG) was used in this assay, demonstrating that the antibody (IgG) did not affect the cells re-epithelialisation (Figure 46&47 A&B). Notably, even though the receptor was blocked with FGFR2 antibodies, there remained a small stimulation of re- epithelialisation by L. rhamnosus GG lysate that was not observed for CXCR2 in the presence of a blocked receptor (P=0.002, n=3) after 18h.

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Figure 46: Representative images of the effect of CXCR2 and FGFR2 antibodies on cells treated with L. rhamnosus GG lysate Keratinocyte monolayers were scratched and then incubated at 37oC for 18h and 24h in basal serum-free keratinocyte medium, either alone (control) or in medium containing(A) anti-CXCR2 or combination of anti-CXCR2+LGGLYS or L. rhamnosusGG lysate alone (LGG LYS). B) In medium containing anti-FGFR2 blocking antibodies or combination of anti-FGFR2+LGGLYS or L. rhamnosusGG lysate (LGG LYS) alone. A negative control antibody (IgG) did not affect the re- epithelialisation of keratinocytes.

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0 C o n tr o l a n ti-F G F R 2 a n ti- L G G ly L G G ly + L G G ly + Ig G a n ti-F G F R 2 a n ti-Ig G T r e a tm e n ts Figure 47: CXCR2 and FGFR2 blocking antibodies reduced the keratinoyte re- epithelialisation in the presence of L. rhamnosus GG lysate A)Combination of CXCR2 antibody with L. rhamnosus GG lysate (anti- CXCR2+LGGLYS) significantly decreased the re-epithelialisation of the scratch compared with culture treated solely with lysate (LGGLYS) (P=0.0003, n=3). In the control scratched culture, a significant difference was observed between the untreated control to cultures treated with the CXCR2 blocking antibody (anti-CXCR2, P=0.004, n=3) at 18h. B) Combination of FGFR2 blocking antibody with L. rhamnosus GG lysate (anti-FGFR2+LGGLYS) decreased significantly the cell re-epithelialisation of

154 the scratch at 18h (P=0.04, n=3), compared with cultures treated solely with lysate (LGGLYS). Additionally, in control scratched culture, a significant difference was observed between control and treated cultures with FGFR2 blocking antibody (anti- FGFR2, P=0.004, n=3) at 18h. There was also a significant change between control with FGFR2 blocking antibody (anti-FGFR2) andcombination of L. rhamnosus GG lysate plus FGFR2 blocking antibody (P=0.002, n=3) at 18h; whereas, this was not noticed in CXCR2 blocking antibody cultures (A). Data are representative of three individual experiments and are expressed as the mean ± SEM, *P<0.05.

5.8 Discussion Non-healing wounds in conditions such as diabetic ulcers or infected wounds are a significant cause of morbidity and mortality for a large proportion of the population (Wysock et al., 2002). Therefore, more effective and safe treatments are urgently required. In particular, treatment agents are needed to encourage or promote wound re-epithelialisation and reduce infection. Over-use of antibiotics has given rise to many antibiotic-resistant infections that are associated frequently with poor outcomes in wound healing, especially following surgery (Falanga et al., 1993; Dow et al., 1999; Wysock et al., 2002). Currently, there is an unmet, clinical need for new therapies in the treatment of wounds. Since probiotics have been demonstrated to promote wound healing in the gut (Lam et al., 2007; Polkset al., 2007; Remus et al., 2011; Polks et al., 2011), the aim of this study was to examine the effects of enteric probiotic lysates in a model skin wound.

In the present study, the lysates of L. rhamnous GG and L. reuteri were able to accelerate the re-epithelialisation of keratinocyte monolayers. However, this was apparent only at 18h post-scratching whilst other lysates from different probiotics either had no effect on re-epithelialisation, (L. plantarum) or inhibited epithelialisation (L. fermentum) (Figures 37). This is probably related to the observation made previously that L. fermentum reduces keratinocyte viability (Chapter 3, Sultana et al., 2013). The present study examined the effect of the L. reuteri and L. rhamnosus GG lysate on keratinocyte proliferation and migration. The enhancement in re- epithelialisation was probably due to elevation in both keratinocyte migration and proliferation observed with both probiotic lysates, although the two lysate were not equally effective in promoting cell migration. L. rhamnosus GG was much more effective at stimulating migration than L. reuteri (Figures 38&39). However, in

155 vitroscratch assays in the presence of the proliferation inhibitor Mitomycin C demonstrated that migration was the dominant mechanism for L. rhamnosus GG lysate because scratches in cell cultures treated with Mitomycin C were still able to re- epithelialise at a significantly faster rate than untreated scratches (Figure 40). Conversely, the dominant mechanism for the L. reuteri lysate was stimulation of proliferation, as inhibition of this using Mitomycin C negated completely the stimulatory effects of the L. reuteri lysate. Thissuggests that bacterial lysates may have different effects on cell functions.

Previously, the spent culture fluid of L. plantarum has been proven to stimulate re- epithelialisation in the scratch assay via a molecule, which the authors names “Plantaricin A”. This stimulates keratinocyte migration and proliferation (Remus et al., 2011). The authors investigated the mechanisms underlying the improved migration and proliferation, suggesting it to be via increased expression of transforming growth factor (TGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-7) (Remus et al., 2011). In the present study, the effect of spent culture fluid of L. plantarum on the stimulation of keratinocyte re- epithelialisation was not measured. Therefore it is not possible to exclude the likelihood that the spent culture fluid from L. plantarum may have been efficacious in this assay. What is clear is that lysates of both L. reuteri and L. rhamnosus GG are more efficacious than the L. plantarum lysate.

Attention focused on understanding and determining the mechanisms behind the stimulation of keratinocyte activities; in particular, by the L. rhamnosus GG lysate, since this was the most efficacious. Microarray analysis for gene expression profiling allowed the measurement of thousands of genes in a single RNA sample extracted from scratched keratinocytes treated with or without L. rhamnosus GG lysate for 12h. This time point was selected because, during normal wound-healing processes, keratinocyte activates, such as migration and proliferation, commence within12-24h (Quinn et al., 1998). Gene profiles have defined a role for the L. rhamnosus GG lysate to up-regulate the expression and secretion of CXCL2 from keratinocytes. CXCL2 has several functions in the wound-healing process, as reported in numerous studies (Nanney et al., 2003; Devalaraja et al., 2005).In vitro studies highlighted that CXCL2 play crucial roles in

156 the wound-healing process, such as the stimulation of human keratinocyte migration, proliferation and adhesion (Nanney et al., 2003; Rousseau et al., 2006). Indeed, it has been revealed that CXCL2 may possibly be chemoattactant for human keratinocytes, and may also stimulate the secretion of IL-8 that works as a chemoattractant for keratinocytes through increasing the expression of CXCR2 receptor in vitro (Zlotnik et al., 2006). In fact, in a vivo model using CXCR2 knockout mice, loss of CXCR2 resulted in delayed wound healing that appeared to be related to a delay in cell recruitment to the wound site; subsequently, this led to a delay in re-epithelialisation (Devalaraja et al., 2005). Moreover, a number of vitro studies have identified that CXCL2 stimulates the production of additional chemokines, such as CXCL12 and CXCL10 which are chemoattractants for keratinocytes in the wound-healing process (Leonard et al., 1993; Rennekampf el at., 2000; Frank et al., 2000; Zlotnik et al., 2006).

Based on these findings and current microarray data sets (Section 5.4), it is hypothesised that the interaction of CXCR2 and CXCL2 may mediate the acceleration of re-epithelialisation of keratinocytes under the effect of L. rhamnosus GG lysate. This hypothesis has been confirmed based on several lines of evidence: I) the data from Q-PCR confirmed that L. rhamnosus GG lysate up-regulated the expression of CXCR2 and CXCL2 in treated cells compared to untreated ones; ii) L. rhamnosus GG lysate stimulated the secretion of CXCL2 from keratinocytes, as demonstrated in the ELISA assay, which was greater than that from untreated keratinocytes and iii) Western blot analysis has shown that L. rhamnosus GG lysate augmented the expression of CXCR2. The data strongly suggest that the stimulation effects observed in the present study rely on up-regulation of CXCR2 and CXCL2 activity.

In addition, the present study also suggests a role for FGF7 and its receptor, FGFR2 following treatment with L. rhamnosus GG lysate. The impact of FGF7/FGFR2 in the context of clinical wound healing has been well investigated (Werner et al., 1998). This growth factor appears to play multiple roles in the wound-healing process, by the stimulation of keratinocyte proliferation that promotes the re-epithelialisation process (Werner et al., 2003). It has been evidenced that strong up-regulation of FGF7 in keratinocytes exists following skin injury and the expression of its receptor in keratinocytes is increased. This suggests that the stimulation of wound re-

157 epithelialisation occurs by stimulating the proliferation of nearby cells (Basilicoet al., 1992; Werner et al., 2003). These findings, combined with the present work on gene expression, suggest a significant role for L. rhamnosus GG lysate in the stimulation of FGF7/FGFR2 that induces activation in the healing process. A number of observations suggest this: i) secretion of FGF7 from keratinocytes treated with L. rhamnosus GG lysate and ii) increases of FGFR2 expression in response to L. rhamnosus GG lysate stimuli. Overall, these results suggested that L. rhamnosus GG lysate increases the rate of re-epithelialisation through modulation of cell migration as the dominant mechanism. This was evaluated in two results: first, Mitomycin C did not affect the ability of the L. rhamnosus GG lysate to stimulate keratinocyte re- epithelialisation in the scratch wound assay (Figure 5.5); second, there is a significant difference between the rates of re-epithelialisation in cultures treated with L. rhamnosus GG lysate combined with FGFR2 blocking antibody (63%), and cultures combined with CXCR2 blocking antibody treated (40%). The fact that there is some migration with FGFR2 blocking antibody suggests that FGF7 is not the only mechanism, possibly the chemokine acting CXCL2. These data suggest that migration may be the dominant mechanism utilized by the L. rhamnosus GG lysate. As demonstrated previously, CXCR2 is a receptor for IL-8 and CXCL2, which are migration stimulators.Therefore, L. rhamnosusGG lysate may modulate keratinocyte motility at least in part, by a chemokine-dependent pathway.

Comparing between the present study and study conducted in 2011, which showed that L. rhamnosus GG derived material regulates intestinal epithelial cell survival and growth in injury conditions through stimulation of epidermal growth factor (EGF) ligand and its receptor in vitro, (Polk et al., 2011), the microarray data in the current study highlight that neither EGF nor its receptor EGFR-α, were significantly stimulated in cells treated with L. rhamnosus GG lysate. However, some of the main genes in the EGF signaling cascade, such as EGFR-β, JNK1, MEK1, STAT3 and ADAM17, were significantly up-regulated in response to L. rhamnosus GG lysate (Appendix 2). However, CXCL2/CXCR2 and FGF7/FGFR2 gene expression are significantly more up-regulated than the expression of genes that are associated with the EGF pathway.Polk’s study also identified specific proteins from L. rhamnosus GGspent medium fluid that promoted cell growth in cultured human and mouse colon epithelial cells. In the current study, the L. rhamnosus GG lysate stimulated

158 keratinocyte migration and proliferation. The spent culture fluid was also tested but found not to be as efficacious as the lysate (data not shown). It is possible that the efficacious molecule(s) may be synthesized but not secreted, suggesting a different efficacious molecule than in the Polk’s study. Alternatively, it is possible that it is the same molecule(s) and that concentration of the spent culture fluid may be required to see the effects. In the Polk’s study, concentration of the spent culture fluid was required to observe the stimulation effects (Polk et al., 2002; Polk et al., 2011).

Additionally, the possibility that the L. rhamnosus GG lysate does stimulate the EGF pathway in keratinocytes cannot be dismissed given that up-regulation of key genes in this pathway (such as EGFR-β, JNK1, MEK1, STAT3 and ADAM17) was observed (Appendix 2). Furthermore, the microarray data identified up regulation of IL-8 (Appendix 2) which is known to be a stimulator for EGF pathway (Mascia et al., 2003; Paul et al., 2014). The downstream signaling from IL-8 induces cell migration (Mascia et al., 2003; Paul et al., 2014). However, the up regulation of IL-8 observed in the micro array was not as great as that of CXCL2, suggesting that a different pathway may also be involved since there is no direct relationship between EGF and CXCL2. However, no association is found between the EGF pathway and FGF7 pathway; each has different genes and different effects on the keratinocyte functions. The first pathway stimulates the keratinocyte growth, migration, adhesion and differentiation (Pastore et al., 2008); whereas the second merely stimulates the keratinocyte proliferation(Turner et al., 2010). These data suggest that probiotic lysate may induce up regulation of novel pathway(s) in keratinocytes thatstimulate migration via CXCL2/CXCR2 interaction and keratinocyte proliferation by FGF7/ FGFR2 interaction. Although the present study data does not exclude a role for EGF, the data suggest that it is not the main mechanisms by which the L. rhamnosus GG lysate stimulates migration in keratinocytes.

In conclusion, these findings suggest a new approach based on use of a bacterial lysate that could be used to stimulate the re-epithelialisation process in wounds through specific signaling pathways that promote keratinocyte migration and proliferation.The present study postulates that L. rhamnosus GG lysate promotes re-epithelialisation processes invitro through stimulation of the CXCL2/CXCR2 and FGF7/FGFR2 pathways. However, further research to understand the various actions of the L.

159 rhamnosus GG lysate will no doubt continue to determine the effects of L. rhamnosus GG lysate on ex vivo models.

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8 Chapter Six

An investigation into the effects of Lactobacillus rhamnosus GG lysate on the re-epithelialisation of ex vivo wounded skin Chapter five demonstrated that L. rhamnosus GG lysate can stimulate the re- epithelialisation of scratched keratinocytes by activating cell migration or proliferation in vitro. However, a more complex model is required to provide additional evidence of the principle that this lysate could increase the rate of wound healing. Human full-thickness ex vivo skin cultures have been employed in several studies associated with human dermatology; for example, identification and preclinical examination of novel therapeutics (Hardman et al., 2008; Paus et al., 2012), cytokine expression in psoriatic skin (Yoshinaga et al., 1995), effects of hormones on wound re-epithelialisation (Paus et al., 2012), infected wounds (Greenwaldet al., 1992; Steinstraesser et al., 2010) and the study of normal epidermal epithelialisation (Krugluger et al., 2005). This chapter describes the investigations conducted into whether L. rhamnosus GG lysate has the capability to stimulate the re-epithelialisation of wounded skin in an ex vivo model. Full-thickness skin was obtained from individuals undergoing elective cosmetic surgery. This skin was wounded using excisional punch and cultured using a serum-freemedium, either in the presence or absence of L. rhamnosus GG lysate. The use of serum-free medium models the “worst-case scenario” that may occur in ulcer where nutrient supply to the wound is sparse. The aims of this investigation were:  To examine the ability of L. rhamnosus GG lysate to promote re-epithelialisation in an ex vivo human skin model of wounding  To determine the effects of L. rhamnosus GG lysate on cell migration, proliferation and differentiation  To examine the production of L. rhamnosus GG induced cytokine expression in the ex vivo skin model.

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6.0L. rhamnosus GG lysate stimulated re-epithelialisation of human skin maintained in organ culture Skin biopsies were wounded in a “punch in a punch” model (Section 2.3.4, Figure 48) and cultured in the serum-free medium in the presence or absence of 500μl ofL. rhamnosus GG lysate (Section 2.1.3). Biopsies were harvested at day 0, 1, 3 and 7 post-wounding. They were then embedded in Cryomatrix, frozen in liquid N2 andthen 5μm sections were cut using a cryostat. Histological staining of the sections was performed with Haematoxylin& Eosin E to quantify “epithelial tongue length”. This is the length of the new epithelial ‘tongue’ which grows and covers the exposed dermis at the inner wound edges; the measurement scale is displayed in the methods section. The results demonstrate clearly that L. rhamnosus GG lysate enhanced significantly the re-epithelialisation of treated wounds compared with that of untreated wounds (P=0.005, n=3).Tongue length, at day 1 was 7.55μm 0.15, at day 3 it was 18.5μm 0.25 and at day 7 was 22.9μm 0.35. These figures can be compared with untreated cultures in which tongue length was 3.25μm 0.35, day 3 was 9.65μm 0.25 and day 7 was 13.5μm 0.15 post-wounding (Figure 49A&B).

Figure 48: Wound-healing assay design Full-thickness wounds were made using a 2-mm in 6-mm biopsy punch. Photographs of “punch in a punch” biopsy injury on human skin at day 0.

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6.1L. rhamnosus GG lysate increases thickness of the epidermis in wounded skin The present study notes a considerable difference in the total thickness of the epidermis between wounds treated with L. rhamnosus GGlysate and the control. To determine whether this increased thickness was due to increased numbers of cells, or increases in stratum corneum thickness, the number of cells in the living layers was counted and the height of the stratum corneum was measured in three different areas of the sections in the wounded area (Images were captured at x40 magnification; bars=100µm, as described in Section 2.3.4). Total epidermal layers thickness was observed as 8.5μm 0.2 in the treated wounds at day 3 and 16μm 0.65 at day 7, compared with 4.2μm 0.55 at day 3 and 5.5μm 0.5 at day 7 in the control (P=0.02, P=0.004 respectively, n=3, Figure 50).

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Figure 50: Epidermis thickness increases in human skin following L. rhamnosus GG lysate treatment with increasing keratinocyte numbers A) Representative H&E-stained sections of human skin punch-wounds following 7 days’ culture with L. rhamnosus GG lysate (LGG LYS) and untreated control (40x magnification, scale bar represents 100μm, the black lines indicated the measurement area). B) The graph displays total thickness measurements for all epidermal layers in the wound area as analysed from H&E-stained human skin sections treated with L. rhamnosus GG lysate. These were significantly thicker at days 3 and 7 compared with control (P=0.02, P=0.004 respectively, n=3). Significance relative to control datadenoted by *P< 0.05.

The thickness of the stratum corneum (SC) in the wound area was 6.5μm 0.2 in treated skin compared with 2.8μm±0.55 at day 7 in control (P=0.003, n=3, Figure 51A). However, graph 51B illustrates that thickness of the living epidermal layers in the wound area following treatment with L. rhamnosus GG lysate was 5.90μm 0.60 and 10μm 0.65, compared with 2.5μm 0.56 and 2.9μm 0.55 at days 3 and 7 respectively in the control (P=0.03, P= 0.04 respectively, n=3).

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0 1 3 7 T im e (D a y s) Figure 51: Epidermis thickness increment in human skin following L. rhamnosus GG lysate treatment A) The graph shows thickness measurements for Stratum Corneum (SC) in the wound area as analysed from H&E-stained human skin sections (Fig. 6.3) following treatment with L. rhamnosus GG lysate,which was thicker than control at day 7 (P=0.003, n=3). B) The graph illustrates thickness measurements for living epidermal layers in the wound area as analysed from H&E-stained human skin sections (Fig. 6.3) after treatment with L. rhamnosus GG lysate. There was a significant increase at days 3 and 7 compared to control (P=0.03, P= 0.04 respectively, n=3).

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Furthermore, L. rhamnosus GG lysate caused a significant increase in cell numbers (P=0.006, P=0.008, n=3), with 68±0.65 cells and 120±0.45 cells at day 3 and day 7 compared with 35±0.45 cells and 90±0.35 cells in the wounded control at day 3 and day 7 (Figure 52). This was calculated from the value (mean ± standard error) of the number cells from at least three sections for each experimental group in three high- powered fields and normalized to the number of cells in the control.

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Figure 52: Increased keratinocyte numbers in wounded skin epidermis following L. rhamnosus GG lysate treatment The graph displays cell numbers in the epidermis in the wound area as analysed from H&E-stained human skin sections (Figure 6.4) following treatment with L. rhamnosus GG lysate. The cell numbers were increased significantly in treated wounds compared with control at days 3 and 7 (P=0.006, P=0.008 respectively, n=3). The measurement area is indicated in Figure 50. Results are expressed as the mean ± SEM, *P<0.05.

6.2L. rhamnosus GG lysate stimulates cell migration in wounded skin The potential effect of L. rhamnosus GG lysate on stimulating cell migration was revealed as a major mechanism in enhancing the re-epithelialisation of keratinocytes in vitro (Chapter 5). Therefore, the expression of phosphorylated cortactin was compared between the treated wounded skin and wounded control skin. Phosphorylated cortactin was measured because it is well recognised as a marker of cell migration in wounded skin (Nassar et al., 2012). Images were captured at 40x magnification in three standard areas of the wounds described in section 2.3.5, and

167 were used to measure the pixel intensity of the phosphorylated cortactin staining (green). The results demonstrate that the expression of phosphorylated cortactin was greater in lysate-treated wounded skin than in control skin at days 1, 3 and 7 post- wounding. The expression level of phosphorylated cortactin at these days was analysed by measuring the pixel intensity of the staining in new, re-epithelisaed tongue and 50µm length behind the new tongue. The expression of phosphorylated cortactin in treated wounded skin was 9.9±1.45 at day 1, 16.35±0.35 at day 3 and 24.9±0.25 at day 7. This was significantly higher (overall P=0.006) than the control, which was 5%±0.5 at day 1, 8.7%±0.15 at day 3 and 12%±0.20 at day 7 (Figure 53A & B).

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P 0 1 3 7 T im e (D a y ) Figure 53: Phosphorylated cortactin expression increased in wounded skin following L. rhamnosus GG lysate treatment A) Representative images of Alexa-Fluor-conjugated anti-cortactin antibody stained sections of human skin punch wound culture treated with or without (control) L. rhamnosus GG lysate at 7 days post-wounding (40x magnification, scale bar represents 100μm). The inset shows higher magnification of the stained cells (100x).B) The graph illustrates the pixel intensity of phosphorylated cortactin in the new tongues and 50µm length behind the new tongue, as determined by immune- fluorescence with anti-cortactin antibodies (green) in human skin sections following treatment with L. rhamnosus GG lysate. The phosphorylated cortactin expressions in treated wounds were significantly higher than in control wounds at 1, 3 and 7 days post-wounding (overall P=0.006, n=3). Blue = cell nuclei, the white lines indicated the measurement area. The results are expressed as the mean ± SEM, *P<0.05, Y axis unit= arbitrary units= arb.uni.

6.3 L. rhamnosus GG lysate stimulates cell proliferation in wounded skin The processes of skin wound repair involve not only migration, but also proliferation, both of which play a role in the initial re-epithelialisation of the wound. Based on this, a comparison was made of the expression of nuclear proliferation marker Ki-67 between the treated wounded skin and wounded control skin by counting the number of cells in which the nuclei was stained red. Images were captured in three different areas of thewound. The results revealed a significant

169 difference in Ki-67 expression between treated wounded skin and wounded control(P=0.002, n=3). An increase in the number of Ki-67-positive cells was observed clearly in the treated wounds at day 3 and day 7. There were 36.25%±0.5 cells and 61.5%±0.5 cells, respectively, compared with the control, which was 26.1%±0.55 cells at day 3 and 37.5%±0.75 cells at day 7 (Figure 54 A&B).

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0 1 3 7 T im e (D a y ) Figure 54: L. rhamnosus GG lysate increased cell proliferation in human skin punch wounds A) Representative images indicated Ki67 (red) expression in control and L. rhamnosus GG lysate treated human skin punch wound sections, 7 days post-wound (40x magnification, scale bar represents 100μm). The inset shows higher magnification of the stained cells (100x).B) Quantification of Ki67 immuno- reactivity in the new epithelial tongues indicated and 50µm length behind the new tongue in A. The number of Ki-67-positive cells was observed clearly in the treated wound at days 3 and 7, compared with the control (P=0.002, n=3). (Blue = cell

170 nuclei, Red= proliferated cells, the white lines indicated the measurement area). The results are expressed as the mean ± SEM, *P<0.05.

6.4L. rhamnosus GG lysate did not affect cell apoptosis in wounded skin Apoptosis is a fundamental process to normal wound healing, particularly in the removal of inflammatory cells. Therefore, this study identified DNA fragmentation in apoptotic cells using the TUNEL assay. The findings demonstrate the presence of very few TUNEL-positive cells at seven days after wounding. No difference was observed in TUNEL positive (apoptotic) cells in the epithelial tongues following treatment, compared with the control skin (Figure 55 A&B).

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0 1 3 7 T im e (D a y ) Figure 55: L. rhamnosus GG lysate did not affect cell apoptosis in human skin punch wounds A) Representative images indicated apoptotic cells (green) in control and L. rhamnosus GG lysate treated human skin punch wound sections, 7 day post- wounding (40x magnifications, scale bar represents 100μm). The inset shows higher

171 magnification of the stained cells (100x).B) Percentage of apoptotic (TUNEL- positive) cells in the wounded area of the control and L. rhamnosus GG lysate treated human skin. (Blue = cell nuclei, Green= apoptotic cells, white lines indicated the measurement area). The results are expressed as the mean ± SEM, *P<0.05.

6.5L. rhamnosus GG lysate up-regulated wound healing-associated differentiation in human skin Next to be examined was the effect ofthe L. rhamnosus GG lysate on differentiation in wounded skin. Keratinocyte differentiation consists of a continuous and complex sequence of biochemical and morphological alterations that lead to terminally- differentiated corneocytes in the stratum corneum layer. This layer is critical to maintaining the epidermal barrier and completing the wound-healing process (Fuchs et al., 1990; Polakowska et al., 1994). In general, the keratinocyte differentiation process is associated with producing a number of proteins including cytokeratin 10 (CK10) that is expressed in the suprabasal layer of the epidermis at the earliest stage of differentiation. Moreover, involucrin is a precursor of the cornified envelope, and is expressed in the outer layer of epidermis at the terminal stage of differentiation (Fuchs et al., 1990; Polakowska et al., 1994). A comparison was made of the expression of CK10 and involucrin between the treated wounded skin and wounded control skin. This was achieved by measuring the pixel intensity of the staining (green) divided by the number of epidermal nuclei in both the suprabasal and outer layers of the epidermis.Images were captured in three randomly selected areas of the wounded area at 60x magnification for new re-epithelialiased tongue and 50µm length behind the new tongue. The results reveal that L. rhamnosus GG lysate increased the expression of CK10 in wounded skin. This was discerned by comparing the expression levels of CK10 expression in the suprabasal layer of the new re-epithelialised tongue and between the untreated and treated wounded skin. The expression of CK10 was found to be induced to a greater extent in treated wounded skin than in untreated wounded skin after 7 days (P=0.003, n=3) (Figure 56 A&B). To determine whether differentiation had occurred in the outermost layer of the skin, the expression of involucrin on untreated and treated wounded skin was examined. An increase in involucrin expression in the epidermal outer layer of the new re-epithelialised tongue and 50µm length behind the new tongue was clearly

172 observed in the treated wound at day 7, compared with the untreated wound (P=0.01, n=3) (Figure 57 A&B).

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Figure 56: L. rhamnosus GG lysate increased cytokeratin10 (CK10) expression on human skin punch wounds A)Representative images indicated CK10 expression (pixel intensity of green staining of differentiation marker in the suprabasal layer) in control and L. rhamnosus GG lysate-treated human skin punch wound sections, 7 days post- wounding (60x magnification, scale bar represents 100μm).The inset shows higher magnification of the stained cells (100x). B) Quantification of CK10 immuno- reactivity in the new epithelial tongues and 50µm length behind the new tongue indicated in A. The expression of CK10 increased significantly in treated culture compared with control culture after 7 days post-wounding (P=0.01, n=3). Significance relative to control data is denoted by * P< 0.05. (Blue = cell nuclei, white lines indicated the measurement area, Y axis unit= arbitrary units= arb.u).

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Figure 57: Effect of L. rhamnosus GG lysate on Involucrin expression in human skin punch wounds A) Representative images indicated involucrin expression in control and L. rhamnosus GG lysate-treated human skin punch wound sections, 7 days post- wounding (40x magnification, scale bar represents 100μm). The inset shows higher magnification of the stained cells (100x).B) Quantification of involucrin immuno- reactivity in the new epithelial tongues and 50µm length behind the new tongue indicated in A. The expression of involucrin in the epidermal outer layer of new, re- epithelised tongues and 50μm behind the tongue was significantly higher in the treated wound than in the untreated wound at day 7 as measured by pixel intensity of staining at wounded area (P=0.04, n=3). Significance relative to control data is denoted by * P< 0.05. (Blue = cell nuclei, black lines indicated the measurement area, Y axis unit= arbitrary units= arb.u).

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6.6 CXCL2 and FGF7 secretion in wounded skin cultures L. rhamnosus GG lysate up-regulated the gene expression of the chemokine receptor and ligands CXCR2 and CXCL2, and stimulated the secretion of the chemokines from keratinocyte cultures as outlined in Chapter 5. Moreover, up-regulation in the gene expression of FGF7 and FGFR2, and stimulation of FGF7 secretion from keratinocytes after treatment with L. rhamnosus GG lysate, was detected in the experiments outlined in Chapter 5. In order to confirm whether this also occurred in ex vivo human skin wounded cultures, the secretion of CXCL2 and FGF7 in response to L. rhamnosus GG lysate after 1, 3 and 7 days post-wounding were measured using ELISA assays (Section 2.6). The results demonstrated that CXCL2 was secreted at a higher level by L. rhamnosus GG lysate with 7.80µg/ml±0.22 and 9.80µg/ml±0.20 in treated skin, compared with 3.4µg/ml±0.32 and 5.10µg/ml±0.12 in untreated skin after 1 and 3 days post-wounding, respectively (P=0.02, P=0.03 respectively, n=3, Figure 58A). At day 7 post-wounding, no significant difference was observed between treated and control cultures in CXCL2 production. Additionally, a significant increase was found in FGF7 production relative to control at 1 day post-wounding in skin treated with L. rhamnosus GG lysate (15.10µg/ml±0.04, P=0.02, n=3) whereas, the control had 10.80µg/ml±0.02 (Figure 58B). Following 3 and 7 days’ incubation, the production of FGF7 was equal in both the control and treated cultures (Figure 58B).

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0 1 3 7 T im e (D a y s) Figure 58: CXCL2 and FGF7 levels in ex vivo wounded skin culture A) Wounded skin exposed to L. rhamnosus GG lysate for 1 day and 3 days had significantly higher concentrations of CXCL2, compared with untreated controls (P=0.02, P=0.03 respectively, n=3). B) FGF7 concentration increased significantly in treated skin (P=0.02) compared with the control after 1 day post-wounding. The results are expressed as the mean ± SEM, *P<0.05.

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6.7 Discussion Topical application of probiotics or their extracts has been suggested recently as a treatment for infected wounds. Indeed, convincing evidence is presented in numerous studies that live probiotics or their spent culture fluid could impact significantly on enhancing the wound-healing process and reducing infection (Gan et al., 2002; Valdéz et al., 2005b; Sullivan et al., 2009; Peral et al., 2009b).

A known technique of serum-free organ culture of full-thickness human skin (Lu et al., 2007; Rizzo et al., 2012) and the punch-in-a-punch design (Katz et al., 1998; Xu et al., 2012) was applied to study the effects of L. rhamnosus GG lysate on the epidermal regeneration of adult human skin in organ culture. This demonstrated that re-epithelialisation of wounded skin can be stimulated with L. rhamnosus GG lysate. L. rhamnosus GG lysate enhanced re-epithelialisation of wounds as measured by tongue length, at days 1, 3 and 7 post-wounding, compared with untreated skin (Figure 49A&B). The stimulation of re-epithelialisation is possible as a consequence of the combination of keratinocyte migration and proliferation (Radice et al., 1980; Falanga et al., 2008). Indeed, as early as 1 day post wounding, human skin explants yielded increased cell migration in the newly-formed epithelial tongues (Figure 54). Therefore, epithelial migration is certainly a factor in this initial re-epithelialisation, in addition to increasing keratinocyte proliferation, which is also stimulated early in the repair process in wounds treated with lysate (Figure 55). In comparison to controls, L. rhamnosus GG lysate notably up-regulated protein expression of the wound healing-associated keratin, CK10 and involucrin (Figures 56 & 57). This suggests that L. rhamnosus GG lysate has a significant impact on keratinocyte terminal differentiation under assay conditions. These correlate with significantly increased epidermal thickness as early as day 3 of organ culture. It is possible to hypothesise that the higher migration and proliferation rate may contribute to the enhancement of epidermal thickness as early as 3 days post-wounding. Moreover, a higher differentiation rate of cells may also potentially contribute to the increase of epidermal thickness. Notably, the epidermal thickness is associated with increased epidermal living layers possibly due to increased migration and proliferation of cells which occurs at the early stages of the wound-healing process; whereas, increases in the stratum corneum layer thickness could be induced by increased cell

177 differentiation (exemplified by higher involucrin expression) that occurs at 7 days post-wounding. The present study speculates that L. rhamnosus GG lysate has the potential to be used as a therapeutic agent to enhance wound healing. However, some considerations should be taken, such as L. rhamnosus GG lysate induced hyper-migration and hyperproliferation in keratinocytes (Krueger et al., 2005; Morhenn et al., 2013). Organ cultured skin usually displays a hyper-proliferative phenotype, with an up-regulation and ectopic expression of differentiation markers (Morhenn et al., 2013). This may be a result of the wound itself, irrespective of the L. rhamnosus GG lysate treatment. Another concern is the epidermal thickening, combined with the high-proliferative activity and high-differentiation activity. While epithelial migration appears to be enhanced, the rapid increase in epidermal thickness could be problematic for patients if it mimics the hyper-proliferation associated with disorders, such as psoriasis. However, it is possible that the epidermal thickening observed in response to the lysate may be just an epiphenomenon of the ex vivo model. In vivo studies have demonstrated that remodeling happens over a long time of period (Henson et al., 2005) and this phase may overlap with the repair phase. Remodeling can begin as early as 1 week after injury or may require 2 years depending on the type of the wound (Ferguson et al., 1998). Hence, if there is some initial thickening, this tends to disappear with time. However, the ex vivo skin does not have the opportunity to remodel because of its short lifetime in culture. This observation is highlighted in other investigations, suggesting that it was hard to culture the human skin ex vivo for more than 2 weeks because the human epidermis lost its barrier function with a continuous degradation of the epidermis that causes a total detachment of the epidermal layer if the tissue is cultured more than 2 weeks (Krugluger et al., 2005; Steinstraesser et al., 2009). Hence at present, it is not possible to be definitive as to whether the thickening observed in response to the lysate will be problematic. Further studies in healthy volunteers will be required to fully elucidate this point.

Conversely, enhanced wound healing by increasing epithelial migration and proliferation is a positive effect of L. rhamnosus GG lysate. For example, in animal models of wound repair in the aged, the rate of healing is delayed significantly in comparison with young animals (Quirinia et al., 1991; Gosain et al., 2004). This delay is associated with increasing the time which keratinocytes take to migrate from the

178 basal layer to the skin surface and begin proliferating, which is the main repair process (Montagan et al., 1990; Swift et al., 2001). Wound repair in the aged is a main clinical and economic problem evident in the US, since wound healing is impaired in elderly people (DuNouy et al., 1991; U.S. Census Bureau., 2000). Therefore, L. rhamnosus GG lysate may offer a new treatment that can speed up the healing process in ageing skin. Moreover, compared with normal skin, the barrier function in the stratum corneum is defected in atopic dermatitis patients who can have delays in wound healing process. In these cases, L. rhamnosus GG lysate may be an effective treatment to stimulate the healing process through simultaneously improving the barrier by augmenting the keratinocyte differentiation to form a strong barrier. However, further investigation is required to determine the effect of L. rhamnosus GG lysate on wounded skin.

Although the idea of utilising enteric probiotics for treatment wounds is not an original one, there are only a limited number of published reports on topical applications of enteric probiotics or their extracts on wounded skin. For example, an ex vivo study conducted by Polk and colleague in 2007 demonstrated that specific components of L. rhamnosus GG spent culture fluid promoted intestinal epithelial cell proliferation and survival in ulcerated tissue. The author identified 2 proteins (p75 and p40) from L. rhamnosus GG that stimulated cell growth, and reduced tumour necrosis factor (TNF-α) production from epithelial cells that induces epithelial cell apoptosis (Polk et al., 2007). The same group analyzed the mechanisms used by purified protein p40 to regulate cellular responses in intestinal epithelial cells from mouse models. The study demonstrated that the p40 protein stimulated EGFR and its pathway which is essential to inhibit cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo (Polket al., 2012). Conversely, the current study identifies the partial mechanism that contribute to stimulating the wound-healing process ex vivo in response to L. rhamnosus GG lysate is an increase in the production of CXCL2 and FGF7 in ex vivo models (Figure 58).High levels of CXCL2 or FGF7 have already been implicated in multiple aspects of stimulation of wound healing through activation of keratinocyte migration and proliferation (Steiling et al., 2003; Kelle et al., 2004; Richmond et al., 2012). However, the present study did not test the effects of L. rhamnosus GG lysate on the CXCL2 and FGF7 receptors in ex vivo models ; therefore, it is not possible to

179 say whether the effect of probiotic lysates is due to stimulation of the CXCL2/CXCR2 pathways and FGF7/FGFR2 pathways in ex vivo cultures. Future work may be carried out to elucidate this point.

In conclusion, probiotic lysates have been applied directly to wounded skin ex vivo in an attempt to improve the re-epithelialisation rate of the wounds. Topical probiotic preparations have been analysed for their use as a potential treatment to stimulate keratinocyte migration, proliferation and differentiation; all of which are involved in wound healing. This suggests that L. rhamnosus GG lysate may have therapeutic potential in terms of epidermal wound healing.

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9 Chapter Seven

Conclusions and Future Work

7.0 Overview The general aim of this doctoral project was to explore the possibility of using enteric probiotics or their extracts for the protection of skin from infection and to stimulate the wound-healing process. Topical application of probiotics and the relationship between the skin infection and probiotics has been investigated in a small numbers of studies previously but in general the mechanisms underlying effects were not investigated (Guéniche et al., 2006b, 2010, Gan et al., 2002). In addition, a few studies have analysed the use of topical probiotics in relation to wounded skin. The small amount of evidence suggests that enteric probiotics could have important therapeutic value in the prevention of infection and promotion of wound healing (Peral et al., 2009, Daniela et al., 2011). This thesis has studied whether probiotic bacteria or extracts can inhibit the growth of S. aureus and the adhesion of S. aureus to keratinocytes (Chapter 3). It has also examined whether probiotic lysates have a specific protective molecular composition that protects keratinocytes from the cytotoxic effects of S. aureus, and the mechanisms behind the protective effects (Chapter 4). Finally, an evaluation was performed of the effect of probiotic lysates on the wound-healing process in vitro and ex vivo models and the mechanisms behind the observed effects were examined (Chapter 5 & Chapter 7).

The data throughout this thesis indicate that probiotics have species dependent effects on keratinocytes. Whilst most species were nontoxic, L. fermentum had a significant negative effect on keratinocyte viability. The reasons for this are currently unclear but it is likely to be due to expression of particular molecules by this species that are not expressed by the other lactobacilli tested. Further investigation of this could begin via a bioinformatics investigation of genes expressed by L. fermentum compared with e.g. L. rhamnosus GG, since the genome sequences of both species are available (Morita et al., 2008; Morita et al., 2009). Genes common to both species are unlikely to be toxic and it is in the genes expressed only by L. fermentum that the toxic moiety is

181 likely to be found. Identification of this could be useful to inform future selection of species suitable for studies in skin.

Species dependent effects were also evident in the assays of wound healing and protection from the effects of S. aureus. In these assays, some probiotics, e.g. L. salivarius were not efficacious whereas others were extremely effective. Of note is L. rhamnosus GG, which proved the most efficacious of the species, tested in all the assays. This organism had ability to protect keratinocytes from pathogen and stimulate the wound healing process both in vitro and ex vivo suggesting that it could have potential utility as a novel therapeutic for skin in health and disease. Furthermore, L. rhamnosus GG was efficacious when applied as a lysate suggesting it may be a very safe treatment for skin. Hence, this organism has been the major focus of the thesis. One mechanism used by L. rhamnosus GG to protect keratinocytes may be to reduce S. aureus growth. The lactobacilli can produce antimicrobial substances, such as organic acids and bacteriocins that can have direct effects on pathogens growth (Dobson et al., 2011). While L. rhamnosus GG is known to produce acid (Asahara et al., 2004) this does not appear to the mechanism involved here because a lysate of the bacterium neutralized to pH 7 also inhibited growth of S. aureus. The genome sequence of L. rhamnosus GG is known (Morita et al., 2009) and hence one might expect that any bactericidal/static molecules might have been identified. However, the function of many of the genes is currently not known and hence it is likely that a protein of currently unknown function is responsible for the inhibition of growth of S. aureus. Furthermore, a second mechanism used by L. rhamnosus GG to protect keratinocytes is inhibition of adhesion and displacement of S. aureus from keratinocyte binding sites. The work described in chapter 4 demonstrates that this may be due to multiple proteins with known ‘moonlighting’ functions. Fractionation of the lysate indicated that at least 4 proteins, elongation factor thermo unstable (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, and triosephosphateisomerase (TPI) were the major proteins present in the fraction with the greatest ability to displace and inhibit adhesion of S. aureus. Further work in this laboratory has also identified the protein SpaC in this fraction by immunoblotting (Dr R Choudray, personal communications). SpaC is a protein component of the pilus structure used by L. rhamnosus GG to attach itself to the epithelium of the gut (Kankainen et al., 2009). Thu, it is possible that the anti-adhesive effects of L.

182 rhamnusus GG are due to multiple proteins some of which have been suggested in this thesis.

The clinical treatment of wounded skin is a major problem in surgical procedures and a new therapeutic treatment selected to treat wounds should improve one or more phases of healing without any harmful side effects. Rodrigues (2004) was the first person who proposed the use of probiotics in cutaneous wound healing. He used Kefir, which is a probiotic mixture of bacteria and yeasts. The study demonstrated that Kefir is able to decrease inflammation and heal the wounded area (Rodrigues et al., 2004). The current project revealed that wound healing was markedly accelerated in L. rhamnous GG lysate treated cultures in vitro and ex vivo. The process of cutaneous wound healing involves the proliferation and migration of epidermal keratinocytes at the wound edges that enhance the re-epithelialisation of treated wounds. In histological analysis, incisions in the human skin in ex vivo showed a marked increase of migration, proliferation and differentiation of cells when compared with that in control, indicating that L. rhamnous GG lysate could play a role in the early stage of the wound healing process.

The current investigation describes some unique features of the therapeutic effect of probiotic lysates, specifically in regards to the wound healing process. In contrast to untreated wounds, wounds treated with L. rhamnous GG lysate showed a significant increase in expression of specific genes and production from keratinocytes of proteins such as CXCL2 and its receptor CXCR2, and FGF7 and its receptor FGFR2. These genes play an important role in the wound-healing process by stimulating keratinocyte migration and proliferation (Werner et al., 2003;Devalaraja et al., 2005). The probiotic lysate may provide a novel approach to stimulation keratinocyte migration by CXCL2/CXCR2 interaction and keratinocyte proliferation induced by FGF7/FGFR2 interaction. The combination of these two pathways may cause significant stimulation in the wound-healing process, as noticed in vitro and ex vivo models as a response to L. rhamnosus GG lysate.

Application of L. rhamnosus GG lysates is a very interesting notion for dermatologists as it may provide alternative and prophylactic treatments to conventional therapies to treat impaired-healing wounds especially those associated with infection. Infection

183 has a negative effect on wound healing and infected wounds are a major cause of morbidity in the UK; treatment charges account for least £1 billion per year (Thomas et al., 2000). Recently, bacteriotherapy has been investigated and the topical application of probiotics is becoming increasingly of interest as a method to reduce infection and improve the wound-healing process (Peral et al., 2009 &2010). However, in situations such as wounded skin, the live probiotic may be just as toxic since live bacteria could invade the bloodstream and be as deadly as pathogenic bacteria. Therefore, the effects of a L. rhamnosus GG lysate could negate these potentially harmful effects. Using probiotic lysates to treat infected skin may also have a comparatively low production cost since the need to preserve viability is not required. This, L. rhamnosus GG lysate may offer new opportunities for treatment of skin infection, improvement of wound healing and possibly cosmetic applications since other work in this laboratory has shown that the lysate can also improve skin barrier function (Sultanna et al., 2013). However, clearly more work will be necessary before the lysate can be used on humans.

7.1 Future work Data presented in the present thesis advocates that the protective effect of L. rhamnosus GG or its extracts is due to anti-adhesion and anti-microbial effects against S. aureus infection Partial purification of proteins from the lysate led to identification of some proteins associated previously with anti-adhesive activity; for example, elongation factor thermo unstable (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, and triosephosphateisomerase (TPI) (Pancholi et al., 1998). Further work could be undertaken to determine the exact protein and apply these isolated adhesion proteins (possibly as recombinant proteins) directly to infected keratinocytes with S. aureus in order to confirm their ability to reduce adhesion. Further work is also required to identify the anti-microbial proteins from L. rhamnosus GG lysate that reduce the viability of S. aureus.

This study evaluated the ability of L. rhamnosus GG lysate to inhibit S. aureus. Many other skin pathogens also cause significant morbidity. For example, Group A streptococci (e.g. S. pyogenes) P. aeruginosa and P. acnes can all cause skin infections such as necrotising fasciitis and severe infections in burn victims

184 respectively (Bek-Thomsen et al., 2008; Tognetti et al., 2012). Hence the activity of the lysate against these organisms should also be evaluated.

The model used in the present study was primary human epidermal keratinocytes, which is a model of the living layers of the epidermis. In whole skin, bacteria first make contact with the stratum corneum; therefore, the ability of probiotic bacteria or extracts to inhibit colonisation of the stratum corneum would be of interest, which would need more complete models of the skin. It could be examined the effect of bacteria or probiotic extracts on ex vivo model.

Topical probiotic lysates therapeutics for wounded skin was investigated in the present study by using two different model cell cultures and ex vivo skin cultures. In both cultures, the L. rhamnosus GG lysate stimulated the re-epithelialisation process by activating cell migration and proliferation. The initial results hint that L. rhamnosus GG lysate induces cell migration and proliferation by stimulating the specific chemokines pathway, such as CXCL2/CXCR2, and specific growth factors, such as FGF7/FGFR2. However, the gene expression profiles for keratinocytes treated with L. rhamnosus GG lysate identified the alteration of numerous gene expressions in response to L. rhamnosus GG lysate, which remain to be determined. For example, the L. rhamnosus GG lysate increased the expression of IL-8, TNF-α, CXCR4, some Toll- like receptors TLR2/TLR3, and some Tight Junction proteins Occludin/Tight Junction protein ZO-1; all of which play important roles in the wound-healing process; therefore, further investigation is required. Furthermore, the effects of the immune system could be investigated, to some extent in the ex vivo skin model since this has its own resident immune cells. Combination models could also be investigated where the ability of the lysate to heal a wound in the presence of S. aureus could be tested.

In conclusion, the L. rhamnosus GG lysate shows promise as a novel treatment for skin. However, many more studies will be required before it can be translated into use for patients. The data presented in this thesis are therefore the first step along the development pathway.

185

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Appendixes

The appendixes 1, 2 and 3 have been arranged as:

Appendix 1: Publication paper Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes. Applied and Environmental Microbiology.

Appendix 2: Full list for Microarray genes.

Appendix 3: Full list for Microarray genes that are related to cell migration and proliferation.

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Lactobacillus rhamnosus GG Inhibits the Toxic Effects of Staphylococcus aureus on Epidermal Keratinocytes

Walaa Mohammedsaeed, Andrew J. McBain, Sheena M. Cruickshank and Catherine A. O'Neill Downloaded from Appl. Environ. Microbiol. 2014, 80(18):5773. DOI: 10.1128/AEM.00861-14. Published Ahead of Print 11 July 2014.

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Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Lactobacillus rhamnosus GG Inhibits the Toxic Effects of Staphylococcus aureus on Epidermal Keratinocytes

Walaa Mohammedsaeed,a Andrew J. McBain,b Sheena M. Cruickshank,c Catherine A. O’Neilla a b c

Institute of Inﬂammation and Repair, School of Pharmacy and Pharmaceutical Sciences, and Faculty of Life Sciences, the University of Manchester, Manchester, United Downloaded from Kingdom

Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 108 CFU/ml of live L. rhamnosus GG, the L. rhamnosus GG lysates and spent culture fluid also provided .(0.01 ؍ viability of the infected keratinocytes increased to 57% (P ؍ ؍ significant protection to keratinocytes, with 65% (P 0.006) and 57% (P 0.01) of cells, respectively, being viable following 24 http://aem.asm.org/ h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable S. aureus infection. However, spent (0.01 ؍ or 12 h after (P (0.005 ؍ form or as cell lysates 2 h before or simultaneously with (P culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but and 0.01, respectively). Furthermore, growth of S. aureus was 0.04 ؍ only viable bacteria or the lysate could displace S. aureus (P inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. on November 21, 2014 by University of Manchester Library

he concept that probiotics are beneficial to gut health has been coccus thermophilus strains to patients suffering from atopic der- Tinvestigated for a number of years. Studies have demonstrated matitis resulted in improved barrier function apparently through that probiotics improve gut function potentially through a num- increasing the level of ceramides in the stratum corneum (26). The ber of mechanisms (1, 2), including increasing epithelial barrier topical application of Lactobacillus plantarum lysate inhibited the function (3) and modulation of the immune response (4–6). pathogenic activity of Pseudomonas aeruginosa in infected burns There is also evidence that probiotics can prevent colonization of (29). In vivo, L. plantarum lysate has also been shown to improve the gut by pathogens. This can be via mechanisms such as down- wound healing in burn patients (30). regulation of virulence factors and inhibition of pathogen ad- Staphylococcus aureus is both a transient colonizer of skin and a herence to the epithelium (7–9). For example, Lactobacillus spe- major opportunistic skin pathogen, causing diseases ranging from cies inhibit the adhesion of Enterobacter sakazakii to intestinal impetigo to life-threatening conditions such as sepsis (31, 32). mucus by competitive exclusion (8, 10). Other studies demon- Previously, our laboratory demonstrated that the probiotic Lacto- strated that some probiotics increase the production of intestinal bacillus reuteri or its lysate could protect epidermal keratinocytes mucin, thus inhibiting pathogen adherence to intestinal epithelial from the toxic effects of S. aureus via competitive exclusion of the cells (11). Probiotics are also able to produce antimicrobial pep- pathogen from keratinocyte binding sites (33). In the present tides (bacteriocins) and acids. Collectively, there are numerous study, we have identified Lactobacillus rhamnosus GG as a second probiotic-mediated mechanisms that limit pathogen colonization probiotic with the ability to protect skin cells from the effects of S. (1, 12–14). aureus. The selection of L. rhamnosus GG was based on the results Since probiotics may have positive impacts on the gut, their of a screening assay testing a range of probiotics for their ability to potential effects on other systems, such as the mouth (15, 16) and protect human keratinocytes from the effects of S. aureus (data not the urogenital tract (17), have also been investigated. For example, shown). In this assay, L. rhamnosus GG proved to be extremely a study in 2002 examined the impact of oral administration of efficacious either live or as a lysate and uses multiple mechanisms Lactobacillus plantarum to patients who had abdominal surgery to protect against infection, including inhibition of S. aureus and showed that this bacterium lowered the incidence of postsur- growth, competitive exclusion, and displacement of the pathogen gical infection (18). Currently, research is also investigating the from keratinocytes. topical use of probiotics to augment the skin barrier function to promote skin health or prevent or treat disease (9, 19–24). The benefits of topical application of probiotics are still speculative, Received 7 April 2014 Accepted 8 July 2014 and researchers are now focusing on this area to improve condi- Published ahead of print 11 July 2014 tions such as excessive skin sensitivity, atopic dermatitis, and pso- Editor: D. W. Schaffner riasis and to stimulate the wound healing process (19, 25–29). Address correspondence to Catherine A. O’Neill, However, an important consideration will be the safety of using [email protected]. live bacteria, especially in situations where the skin barrier is Copyright © 2014, American Society for Microbiology. All Rights Reserved. breached. For this reason, many investigators have used bacterial doi:10.1128/AEM.00861-14 lysates in their studies. Topical application of sonicated Strepto-

September 2014 Volume 80 Number 18 Applied and Environmental Microbiology p. 5773–5781 aem.asm.org 5773 Mohammedsaeed et al.

MATERIALS AND METHODS A volume of 50 ␮l of live organisms or 50 ␮l of lysate extracted from 108 Mammalian cell culture. Normal human epidermal keratinocytes CFU/ml of L. rhamnosus GG or L. reuteri cultures was spotted onto an S. (NHEK) cultured in keratinocyte basal medium (Promocell, Heidelberg, aureus lawn. The inhibition zone was evaluated after overnight incubation Germany) containing a supplement mix (bovine pituitary extract, 0.004 by measuring the diameter of the zone in millimeters using a ruler. mg/ml; epidermal growth factor [recombinant human], 0.125 ng/ml; in- Determination of the outcome of coculture (competition assays). ␮ ␮ 6 sulin [recombinant human], 5 ␮g/ml; hydrocortisone, 0.33 ␮g/ml; epi- Aliquots (100 l) of L. rhamnosus GG lysates and 100 lof10 -CFU/ml S. nephrine, 0.39 ␮g/ml; and holo-transferrin [human], 10 ␮g/ml) and 0.06 aureus were inoculated into 10-ml Wilkins-Chalgren broth. The pH and optical density of cultures were measured at 0 and 24 h. At regular inter- mM CaCl2 (Promocell) were used as a model system. These were cultured

vals (indicated below), bacteria were counted by serial dilution plate Downloaded from routinely at 37°C in a humid atmosphere of 5% CO2 in T-75 culture flasks as described previously (33). counts using selective agar. Bacterial cell culture. Lactobacillus rhamnosus Goldin and Gorbach Statistical analyses. All experiments were performed a minimum of (L. rhamnosus GG; ATCC 53103), Lactobacillus reuteri (ATCC 55730), three times, with three replicates within each experiment. Data generated and Lactobacillus salivarius (UCC118) (ATCC, Middlesex, United King- were analyzed by one-way analysis of variance (ANOVA) and post hoc dom) were grown routinely in Wilkins-Chalgren broth or agar (Oxoid, Tukey test using SPSS (IBM SPSS Statistics version 16.0) program. Results Ͻ Basingstoke, United Kingdom) at 37°C in an anaerobic cabinet (atmo- were considered significant if the P value was 0.05. Data are expressed as means Ϯ standard errors of the means (SEM). sphere,10:10:80 H2-CO2-N2). Staphylococcus aureus was grown aerobically at 37°C in nutrient broth (Oxoid) as described previously (33). Treatment of keratinocytes with bacteria. Bacteria (108 CFU/ml of RESULTS http://aem.asm.org/ 6 probiotics and 10 CFU/ml of S. aureus) were centrifuged at 15,000 ϫ g, L. rhamnosus GG protects keratinocytes from the pathogenic washed twice in 0.85% NaCl, and resuspended in keratinocyte basal me- effects of S. aureus. Initially, we investigated whether the viability ϫ 3 2 dium. This suspension was added directly to 5 10 cells/cm of NHEK of keratinocytes was affected by incubation with L. rhamnosus GG. growing in 24-well plates. For experiments using a probiotic lysate, 100 ml However, following 24 h of incubation, there was no difference of 108-CFU/ml L. rhamnosus GG was centrifuged, washed, resuspended in 25 ml of phosphate-buffered saline (PBS, pH 7.4; Invitrogen, Life Tech- in the viability of keratinocytes incubated with the probiotic nologies Ltd., Paisley, United Kingdom), and lysed using an MSE Soni- bacteria versus the control of untreated keratinocytes (data not prep 150. Samples were filtered using a 0.22-␮m-pore filter (Millipore, shown). Next, the ability of L. rhamnosus GG to protect kera-

Billerica, MA) to remove any whole bacteria remaining. Approximately tinocytes from the effects of S. aureus was investigated. In agree- on November 21, 2014 by University of Manchester Library 100 ␮l of this lysate was used to treat keratinocytes (5 ϫ 105 cells/cm2). In ment with our previous findings (33), 24 h of exposure of kerati- some experiments, cells were sedimented in a centrifuge at 15,000 ϫ g for nocytes to 106 CFU/ml of S. aureus resulted in significant 5 min, and the cell-free supernatant (spent culture fluid) was collected and keratinocyte death. However, keratinocytes incubated simultane- filtered using a 0.22-␮m-pore filter (Millipore) to remove any whole bac- ously with the pathogen and L. rhamnosus GG had a significantly teria remaining. In other experiments, keratinocyte monolayers were higher viability (57%; P ϭ 0.01) than monolayers infected with coinfected with pathogen plus probiotics or lysates simultaneously. In the pathogen alone (Fig. 1A). separate experiments, cells were exposed to L. rhamnosus GG lysate for 2, We investigated whether viable bacteria were essential for the 4, 6, 8, and 12 h after S. aureus infection had commenced. In all experiments, keratinocytes were detached and cell viability was determined us- protective effect of L. rhamnosus GG by examining the effect of ing trypan blue exclusion assays as described in reference 33. In other probiotic lysate and spent culture fluid on S. aureus-infected ke- experiments using heated lysates, these were heat inactivated by placing ratinocytes. Neither lysate nor spent culture fluid significantly af- them in a boiling water bath at 100°C for 5 min. fected the viability of keratinocytes (P Ͼ 0.05) (data not shown). Measurement of S. aureus viability in cell culture. To determine However, both the lysate and spent culture fluid reduced the tox- whether L. rhamnosus GG lysates or spent culture fluid was able to inhibit icity of S. aureus such that the viabilities of treated keratinocytes the growth of S. aureus in cell culture, keratinocytes were grown to con- were 65% and 55.93%, respectively, compared to 25% in kerati- fluence in a 24-well plate. These were exposed to 100 ␮lof106-CFU/ml S. nocytes infected with S. aureus alone (P ϭ 0.006 and P ϭ 0.01, ␮ ␮ aureus alone or S. aureus plus 100 lofL. rhamnosus GG lysates or 100 l respectively) (Fig. 1B). This is in contrast to the effects observed of spent culture fluid. In separate experiments, cells were exposed to L. with L. reuteri, which we showed previously to be protective to rhamnosus GG lysates for 2, 4, 6, 8, and 12 h after infection with S. aureus. pathogen-infected keratinocytes (33). L. reuteri provides protec- The total number of viable staphylococci was determined by counting the colonies as described previously (33). tion only when added either live or as a lysate, but the spent culture Measurement of bacterial adhesion to keratinocytes. Confluent ke- fluid has no ability to protect keratinocytes from the effects of S. ratinocytes were exposed to 106 CFU/ml of S. aureus and 108 CFU/ml of L. aureus (Fig. 1C). rhamnosus GG for 1 h. Cells were then washed three times in PBS (pH 7.4) L. rhamnosus GG lysate but not spent culture fluid rescues to remove nonadherent bacteria. The cells were trypsinized and serial keratinocytes from S. aureus toxicity. We next investigated the dilution plate counts performed to assess the number of adherent bacte- timing of the protective effect of L. rhamnosus GG by adding the ria. Selective agar was used for growth of staphylococci. Additionally, live bacteria, the lysate, or the spent culture fluid either before or keratinocytes were exposed to 106 log CFU/ml of S. aureus combined with after infection of keratinocytes with S. aureus. The percentage of 100 ␮l of lysate or spent culture fluid of L. reuteri or Lactobacillus salivarius keratinocytes remaining viable was significantly greater in mono- UCC118. The experiment was carried out three times and results were layers exposed to L. rhamnosus GG for 2 h prior to infection with taken as triplicates. S. aureus than in monolayers infected with S. aureus alone (P ϭ In separate experiments, cells were exposed to 100 ␮lof108-CFU/ml 0.006). The lysate and spent culture fluid afforded similar levels of probiotic bacteria or lysates or spent culture fluid for 1 h before the addi- ϭ ϭ tion of 100 ␮lof106-CFU/ml S. aureus at the same time or 2, 4, 6, 8, and protection (P 0.005 and P 0.004) (Fig. 2A). In postexposure 12 h after infection with S. aureus. experiments, keratinocytes were exposed to S. aureus for2h,4h, Determination of bacterial antagonism. A 10-␮l aliquot of an over- 6 h, 8 h, and 12 h before addition of the live L. rhamnosus GG, night culture of S. aureus was inoculated into 7 ml of soft agar medium lysate, or spent culture fluid. The viability of the keratinocytes was (0.7% agar) and was added directly onto plates prepoured with agar base. then measured at 24 h after infection with S. aureus. The data in

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FIG 1 (A) L. rhamnosus GG, lysate, or spent culture fluid protects keratinocytes from the toxic effects of S. aureus. A combination of S. aureus (SA) and L. rhamnosus GG (LGGϩSA) resulted in a significantly higher (P ϭ 0.01) percentage of viable keratinocytes after 24 h than in monolayers infected with S. aureus alone. The data were compared to those produced by uninfected control cells (control). (B) The viability of S. aureus-infected keratinocytes treated with L. rhamnosus GG lysate (SAϩLGG LYS) or spent culture fluid (SAϩLGG CM) was significantly increased compared to that of keratinocytes infected with S. aureus (SA) alone. (C) Monolayers exposed to S. aureus and a lysate of L. reuteri (SAϩLR LYS) had a significantly higher percentage of viable keratinocytes than those infected with pathogen alone, but the same effect was not found with the spent culture fluid of L. reuteri (SAϩLR CM). Data are representative of three individual experiments, and all values represent means Ϯ SEM. *, P Ͻ 0.05.

Fig. 2B and C show that both live probiotic and its lysate could species specific, because the lysate from L. reuteri made in exactly protect the keratinocytes when added after S. aureus. Even at 12 h the same way had no effect on the growth of S. aureus (Fig. 3D). after S. aureus infection, L. rhamnosus GG or lysate still afforded We determined the numbers of viable staphylococci following protection to the keratinocytes such that 58% and 55%, respec- 24 h of incubation with keratinocytes in the presence or absence of tively, of cells remained viable, compared to 25% when exposed to the L. rhamnosus GG lysate. When S. aureus was added to kerati- S. aureus alone (P ϭ 0.003 and P ϭ 0.01, respectively). However, nocytes at the same time as the L. rhamnosus GG lysate, the total the spent culture fluid from L. rhamnosus GG had no protective number of viable staphylococci was also significantly reduced, to 5 effect on keratinocytes when added after S. aureus (Fig. 2D). log10 CFU/ml (Fig. 4), compared to 8 log10 CFU/ml for S. aureus L. rhamnosus GG lysate, but not spent culture fluid, inhibits alone (P ϭ 0.02) (Fig. 5). Furthermore, when the L. rhamnosus GG the growth of S. aureus. We investigated whether the probiotic lysate was added 12 h after infection of the keratinocytes, a reduc- lysate had direct effects on the growth of the pathogen by growing tion in number of viable S. aureus organisms was observed when them simultaneously in culture. Competition assays showed a sig- these were counted 24 h later (Fig. 4). These effects were not seen nificant reduction in S. aureus growth over a period of 24 h in the with either the spent culture fluid from L. rhamnosus GG or a presence of 100 ␮lofL. rhamnosus GG lysate compared to un- lysate from L. reuteri (data not shown). Since lactobacilli can pro- treated cultures (P ϭ 0.02) (Fig. 3A). This effect was specific to the duce organic acids, we measured the pH of keratinocyte media lysate, because the spent culture fluid from L. rhamnosus GG had infected for 24 h with S. aureus, L. rhamnosus GG lysate, or both no effect on the growth of S. aureus (Fig. 3B). Furthermore, the simultaneously. However, there was no significant difference in ability of the lysate to inhibit pathogenic growth was negated by the pH between treatment groups (data not shown). We also mea- heating the lysate to 100°C for 10 min (Fig. 3C). Finally, this direct sured the pH of lysate alone and found it be 7.2, thus suggesting effect of L. rhamnosus GG on pathogenic growth appeared to be that acid-mediated effects were not likely to be the mechanism

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FIG 2 L. rhamnosus GG protects and rescues keratinocytes from infection with S. aureus. (A) The percent viability of infected keratinocytes was significantly higher in cells that were preexposed to L. rhamnosus GG (LGGϩSA), lysate (LGG LYSϩSA), or spent culture fluid (LGG CMϩ SA) than that of S. aureus (SA)-infected cells. (B) The viability of S. aureus-infected keratinocytes was significantly higher in cells exposed to L. rhamnosus GG 12 h after infection with S. aureus (“Post-exposed”). A similar effect was observed with lysate (C). However, cells postexposed to L. rhamnosus GG spent culture fluid (CM) did not have significant protection (D). Data are representative of three individual experiments, and all values represent means Ϯ SEM (n ϭ 3). *, P Ͻ 0.05.

underlying inhibition of pathogenic growth. The antimicrobial grown anaerobically (Table 1). In contrast, live L. reuteri or lysate properties of L. rhamnosus GG and lysate were evaluated using a did not induce zones of inhibition in this assay (Table 1). spot-on-lawn assay. This assay showed signiﬁcant inhibition of S. L. rhamnosus GG inhibits adhesion of S. aureus to keratino- aureus growth (as evidenced by the presence of zones of inhibi- cytes. Another mechanism by which live bacteria, lysate, or spent tion) by anaerobic live cultures or lysates of L. rhamnosus GG culture ﬂuid of L. rhamnosus GG may protect keratinocytes is by

5776 aem.asm.org Applied and Environmental Microbiology L. rhamnosus GG Inhibits S. aureus

A) B) 1.60 SA SA+LGG LYS * 1.40 1.40 * SA SA+LGG CM 1.20 * * * 1.20 1.00 * 1.00 0.80 * 0.80 0.60 0.60 0.40 0.40 Downloaded from

Absorbance 660nm Absorbance 0.20 0.20 Absorbance 660nm Absorbance 0.00 0.00 0h 1h 2h 4h 6h 8h 10h 12h 18h 20h 22h 24h 0h 1h 2h 4h 6h 18h 20h 22h 24h

C) 1.40 D) SA SA+ heated LGG LYS 1.40 SA SA+LRLYS 1.20 1.20

1.00 1.00

0.80 0.80 http://aem.asm.org/

0.60 0.60

0.40 0.40

Absorbance 660nm Absorbance 0.20

Absorbance 660nm 0.20

0.00 0.00 0h 1h 2h 4h 6h 8h 10h 12h 18h 20h 22h 24h 0h 1h 2h 4h 6h 8h 10h 12h 18h 20h 22h 24h

FIG 3 Effect of L. rhamnosus GG or L. reuteri lysates and spent culture ﬂuid on S. aureus growth in a competition assay. The optical densities of cultures of S.

aureus (SA) growing in the presence of L. rhamnosus GG lysate (LGG LYS) (A) or spent culture fluid (LGG CM) (B) or heated L. rhamnosus GG lysate (heated on November 21, 2014 by University of Manchester Library LGG LYS) (C) or L. reuteri lysate (LRLYS) (D) were determined every hour to monitor the growth of the bacteria. In the presence of the L. rhamnosus GG lysate, the growth of S. aureus was significantly lower than when it was grown alone (P ϭ 0.02; n ϭ 3), whereas the heated L. rhamnosus GG lysate or spent culture fluid had no significant effect (P Ͼ 0.05; n ϭ 3). Furthermore, a lysate of L. reuteri had no effects on the growth of S. aureus. Data are representative of three individual experiments, and all values represent means Ϯ SEM (n ϭ 3). *, P Ͻ 0.05. inhibition of pathogenic adhesion. Previously, we showed that performed to determine whether inhibition was due to competi- agents that reduce adhesion of S. aureus to keratinocytes also re- tion, exclusion, or displacement of pathogen from binding sites on duce its toxicity (33). Hence, we considered that inhibition of keratinocytes. L. rhamnosus GG, either as viable cells or as lysate, adhesion may also be part of the protective mechanism of L. rh- was able to inhibit pathogen adhesion if keratinocytes were coin- amnosus GG, lysate, or spent culture fluid. Adhesion assays were fected (competition; P ϭ 0.03), preexposed (exclusion; P ϭ 0.04), or applied 12 h after infection with S. aureus had begun (displacement; P ϭ 0.01) (Fig. 5A and B). By comparison, and as shown previously, live L. reuteri or its lysate could reduce staphylococcal adhesion if it was added at the same time as the pathogen (33)(Fig. 5D). However, the spent culture fluid did not reduce S. aureus adhesion. Interestingly, the spent culture fluid from L. rhamnosus GG inhibited pathogen adhesion only if it was added to keratinocytes either before or at the same time as the pathogen, in keeping with the data on viability (Fig. 5C). Finally, L. salivarius, its lysate, or spent culture fluid did not affect the adhesion of S. aureus to keratinocytes (Fig. 5D).

DISCUSSION This study explored whether an enteric probiotic, L. rhamnosus GG, could protect keratinocytes from the pathogenic effects of S. aureus. Our data indicate that L. rhamnosus GG, in the form of viable cells, a cell-free lysate, or spent culture fluid, enhanced keratinocyte viability in the presence of the pathogen. FIG 4 L. rhamnosus GG lysate, but not spent culture fluid, reduced the numbers of viable staphylococci. The number of viable S. aureus organisms (SA) The timing of application of L. rhamnosus GG cells or lysate did was 8 log CFU/ml, whereas 5 log CFU/ml of S. aureus were viable in the not affect the degree of protection conferred by the probiotic or presence of L. rhamnosus GG lysate (“Co-exposed”). Additionally, the total lysate because keratinocytes pre-, post-, or coexposed to L. rham- number of viable staphylococci in keratinocyte culture was reduced by the nosus GG or lysate were protected from S. aureus-induced cell L. rhamnosus GG lysate when this was added 2, 4, 6, 8, and 12 h after infection of the keratinocytes with pathogen (“Post-exposed”; P ϭ 0.05; death. However, the probiotic spent culture fluid protected kera- n ϭ 3). Data are representative of three individual experiments, and all tinocytes only if it was added either before or at the same time as values represent means Ϯ SEM (n ϭ 3). *, P Ͻ 0.05. the pathogen. These data contrast with those for L. reuteri and L.

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FIG 5 Live L. rhamnosus GG, lysate, or spent culture fluid inhibited S. aureus adhesion to keratinocytes. (A) Live L. rhamnosus GG (LGG) inhibited S. aureus adhesion when added at the same time (LGGϩCo), before (LGGϩPre), or after (LGGϩPost) infection of cells with S. aureus. (B) A similar effect was also observed with the lysate. (C) Spent culture fluid (LGG CM) reduced the adhesion of S. aureus but only when added at the same time or before infection with pathogen. (D) The L. reuteri lysate (SAϩLR LYS) reduced the adhesion of S. aureus to keratinocytes when added simultaneously, but the L. reuteri spent culture fluid (SAϩLRCM) did not. L. salivarius lysate (SAϩLS LYS) or spent culture fluid (SAϩLS CM) had no effect on the adhesion of S. aureus to keratinocytes. Data are representative of three individual experiments, and all values represent means Ϯ SEM (n ϭ 3). *, P Ͻ 0.05.

TABLE 1 L. rhamnosus GG bacteria or lysate reduces the growth of S. aureus in a spot-on-lawn assaya salivarius, since L. reuteri can protect as a live organism or lysate only when added before or at the same time as the pathogen and L. Zone of inhibition (mm) salivarius has no ability to protect keratinocytes (33). Aerobic Anaerobic The current investigation suggests that there are at least two, Bacteria condition condition possibly separate, activities involved in the protective effects of L. S. aureus ϩ L. rhamnosus GG No inhibition 11 Ϯ 1.3 rhamnosus GG. These are likely to be inhibition of pathogen ad- S. aureus ϩ L. rhamnosus GG lysate No inhibition 18.38 Ϯ 0.7 hesion and inhibition of pathogen growth. We showed previously S. aureus ϩ L. reuteri No inhibition No inhibition that agents that reduce adhesion of S. aureus to keratinocytes also S. aureus ϩL. reuteri lysate No inhibition No inhibition reduce its toxicity in our viability assay (33). In keeping with this, a Spot-on-lawn assay demonstrating zones of inhibition produced by L. rhamnosus GG the ability of the lysate and spent culture ﬂuid to enhance viability and GG lysate under the anaerobic condition but not under the aerobic condition. However, neither live L. reuteri nor L. reuteri lysate inhibited S. aureus growth under mirrors directly the ability of each to inhibit pathogen adhesion; either condition. The inhibition zone was evaluated after overnight incubation by i.e., while the L. rhamnosus GG lysate protects viability and inhib- measuring the diameter of zone sizes using a ruler. Results are expressed as the its adhesion when added pre- or postinfection, the spent culture means Ϯ SEM of three individual experiments.

5778 aem.asm.org Applied and Environmental Microbiology L. rhamnosus GG Inhibits S. aureus

fluid protects viability only when added before the pathogen and However, again, we cannot rule out the possibility that such mol- has no ability to inhibit adhesion or protect when added after the ecules may be secreted but diluted once contained in the spent pathogen. Thus, we suggest that the live organism or the lysate culture fluid. If L. rhamnosus GG contains bacteriostatic sub- protects against the effects of S. aureus by exclusion and displace- stances, then this may also, at least partially, explain the protective ment, whereas the spent culture fluid can only exclude pathogens. effect of the probiotic in keratinocyte survival assays. Probiotics, In contrast, L. salivarius, which cannot protect keratinocytes from especially lactobacilli, have previously been shown to exert a S. aureus, does not inhibit adhesion as either a live organism, a strong inhibitory effect on S. aureus growth. Certain Lactobacillus lysate, or spent culture fluid. Taken together, all these data point to strains have been reported to be highly antagonistic to biofilm- species-specific effects in the abilities of different lactobacilli to forming S. aureus (3). Other studies have reported that probiotics Downloaded from protect keratinocytes from the toxic effects of S. aureus. Our data can improve gut health by inhibiting growth of pathogens through may also suggest that the antiadhesive effects contained within the production of bacteriocins or lactic acid (36, 54–56). However, in L. rhamnosus GG lysate and spent culture fluid are mediated by the present study, we could find no evidence of the involvement of different molecules. However, we cannot rule out the possibility acid production as part of the protective effects of L. rhamnosus that the same molecule(s) may be involved but that the concen- GG. Indeed, the lysate from this organism was neutral (pH 7.2) tration in the spent culture fluid is too low for some of the effects but was still able to inhibit S. aureus growth. Furthermore, neither to be observed. L. reuteri nor L. salivarius showed any inhibitory activity on the The ability of species of Lactobacillus species to inhibit certain growth of S. aureus even though both these bacteria are also able to http://aem.asm.org/ pathogens from binding to epithelial cells has been demonstrated produce acid (57, 58). previously in models of the gut epithelium (8, 10, 34). For exam- In conclusion, we have shown that L. rhamnosus GG uses mul- ple, in an in vitro study, probiotics (alone or in combinations), tiple mechanisms to protect keratinocytes from S. aureus. These including L. rhamnosus NCC4007 and Lactobacillus paracasei include exclusion of pathogens, inhibition of pathogen growth, NCC2461, were shown to inhibit E. sakazakii adhesion to intesti- and displacement of pathogen from keratinocytes. Of course, it is nal mucus through competitive exclusion and displacement from possible that this displacement activity may be related to the abil- the binding sites (8, 10, 35). Another study by Vesterlund and ity of L. rhamnosus GG to inhibit growth, and further studies will

colleagues (36) showed that certain lactic acid bacteria, including be required to clarify this point. A number of studies have sug- on November 21, 2014 by University of Manchester Library L. rhamnosus GG, were able to reduce the adhesion of S. aureus to gested the utility of probiotic species of lactobacilli for use topi- intestinal cells by as much as 44%. In keeping with our study, the cally. In keeping with these studies, we suggest that L. rhamnosus mechanisms involved included competition, exclusion, and dis- GG is a potential new agent to inhibit the pathogenicity of S. placement. Interestingly, in the study of Vesterlund et al., the au- aureus to keratinocytes. Furthermore, our data show that the util- thors also noted reduced staphylococcal viability in the presence ity of L. rhamnosus GG on skin will not be limited by whether it of some of the probiotic organisms (36). can grow and survive on skin, because a lysate of the organisms is The molecules mediating the inhibitory effects of probiotics just as efficacious at preventing S. aureus colonization as live bac- against pathogens have been investigated in a number of studies. teria. We suggest that the use of bacterial lysates will enhance the In some cases, the molecules mediating antiadhesive activity are utility of lactobacilli since the need to produce formulations that largely associated with other functions, i.e., the so-called “moon- maintain bacterial viability is negated. Furthermore, lysates po- lighting proteins” (37–49). For example, enolase from Lactobacil- tentially offer a safer option than live bacteria for treatment of lus crispatus can bind to laminin and collagen I, which reduces the damaged skin. adhesion of S. aureus to epithelial cell lines through these binding sites (50). Similarly, enolase from L. plantarum has been reported ACKNOWLEDGMENT as binding to fibronectin to prevent S. aureus adhesion to epithe- This work was supported by a Scholarship from Ministry of Higher Edu- lial cell lines (7, 51). Other moonlighting proteins contributing to cation of Saudi Arabia to Walaa Mohammedsaeed. bacterial adhesion have been found in lactobacilli. For example, REFERENCES triosephosphate isomerase (TPI) from L. plantarum plays a role in the adhesion of lactobacilli to Caco-2 cells and has the ability to 1. Rolfe RD. 2000. The role of probiotic cultures in the control of gastrointestinal health. J. Nutr. 130:396S–402S. compete with pathogens such as Clostridium sporogenes and En- 2. Sanders ME. 1999. Probiotics. Food Technol. 53:67–77. terococcus faecalis by excluding and displacing them from the cell- 3. Ewaschuk JB, Diaz H, Meddings L, Diederichs B, Dmytrash A, Backer binding sites (12, 52, 53). However, thus far, the molecules medi- J, Looijer-van Langen M, Madsen KL. 2008. Secreted bioactive factors ating the effects preventing adhesion of L. rhamnosus GG to from Bifidobacterium infantis enhance epithelial cell barrier function. Am. J. Physiol. Gastrointest. Liver Physiol. 295:G1025–G1034. http://dx.doi keratinocytes remain to be identified. .org/10.1152/ajpgi.90227.2008. L. rhamnosus GG lysate may also protect keratinocytes is via 4. Isolauri E, Sütas Y, Kankaanpää P, Arvilommi H, Salminen S. 2001. inhibition of S. aureus growth. Two lines of evidence suggest that Probiotics: effects on immunity. Am. J. Clin. Nutr. 73:444S–450S. this is the case: first, a reduction in the total number of viable 5. Kaila M, Isolauri E, Soppi E, Virtanen E, Laine S, Arvilommi H. 1992. staphylococci in the presence of the L. rhamnosus GG lysate and Enhancement of the circulating antibody secreting cell response in human diarrhoea by a human Lactobacillus strain. Pediatr. Res. 32:141–144. http: inhibition assays demonstrating zones of inhibition when S. au- //dx.doi.org/10.1203/00006450-199208000-00002. reus was challenged with lysates from the probiotic grown anaer- 6. Lai Y, Cogen AL, Radek KA, Park HJ, Macleod DT, Leichtle A, Ryan obically (Table 1). This could be due to the presence of a toxic AF, Di Nardo A, Gallo RL. 2010. Activation of TLR2 by a small molecule molecule(s) within the probiotic that is able to directly inhibit S. produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections. J. Invest. Dermatol. 130:2211–2221. http: aureus growth and/or viability. It is possible that this molecule(s) //dx.doi.org/10.1038/jid.2010.123. may be synthesized but not secreted because there was no effect of 7. Di Cagno R, De Angelis M, Calasso M, Vincentini O, Vernocchi P, L. rhamnosus GG spent culture fluid on the viability of S. aureus. Ndagijimana M, De Vincenzi M, Dessi MR, Guerzoni ME, Gobbetti M.

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September 2014 Volume 80 Number 18 aem.asm.org 5781 Microarray analysis of scratched keratinocytes treated vs untreated with L. rhamnosus GG lysate was chosen as a starting point for understanding mechanisms of action. Keratinocyte monolayers were scratched and treated with L. rhamnosus GG lysate for 12h and RNA was extracted and processed using an RNA extraction assay kit. Samples analysed using Affymetrix gene array chips for three individual expermints. A full list of genes whose expression was changed by treatment with the lysate. The p values associated with a change in expression of these genes were generated using the Enrichr tool (Chen et al., 2013). The data was listed acording to the p values <0.05.

Symbol p-value Location AAK1 2.83E-02 Cytoplasm AARS 1.90E-03 Cytoplasm AARS2 1.18E-03 Cytoplasm AASDH 1.22E-02 Other AASDHPPT 8.86E-07 Cytoplasm ABAT 1.21E-05 Cytoplasm ABCA1 1.46E-02 Plasma Membrane ABCB8 1.09E-02 Cytoplasm ABCC2 3.20E-03 Plasma Membrane ABCC3 5.62E-04 Plasma Membrane ABCC4 8.56E-03 Plasma Membrane ABCC5 1.76E-02 Plasma Membrane ABCF1 5.01E-03 Cytoplasm ABCG1 1.08E-05 Plasma Membrane ABCG4 1.19E-02 Plasma Membrane ABHD12 3.36E-03 Other ABHD5 8.97E-03 Cytoplasm ABHD6 1.26E-03 Cytoplasm ABI1 6.10E-06 Cytoplasm ABI2 4.44E-03 Cytoplasm ABL2 2.25E-03 Cytoplasm ABLIM2 3.87E-02 Cytoplasm ACACA 4.89E-02 Cytoplasm ACACB 6.23E-04 Cytoplasm ACAD8 7.74E-03 Cytoplasm ACAD9 6.99E-03 Cytoplasm ACADSB 1.95E-06 Cytoplasm ACADVL 3.27E-03 Cytoplasm ACAN 2.84E-02 Extracellular Space ACE2 2.98E-02 Plasma Membrane ACHE 2.34E-02 Plasma Membrane ACIN1 2.13E-02 Nucleus ACLY 2.79E-02 Cytoplasm ACO1 1.91E-03 Cytoplasm ACO2 2.33E-02 Cytoplasm ACOT4 7.34E-03 Cytoplasm ACOT7 8.69E-03 Cytoplasm ACOX1 1.99E-03 Cytoplasm ACP1 3.14E-03 Cytoplasm ACP2 8.57E-03 Cytoplasm ACP5 3.67E-02 Cytoplasm ACPL2 1.64E-03 Other ACSBG1 3.60E-02 Cytoplasm ACSL3 3.18E-03 Cytoplasm ACSL4 5.09E-04 Cytoplasm ACSL5 3.35E-03 Cytoplasm ACSS1 2.53E-02 Cytoplasm ACTA2 8.14E-04 Cytoplasm ACTC1 1.39E-02 Cytoplasm ACTG1 5.79E-03 Cytoplasm ACTN1 2.56E-04 Cytoplasm ACTN2 8.65E-03 Nucleus ACTN4 5.07E-03 Cytoplasm ACTR2 7.62E-06 Plasma Membrane ACTR3 3.69E-04 Plasma Membrane ACVR1B 6.83E-03 Plasma Membrane ACVR1C 1.33E-08 Plasma Membrane ACVR2A 2.75E-02 Plasma Membrane ACVR2B 2.12E-03 Plasma Membrane ADA 1.15E-04 Cytoplasm ADAL 2.03E-03 Cytoplasm ADAM10 1.45E-04 Plasma Membrane ADAM12 3.77E-02 Plasma Membrane ADAM15 2.78E-04 Plasma Membrane ADAM17 4.97E-02 Plasma Membrane ADAM22 4.83E-03 Plasma Membrane ADAM23 3.37E-02 Plasma Membrane ADAM30 2.48E-02 Plasma Membrane ADAM8 4.97E-02 Plasma Membrane ADAM9 1.19E-04 Plasma Membrane ADAMTS1 1.56E-05 Extracellular Space ADAMTS6 1.22E-05 Extracellular Space ADAMTS9 1.04E-02 Extracellular Space ADAMTSL4 1.11E-06 Extracellular Space ADAT3 4.59E-02 Other ADCY1 3.78E-02 Plasma Membrane ADCY10 3.09E-02 Cytoplasm ADCY3 7.75E-04 Plasma Membrane ADCY6 5.08E-03 Plasma Membrane ADCY7 2.61E-03 Plasma Membrane ADCY9 9.80E-03 Plasma Membrane ADD1 3.34E-02 Cytoplasm ADD2 1.43E-02 Cytoplasm ADD3 1.07E-06 Cytoplasm ADH5 2.91E-02 Cytoplasm ADI1 2.36E-02 Nucleus ADIPOQ 2.05E-02 Extracellular Space ADIPOR2 4.70E-03 Plasma Membrane ADK 1.47E-02 Nucleus ADM 3.83E-10 Extracellular Space ADORA2A 1.82E-04 Plasma Membrane ADORA2B 2.73E-03 Plasma Membrane ADRB1 1.18E-03 Plasma Membrane ADRB2 1.96E-04 Plasma Membrane ADRBK2 3.89E-05 Cytoplasm ADSS 6.70E-03 Cytoplasm ADSSL1 4.29E-06 Cytoplasm AES 3.67E-03 Nucleus AFAP1L1 1.18E-02 Other AFAP1L2 2.60E-02 Cytoplasm AGA 3.06E-02 Cytoplasm AGGF1 1.78E-06 Cytoplasm AGO2 1.08E-03 Cytoplasm AGO4 4.79E-05 Cytoplasm AGPAT3 7.60E-03 Cytoplasm AGPAT4 5.32E-04 Cytoplasm AGPAT5 7.31E-03 Cytoplasm AGPAT6 1.41E-05 Cytoplasm AGRN 6.57E-05 Plasma Membrane AHCY 3.79E-02 Cytoplasm AHCYL1 2.25E-04 Cytoplasm AHNAK 5.15E-07 Nucleus AHR 4.97E-03 Nucleus AICDA 1.23E-04 Cytoplasm AIFM2 1.26E-02 Cytoplasm AIFM3 3.37E-02 Cytoplasm AK1 5.38E-03 Cytoplasm AK2 1.99E-03 Cytoplasm AK4 2.27E-04 Cytoplasm AK8 1.51E-02 Other AKAP1 2.26E-04 Cytoplasm AKAP10 5.25E-06 Cytoplasm AKAP11 5.72E-03 Cytoplasm AKAP13 1.35E-02 Cytoplasm AKAP2 8.71E-05 Plasma Membrane AKAP6 7.01E-03 Nucleus AKAP7 3.13E-05 Plasma Membrane AKAP8 2.15E-02 Nucleus AKAP9 1.31E-04 Cytoplasm AKR1A1 1.98E-03 Cytoplasm AKR1B1 4.63E-02 Cytoplasm AKR1C1/AKR1C2 1.07E-04 Cytoplasm AKR1C3 4.50E-04 Cytoplasm AKR7A2 2.98E-02 Cytoplasm AKT1 4.60E-03 Cytoplasm AKT1S1 5.47E-05 Cytoplasm AKT2 5.04E-03 Cytoplasm AKT3 4.74E-05 Cytoplasm ALAS1 6.15E-03 Cytoplasm ALAS2 4.14E-02 Cytoplasm ALCAM 2.08E-03 Plasma Membrane ALDH16A1 1.17E-04 Other ALDH1A2 1.47E-02 Cytoplasm ALDH1A3 9.67E-03 Cytoplasm ALDH1L2 7.33E-07 Cytoplasm ALDH2 9.14E-03 Cytoplasm ALDH3A2 1.35E-03 Cytoplasm ALDH4A1 6.03E-05 Cytoplasm ALDH5A1 1.99E-02 Cytoplasm ALDH6A1 2.48E-03 Cytoplasm ALDH7A1 4.33E-02 Cytoplasm ALDH8A1 1.90E-06 Other ALDH9A1 1.93E-02 Cytoplasm ALDOA 1.09E-02 Cytoplasm ALDOB 1.25E-02 Cytoplasm ALDOC 2.61E-04 Cytoplasm ALG1 7.66E-03 Cytoplasm ALG13 2.68E-02 Cytoplasm ALKBH8 1.38E-02 Cytoplasm ALOX12B 1.99E-04 Other ALOX15B 2.01E-02 Cytoplasm ALOX5AP 1.61E-02 Plasma Membrane ALS2 7.00E-05 Cytoplasm ALYREF 3.11E-03 Nucleus AMACR 4.61E-02 Cytoplasm AMD1 8.72E-06 Cytoplasm AMDHD2 2.31E-02 Other AMFR 1.57E-02 Plasma Membrane AMIGO2 1.15E-04 Plasma Membrane AMOTL1 1.30E-03 Plasma Membrane AMT 3.89E-02 Cytoplasm ANAPC10 2.47E-02 Nucleus ANAPC11 5.57E-03 Cytoplasm ANAPC13 2.09E-02 Nucleus ANAPC2 3.31E-03 Nucleus ANAPC4 4.23E-02 Nucleus ANAPC5 7.05E-07 Nucleus ANAPC7 5.43E-03 Nucleus ANG 8.33E-09 Extracellular Space ANGPTL4 5.55E-05 Extracellular Space ANK3 8.99E-03 Plasma Membrane ANLN 1.26E-07 Cytoplasm ANP32A 4.16E-04 Nucleus ANTXR1 1.49E-03 Plasma Membrane ANXA1 4.20E-07 Plasma Membrane ANXA11 3.50E-02 Nucleus ANXA2 2.13E-02 Plasma Membrane AOC3 9.05E-08 Plasma Membrane AP1B1 1.12E-03 Cytoplasm AP1G1 6.16E-03 Cytoplasm AP1G2 3.40E-02 Cytoplasm AP1S1 9.02E-06 Cytoplasm AP1S2 3.37E-03 Cytoplasm AP1S3 3.56E-04 Cytoplasm AP2A2 7.14E-03 Cytoplasm AP2B1 3.25E-02 Cytoplasm AP2S1 1.55E-02 Cytoplasm APAF1 2.64E-03 Cytoplasm APC 1.04E-06 Nucleus APCDD1 1.98E-02 Plasma Membrane APH1A 8.19E-03 Cytoplasm APH1B 1.80E-04 Plasma Membrane API5 8.01E-08 Cytoplasm APLF 2.04E-03 Cytoplasm APLN 7.09E-04 Extracellular Space APOA2 1.94E-02 Extracellular Space APOBEC3B 1.90E-02 Cytoplasm APOBEC3G 3.30E-02 Nucleus APOC1 1.94E-02 Extracellular Space APOC3 2.02E-02 Extracellular Space APOD 1.82E-02 Extracellular Space APOE 3.38E-03 Extracellular Space APP 2.83E-03 Plasma Membrane APPL1 1.09E-02 Cytoplasm APPL2 4.35E-04 Cytoplasm APTX 5.35E-03 Nucleus AQP3 2.30E-03 Plasma Membrane AQP9 3.15E-03 Plasma Membrane ARAP1 4.10E-03 Cytoplasm AREG/AREGB 1.43E-10 Extracellular Space ARF1 2.42E-05 Cytoplasm ARF3 3.37E-04 Cytoplasm ARF4 8.34E-03 Cytoplasm ARF6 5.68E-03 Plasma Membrane ARFGEF2 2.52E-02 Cytoplasm ARG1 1.26E-02 Cytoplasm ARG2 2.11E-07 Cytoplasm ARHGAP1 1.80E-03 Cytoplasm ARHGAP10 3.62E-03 Cytoplasm ARHGAP12 2.04E-05 Cytoplasm ARHGAP18 1.44E-04 Cytoplasm ARHGAP20 3.96E-02 Cytoplasm ARHGAP22 2.14E-03 Cytoplasm ARHGAP24 2.32E-03 Cytoplasm ARHGAP26 8.99E-06 Cytoplasm ARHGAP28 1.37E-04 Cytoplasm ARHGAP35 1.96E-03 Nucleus ARHGAP5 2.35E-05 Cytoplasm ARHGAP8/PRR5-ARHGAP88.79E-03 Cytoplasm ARHGDIA 1.12E-04 Cytoplasm ARHGDIB 1.21E-03 Cytoplasm ARHGEF10 1.03E-05 Cytoplasm ARHGEF12 4.77E-04 Cytoplasm ARHGEF2 2.34E-04 Cytoplasm ARHGEF26 2.93E-02 Plasma Membrane ARHGEF3 1.25E-04 Cytoplasm ARHGEF4 9.27E-03 Cytoplasm ARHGEF6 2.76E-04 Cytoplasm ARHGEF7 2.85E-04 Cytoplasm ARID4B 4.15E-04 Nucleus ARID5B 3.32E-03 Nucleus ARL1 2.24E-02 Cytoplasm ARL6IP5 2.80E-04 Cytoplasm ARNT 6.95E-03 Nucleus ARNTL 1.83E-03 Nucleus ARPC1B 7.74E-03 Cytoplasm ARPC2 1.89E-02 Cytoplasm ARPC4 5.62E-04 Other ARPC5 2.17E-02 Cytoplasm ARPC5L 1.07E-05 Other ARRB1 3.58E-02 Cytoplasm ART1 5.70E-04 Plasma Membrane ARTN 4.36E-04 Extracellular Space AS3MT 1.62E-04 Cytoplasm ASAH1 1.18E-03 Cytoplasm ASAP1 1.19E-04 Plasma Membrane ASB2 1.70E-08 Nucleus ASCL1 2.61E-02 Nucleus ASGR1 7.88E-03 Plasma Membrane ASIP 2.10E-02 Extracellular Space ASL 1.38E-03 Cytoplasm ASNS 2.80E-02 Cytoplasm ASPH 2.26E-05 Cytoplasm ASRGL1 3.72E-02 Cytoplasm ASS1 7.78E-05 Cytoplasm ATF1 1.02E-02 Nucleus ATF2 3.16E-06 Nucleus ATF3 7.82E-07 Nucleus ATF4 3.57E-02 Nucleus ATF5 2.90E-04 Nucleus ATF6 3.30E-03 Cytoplasm ATF6B 2.56E-02 Nucleus ATF7 3.50E-03 Nucleus ATG13 5.90E-05 Cytoplasm ATG5 1.07E-04 Cytoplasm ATG7 1.50E-02 Cytoplasm ATM 2.11E-03 Nucleus ATMIN 1.15E-04 Nucleus ATN1 3.09E-02 Nucleus ATP13A2 6.51E-05 Cytoplasm ATP1B1 2.84E-02 Plasma Membrane ATP2A2 2.52E-05 Cytoplasm ATP2A3 2.41E-02 Cytoplasm ATP2B1 1.36E-05 Plasma Membrane ATP2B3 1.44E-02 Plasma Membrane ATP2C1 3.42E-03 Cytoplasm ATP2C2 6.28E-03 Cytoplasm ATP5B 4.71E-02 Cytoplasm ATP5C1 1.71E-04 Cytoplasm ATP5G2 3.80E-02 Cytoplasm ATP5G3 1.32E-02 Cytoplasm ATP6V0C 9.58E-03 Cytoplasm ATP6V0D1 3.80E-02 Cytoplasm ATP6V0E1 3.78E-02 Cytoplasm ATP8A2 7.15E-03 Plasma Membrane ATP8B1 1.66E-03 Plasma Membrane ATPAF1 3.55E-02 Cytoplasm ATPAF2 4.88E-02 Cytoplasm ATRX 3.92E-04 Nucleus ATXN1 5.09E-08 Nucleus ATXN2 3.54E-03 Nucleus ATXN3 2.74E-02 Nucleus ATXN7 5.09E-05 Nucleus AUH 5.09E-06 Cytoplasm AURKB 7.76E-06 Nucleus AUTS2 1.01E-03 Nucleus AVEN 8.91E-03 Nucleus AXIN1 1.01E-02 Cytoplasm AXIN2 1.51E-03 Cytoplasm AXL 1.26E-03 Plasma Membrane AZI2 1.55E-04 Cytoplasm B2M 2.12E-08 Plasma Membrane B3GALT6 4.04E-02 Cytoplasm B4GALT2 3.41E-03 Cytoplasm B4GALT7 3.61E-02 Cytoplasm BACH1 1.96E-05 Nucleus BACH2 4.89E-09 Nucleus BAG1 1.06E-04 Cytoplasm BAG3 7.76E-05 Cytoplasm BAG4 2.70E-03 Cytoplasm BAG5 2.50E-06 Cytoplasm BAG6 6.44E-03 Nucleus BAK1 2.93E-04 Cytoplasm BAMBI 1.41E-02 Plasma Membrane BAP1 1.98E-04 Nucleus BARD1 5.32E-06 Nucleus BAX 4.98E-04 Cytoplasm BBX 9.08E-05 Nucleus BCAP31 4.03E-02 Cytoplasm BCAR1 2.15E-05 Plasma Membrane BCAT2 1.93E-02 Cytoplasm BCHE 1.59E-04 Plasma Membrane BCKDHB 1.17E-02 Cytoplasm BCL11A 9.16E-08 Nucleus BCL2 1.11E-03 Cytoplasm BCL2A1 2.95E-02 Cytoplasm BCL2L1 6.61E-04 Cytoplasm BCL2L11 1.51E-05 Cytoplasm BCL2L13 8.68E-03 Cytoplasm BCL2L14 3.42E-02 Cytoplasm BCL2L2 2.27E-02 Cytoplasm BCL3 4.28E-04 Nucleus BCL6 3.34E-04 Nucleus BCLAF1 3.27E-05 Nucleus BCR 4.65E-04 Cytoplasm BDH1 1.41E-02 Cytoplasm BDH2 2.54E-02 Cytoplasm BDKRB2 2.30E-03 Plasma Membrane BDNF 4.69E-03 Extracellular Space BDP1 6.53E-06 Nucleus BECN1 1.77E-03 Cytoplasm BET1L 1.20E-05 Cytoplasm BEX1 4.11E-02 Other BEX2 3.51E-05 Nucleus BFAR 2.32E-02 Cytoplasm BHLHE40 1.48E-07 Nucleus BHLHE41 2.35E-07 Nucleus BHMT2 4.45E-02 Cytoplasm BID 1.44E-02 Cytoplasm BIK 2.25E-05 Cytoplasm BIN1 1.37E-02 Nucleus BIN3 4.83E-05 Cytoplasm BIRC2 7.47E-05 Cytoplasm BIRC3 1.44E-09 Cytoplasm BIRC5 1.09E-02 Cytoplasm BLNK 1.47E-04 Cytoplasm BLVRB 1.45E-05 Cytoplasm BLZF1 2.93E-03 Cytoplasm BMP1 8.60E-04 Extracellular Space BMP2 6.56E-10 Extracellular Space BMPER 5.38E-04 Extracellular Space BMPR1A 7.83E-03 Plasma Membrane BMPR1B 3.55E-05 Plasma Membrane BMPR2 2.26E-03 Plasma Membrane BNC1 3.33E-02 Nucleus BNIP1 2.36E-03 Cytoplasm BNIP2 1.58E-02 Cytoplasm BNIP3 1.33E-08 Cytoplasm BNIP3L 3.51E-07 Cytoplasm BOP1 1.13E-02 Nucleus BPI 4.77E-02 Plasma Membrane BPNT1 1.50E-02 Nucleus BRAF 1.08E-05 Cytoplasm BRAT1 3.10E-05 Other BRCA2 2.75E-03 Nucleus BRE 4.24E-02 Cytoplasm BRF2 1.08E-02 Nucleus BRIP1 2.33E-06 Nucleus BRK1 1.33E-02 Other BSG 2.45E-04 Plasma Membrane BTD 2.21E-02 Extracellular Space BTG1 1.00E-07 Nucleus BTG2 5.91E-06 Nucleus BTG3 1.67E-06 Nucleus BUB1 3.67E-03 Nucleus BUB1B 2.88E-02 Nucleus C12orf5 8.48E-04 Other C15orf37 9.84E-03 Other C17orf70 1.62E-04 Nucleus C1QBP 4.88E-03 Cytoplasm C1S 4.36E-04 Extracellular Space C3 1.32E-03 Extracellular Space C5 2.32E-03 Extracellular Space CA4 5.00E-03 Plasma Membrane CA9 4.69E-08 Nucleus CAB39 1.91E-02 Cytoplasm CABLES1 8.45E-03 Nucleus CACNA1E 6.24E-03 Plasma Membrane CACNB2 1.67E-02 Plasma Membrane CACNB3 2.05E-02 Plasma Membrane CACNG5 4.40E-02 Plasma Membrane CACUL1 4.71E-03 Other CAD 2.07E-06 Cytoplasm CALB1 4.83E-06 Cytoplasm CALCA 2.33E-02 Plasma Membrane CALM1 (includes others)7.49E-05 Cytoplasm CALML5 4.44E-03 Cytoplasm CALR 1.01E-02 Cytoplasm CAMK2D 2.16E-02 Cytoplasm CAMK2G 1.55E-03 Cytoplasm CAMK4 1.05E-03 Nucleus CAMKK2 6.58E-03 Cytoplasm CAND1 9.37E-06 Cytoplasm CANT1 2.08E-02 Extracellular Space CANX 4.28E-04 Cytoplasm CAPN1 7.17E-06 Cytoplasm CAPN2 1.32E-04 Cytoplasm CAPN3 6.29E-05 Cytoplasm CAPN7 2.25E-03 Cytoplasm CAPNS1 2.17E-03 Cytoplasm CAPZA1 1.90E-02 Cytoplasm CARD10 8.99E-05 Cytoplasm CARD11 4.28E-02 Cytoplasm CARD8 1.22E-02 Nucleus CARS 4.91E-04 Cytoplasm CASC3 7.27E-03 Nucleus CASK 4.29E-04 Plasma Membrane CASP10 3.23E-02 Cytoplasm CASP2 4.82E-05 Cytoplasm CASP3 2.55E-02 Cytoplasm CASP4 2.53E-02 Cytoplasm CASP6 9.79E-04 Cytoplasm CASP8 1.94E-03 Nucleus CAST 3.69E-03 Cytoplasm CAT 1.15E-03 Cytoplasm CATSPER2 2.92E-03 Plasma Membrane CAV1 3.72E-02 Plasma Membrane CAV2 3.47E-02 Plasma Membrane CBFA2T3 1.12E-02 Nucleus CBL 9.93E-04 Nucleus CBLL1 5.23E-06 Nucleus CBR1 2.16E-02 Cytoplasm CBX5 4.44E-03 Nucleus CCBL1 1.23E-02 Cytoplasm CCL20 1.18E-10 Extracellular Space CCL27 3.71E-04 Extracellular Space CCL28 3.95E-06 Extracellular Space CCL5 4.33E-07 Extracellular Space CCNA1 1.59E-02 Nucleus CCNB1 6.23E-04 Cytoplasm CCNB1IP1 4.16E-04 Nucleus CCNB2 1.48E-04 Cytoplasm CCNC 6.66E-06 Nucleus CCND1 2.99E-06 Nucleus CCND2 1.56E-05 Nucleus CCNE1 4.09E-04 Nucleus CCNF 2.79E-02 Nucleus CCNG1 5.50E-03 Nucleus CCNG2 1.87E-06 Nucleus CCNI 2.46E-07 Other CCNL2 9.93E-03 Nucleus CCNT1 2.22E-04 Nucleus CCNY 2.35E-05 Nucleus CCRN4L 7.64E-05 Nucleus CCS 5.95E-03 Cytoplasm CCT2 5.61E-07 Cytoplasm CCT7 3.64E-02 Cytoplasm CD163 1.83E-02 Plasma Membrane CD177 3.70E-02 Cytoplasm CD247 1.21E-02 Plasma Membrane CD28 6.00E-02 Plasma Membrane CD2AP 3.91E-02 Cytoplasm CD2BP2 2.77E-03 Cytoplasm CD300A 7.94E-03 Plasma Membrane CD3E 4.28E-02 Plasma Membrane CD4 4.44E-02 Plasma Membrane CD40 5.46E-03 Plasma Membrane CD44 7.88E-08 Plasma Membrane CD46 2.66E-02 Plasma Membrane CD47 2.15E-07 Plasma Membrane CD53 3.75E-02 Plasma Membrane CD55 1.16E-07 Plasma Membrane CD59 1.02E-02 Plasma Membrane CD6 1.45E-02 Plasma Membrane CD68 2.77E-02 Plasma Membrane CD69 1.89E-05 Plasma Membrane CD70 3.14E-02 Extracellular Space CD74 4.17E-02 Plasma Membrane CD79A 8.05E-03 Plasma Membrane CD82 8.28E-03 Plasma Membrane CD83 6.83E-04 Plasma Membrane CD84 4.82E-02 Plasma Membrane CD86 2.17E-04 Plasma Membrane CD9 5.77E-07 Plasma Membrane CD96 7.54E-03 Plasma Membrane CD97 3.62E-04 Plasma Membrane CD99 1.77E-02 Plasma Membrane CDC123 3.90E-04 Cytoplasm CDC14A 8.49E-04 Nucleus CDC14B 8.14E-07 Nucleus CDC16 3.09E-02 Nucleus CDC20 1.34E-04 Nucleus CDC23 4.66E-02 Nucleus CDC25A 3.52E-07 Nucleus CDC25B 1.95E-03 Nucleus CDC25C 6.44E-04 Nucleus CDC26 4.72E-04 Nucleus CDC27 8.45E-05 Nucleus CDC34 4.51E-02 Nucleus CDC37 1.05E-03 Cytoplasm CDC42 4.14E-02 Cytoplasm CDC42BPA 5.39E-05 Cytoplasm CDC42EP3 2.10E-03 Cytoplasm CDC42EP5 6.29E-04 Cytoplasm CDC6 1.14E-03 Nucleus CDCA7 6.18E-08 Nucleus CDH1 6.34E-04 Plasma Membrane CDH11 7.89E-03 Plasma Membrane CDH13 7.30E-04 Plasma Membrane CDH16 9.89E-05 Plasma Membrane CDH4 9.55E-03 Plasma Membrane CDH8 2.06E-02 Plasma Membrane CDIP1 2.81E-04 Nucleus CDK10 3.11E-03 Nucleus CDK11A/CDK11B 6.77E-03 Nucleus CDK13 1.05E-03 Nucleus CDK19 7.01E-07 Nucleus CDK3 3.94E-02 Other CDK4 3.48E-03 Nucleus CDK6 2.64E-03 Nucleus CDK8 1.63E-04 Nucleus CDK9 1.62E-03 Nucleus CDKL2 8.35E-04 Nucleus CDKN1B 2.78E-09 Nucleus CDKN1C 6.77E-09 Nucleus CDKN2AIP 7.94E-03 Nucleus CDKN2B 1.64E-03 Nucleus CDKN2C 1.28E-04 Nucleus CDO1 9.17E-04 Cytoplasm CDS2 4.95E-02 Cytoplasm CDYL 5.91E-03 Nucleus CEACAM1 8.10E-03 Plasma Membrane CEBPA 1.48E-03 Nucleus CEBPD 2.82E-05 Nucleus CEBPZ 5.34E-06 Nucleus CECR1 6.78E-03 Extracellular Space CELF1 2.57E-03 Nucleus CELF2 1.93E-07 Nucleus CENPA 4.23E-02 Nucleus CENPF 3.21E-03 Nucleus CEP350 3.20E-06 Cytoplasm CEPT1 2.13E-07 Cytoplasm CES2 5.41E-03 Cytoplasm CFB 3.01E-04 Extracellular Space CFD 4.68E-03 Extracellular Space CFH 1.82E-02 Extracellular Space CFHR1 1.44E-02 Extracellular Space CFL1 5.35E-03 Nucleus CFL2 3.12E-02 Other CFLAR 6.66E-07 Cytoplasm CGA 2.61E-02 Extracellular Space CHAF1A 1.67E-02 Nucleus CHD1L 2.57E-03 Nucleus CHD4 1.45E-04 Nucleus CHEK1 3.43E-04 Nucleus CHERP 9.05E-05 Cytoplasm CHI3L1 1.41E-02 Extracellular Space CHMP1A 2.25E-04 Extracellular Space CHMP2B 3.77E-03 Cytoplasm CHMP3 2.30E-02 Cytoplasm CHMP4B 5.37E-03 Cytoplasm CHMP6 1.10E-03 Cytoplasm CHP1 9.19E-03 Cytoplasm CHPF2 5.04E-04 Cytoplasm CHPT1 1.70E-04 Cytoplasm CHRM4 3.57E-03 Plasma Membrane CHRNA3 2.15E-02 Plasma Membrane CHRNA5 3.65E-02 Plasma Membrane CHRNB1 2.99E-02 Plasma Membrane CHST11 8.29E-03 Cytoplasm CHST12 8.03E-03 Cytoplasm CHST14 1.03E-02 Cytoplasm CHST2 8.31E-03 Cytoplasm CHST3 1.28E-03 Cytoplasm CHST4 1.94E-02 Cytoplasm CHST7 1.24E-06 Cytoplasm CHSY1 9.67E-05 Cytoplasm CHTOP 9.44E-03 Nucleus CHUK 1.19E-02 Cytoplasm CIAO1 1.46E-03 Nucleus CIAPIN1 1.46E-06 Cytoplasm CIRBP 4.89E-06 Nucleus CISH 6.14E-03 Cytoplasm CITED2 5.68E-07 Nucleus CITED4 7.60E-05 Nucleus CKAP5 2.19E-03 Nucleus CKB 1.25E-04 Cytoplasm CKLF 1.26E-02 Extracellular Space CKS1B 4.03E-02 Other CKS2 7.95E-05 Other CLASP1 3.59E-03 Cytoplasm CLCF1 2.48E-02 Extracellular Space CLCN7 1.79E-02 Plasma Membrane CLDN11 4.77E-03 Plasma Membrane CLDN15 9.97E-05 Plasma Membrane CLDN18 1.91E-02 Plasma Membrane CLDN4 4.77E-02 Plasma Membrane CLEC11A 3.96E-04 Extracellular Space CLEC2B 6.94E-09 Plasma Membrane CLEC7A 1.32E-04 Plasma Membrane CLECL1 3.26E-02 Plasma Membrane CLIC2 1.79E-03 Cytoplasm CLIC4 1.60E-04 Plasma Membrane CLINT1 2.20E-02 Cytoplasm CLIP1 4.54E-05 Cytoplasm CLK1 1.00E-04 Nucleus CLOCK 7.28E-04 Nucleus CLTB 1.31E-03 Plasma Membrane CLTC 4.12E-02 Plasma Membrane CMC4 2.42E-04 Cytoplasm CNGA1 6.71E-06 Plasma Membrane CNKSR3 4.51E-03 Plasma Membrane CNOT3 7.42E-04 Cytoplasm CNOT7 4.83E-06 Nucleus CNOT8 8.21E-09 Nucleus CNR2 2.07E-02 Plasma Membrane CNTNAP1 5.79E-03 Plasma Membrane COASY 2.20E-03 Cytoplasm COL16A1 2.94E-02 Extracellular Space COL17A1 1.42E-02 Plasma Membrane COL18A1 7.83E-03 Extracellular Space COL1A1 1.93E-02 Extracellular Space COL1A2 1.23E-02 Extracellular Space COL4A1 9.85E-04 Extracellular Space COL4A2 2.14E-03 Extracellular Space COL4A3 2.52E-02 Extracellular Space COL5A1 3.86E-04 Extracellular Space COLGALT1 4.28E-02 Cytoplasm COPA 3.53E-03 Cytoplasm COPB1 6.54E-05 Cytoplasm COPE 3.34E-03 Cytoplasm COPG2 2.00E-02 Cytoplasm COQ2 7.84E-03 Cytoplasm CORIN 1.78E-05 Plasma Membrane CORO1A 4.22E-02 Cytoplasm CORO6 1.34E-02 Extracellular Space COX15 4.30E-02 Cytoplasm COX17 1.21E-02 Cytoplasm COX4I1 3.39E-02 Cytoplasm COX5B 1.83E-03 Cytoplasm COX6B1 3.32E-02 Cytoplasm COX7B 5.12E-03 Cytoplasm COX7C 3.81E-02 Cytoplasm CP 2.23E-02 Extracellular Space CPE 1.04E-03 Cytoplasm CPOX 6.18E-06 Cytoplasm CPS1 1.81E-04 Cytoplasm CPSF2 2.43E-04 Nucleus CPSF6 1.27E-05 Nucleus CPT1A 2.76E-02 Cytoplasm CPT1B 5.37E-05 Cytoplasm CPT2 1.49E-05 Cytoplasm CRABP1 3.00E-03 Cytoplasm CRCP 2.06E-04 Plasma Membrane CREB1 4.90E-05 Nucleus CREB3 3.35E-03 Nucleus CREBBP 1.40E-02 Nucleus CREBL2 7.84E-03 Nucleus CREBZF 3.27E-05 Nucleus CREM 4.40E-02 Nucleus CRHR1 2.44E-02 Plasma Membrane CRK 8.81E-04 Cytoplasm CRKL 1.36E-02 Cytoplasm CRTAM 1.10E-05 Plasma Membrane CRTC2 8.25E-01 Nucleus CRTC3 3.16E-04 Other CRYL1 1.22E-04 Cytoplasm CS 3.66E-02 Cytoplasm CSAD 6.28E-04 Cytoplasm CSF1 1.25E-02 Extracellular Space CSF2 8.50E-07 Extracellular Space CSF2RB 1.27E-02 Plasma Membrane CSF3 5.73E-01 Extracellular Space CSGALNACT2 9.85E-09 Cytoplasm CSK 9.05E-05 Cytoplasm CSNK1A1 1.32E-05 Cytoplasm CSNK1D 2.02E-05 Cytoplasm CSNK1E 4.47E-02 Cytoplasm CSNK1G1 3.84E-06 Cytoplasm CSNK1G2 1.71E-02 Cytoplasm CSNK2A1 3.87E-06 Cytoplasm CSNK2A2 2.70E-02 Cytoplasm CSRP2 8.28E-09 Nucleus CST3 9.06E-03 Extracellular Space CST6 2.87E-02 Extracellular Space CSTF1 8.37E-05 Nucleus CSTF2 1.14E-02 Nucleus CTBP2 2.15E-02 Nucleus CTGF 1.33E-07 Extracellular Space CTLA4 1.77E-02 Plasma Membrane CTNNA1 5.03E-04 Plasma Membrane CTNNA2 4.42E-02 Plasma Membrane CTNNA3 4.65E-02 Plasma Membrane CTNNB1 3.11E-04 Nucleus CTPS1 4.69E-04 Nucleus CTPS2 2.61E-03 Cytoplasm CTSB 4.25E-03 Cytoplasm CTSC 1.41E-03 Cytoplasm CTSD 2.99E-02 Cytoplasm CTSF 1.55E-02 Cytoplasm CTSK 1.51E-06 Cytoplasm CTSS 4.14E-04 Cytoplasm CTSZ 4.98E-03 Cytoplasm CTTN 6.56E-03 Plasma Membrane CUL1 2.96E-02 Nucleus CUL3 6.15E-07 Nucleus CUL4B 9.99E-05 Nucleus CUL5 1.10E-06 Nucleus CUL9 6.95E-04 Cytoplasm CUX1 1.62E-04 Nucleus CXADR 8.52E-06 Plasma Membrane CXCL1 5.24E-09 Extracellular Space CXCL10 3.10E-07 Extracellular Space CXCL11 1.45E-05 Other CXCL2 9.12E-10 Extracellular Space CXCL3 8.75E-10 Extracellular Space CXCL5 5.14E-05 Extracellular Space CXCL6 8.41E-05 Extracellular Space CXCR2 1.48E-04 Plasma Membrane CXCR7 2.09E-09 Plasma Membrane CYB5R3 5.35E-03 Cytoplasm CYBB 3.91E-02 Cytoplasm CYCS 9.59E-05 Cytoplasm CYLD 3.16E-07 Nucleus CYP1A1 2.27E-03 Cytoplasm CYP1A2 8.91E-03 Cytoplasm CYP1B1 4.34E-04 Cytoplasm CYP21A2 1.83E-02 Cytoplasm CYP24A1 1.14E-06 Cytoplasm CYP26A1 2.73E-03 Cytoplasm CYP26B1 6.56E-05 Cytoplasm CYP27A1 2.77E-02 Cytoplasm CYP27B1 1.20E-02 Cytoplasm CYP2C9 1.52E-02 Cytoplasm CYP2E1 6.44E-04 Cytoplasm CYP2J2 2.40E-03 Cytoplasm CYP2R1 1.61E-03 Cytoplasm CYP2U1 5.60E-05 Cytoplasm CYP39A1 1.18E-02 Cytoplasm CYP3A5 4.76E-03 Cytoplasm CYP4F3 1.62E-03 Cytoplasm CYP51A1 2.42E-04 Cytoplasm CYP7B1 1.58E-02 Cytoplasm CYR61 2.01E-02 Extracellular Space DAAM1 1.48E-05 Cytoplasm DAG1 7.15E-04 Plasma Membrane DAP3 2.53E-02 Cytoplasm DAPK1 3.41E-04 Cytoplasm DAPK2 2.90E-02 Cytoplasm DAPK3 1.95E-03 Cytoplasm DARS 3.95E-04 Cytoplasm DAXX 2.10E-02 Nucleus DBF4 1.16E-02 Nucleus DBF4B 1.09E-02 Nucleus DBN1 1.49E-04 Cytoplasm DBT 4.53E-04 Cytoplasm DCBLD2 6.90E-06 Plasma Membrane DCLRE1B 7.66E-03 Nucleus DCLRE1C 1.50E-05 Nucleus DCN 4.57E-03 Extracellular Space DCT 4.02E-02 Cytoplasm DCTN1 2.80E-04 Cytoplasm DDB1 1.09E-03 Nucleus DDC 2.76E-02 Cytoplasm DDIT3 3.76E-07 Nucleus DDIT4 1.87E-04 Cytoplasm DDR2 1.29E-08 Plasma Membrane DDT 3.39E-03 Cytoplasm DDX11 4.56E-04 Cytoplasm DDX17 9.09E-06 Nucleus DDX3X 7.54E-04 Cytoplasm DDX56 1.45E-04 Nucleus DDX58 1.15E-03 Cytoplasm DDX6 1.82E-05 Nucleus DEDD2 1.18E-04 Nucleus DEFB4A/DEFB4B 3.70E-02 Extracellular Space DEPTOR 1.09E-02 Other DET1 5.35E-03 Nucleus DFFA 1.25E-04 Nucleus DFFB 1.05E-04 Nucleus DGAT2 1.76E-03 Cytoplasm DGKA 1.97E-04 Cytoplasm DGKZ 4.09E-02 Cytoplasm DHCR24 5.49E-03 Cytoplasm DHCR7 2.64E-02 Cytoplasm DHDDS 2.38E-05 Other DHFR 2.90E-03 Nucleus DHFRL1 1.83E-04 Cytoplasm DHPS 4.31E-02 Cytoplasm DHRS3 1.13E-03 Cytoplasm DHRS9 4.99E-03 Cytoplasm DHTKD1 1.35E-03 Other DHX9 2.74E-05 Nucleus DIAPH1 1.49E-03 Plasma Membrane DIAPH2 3.16E-02 Cytoplasm DIAPH3 2.70E-04 Cytoplasm DICER1 4.58E-07 Cytoplasm DIO2 8.20E-03 Cytoplasm DIP2A 3.97E-06 Nucleus DISC1 9.61E-07 Cytoplasm DKC1 2.01E-06 Nucleus DKK1 3.99E-05 Extracellular Space DKK2 1.92E-02 Extracellular Space DKK3 4.81E-02 Extracellular Space DKKL1 3.27E-02 Extracellular Space DLC1 2.68E-05 Cytoplasm DLD 1.24E-02 Cytoplasm DLG1 1.40E-04 Plasma Membrane DLG2 2.88E-02 Plasma Membrane DLG4 3.90E-02 Plasma Membrane DLG5 3.30E-06 Plasma Membrane DLGAP5 9.00E-04 Nucleus DLK1 1.40E-02 Extracellular Space DLL1 8.40E-09 Plasma Membrane DLL3 8.13E-04 Extracellular Space DLST 1.75E-02 Cytoplasm DLX4 1.97E-02 Nucleus DMD 1.51E-03 Plasma Membrane DNAJA1 1.20E-04 Nucleus DNAJA2 2.39E-03 Nucleus DNAJA3 4.26E-02 Cytoplasm DNAJA4 3.44E-02 Nucleus DNAJB1 3.41E-06 Nucleus DNAJB11 1.53E-02 Cytoplasm DNAJB12 1.01E-02 Cytoplasm DNAJB5 1.33E-03 Cytoplasm DNAJB6 4.44E-05 Nucleus DNAJB9 2.77E-04 Nucleus DNAJC1 5.04E-03 Cytoplasm DNAJC10 1.00E-02 Cytoplasm DNAJC11 4.81E-04 Cytoplasm DNAJC13 5.16E-03 Cytoplasm DNAJC14 1.13E-02 Cytoplasm DNAJC16 1.14E-03 Other DNAJC17 2.72E-03 Other DNAJC18 6.09E-03 Other DNAJC2 9.15E-04 Nucleus DNAJC21 7.43E-03 Other DNAJC24 3.31E-02 Other DNAJC25 2.65E-03 Other DNAJC27 9.09E-03 Cytoplasm DNAJC28 1.04E-03 Other DNAJC3 5.86E-06 Cytoplasm DNAJC30 3.89E-04 Cytoplasm DNAJC4 3.86E-02 Cytoplasm DNAJC5 2.28E-04 Plasma Membrane DNAJC7 1.48E-03 Cytoplasm DNAJC9 7.15E-03 Other DNASE1 3.49E-02 Extracellular Space DNM2 1.11E-02 Plasma Membrane DNM3 2.46E-05 Cytoplasm DNMBP 1.40E-05 Cytoplasm DNMT3A 4.38E-02 Nucleus DNTT 1.34E-02 Nucleus DOCK1 4.68E-05 Cytoplasm DOCK10 1.58E-04 Other DOK3 5.16E-05 Cytoplasm DOK4 7.56E-04 Plasma Membrane DOLK 4.70E-05 Cytoplasm DOT1L 2.81E-02 Nucleus DPH1 8.77E-03 Cytoplasm DPH5 1.71E-02 Other DPM2 1.58E-02 Cytoplasm DPP4 1.53E-03 Plasma Membrane DPYD 5.03E-05 Cytoplasm DPYSL2 9.89E-05 Cytoplasm DR1 1.62E-02 Nucleus DRAM1 2.86E-04 Cytoplasm DRAM2 1.20E-05 Cytoplasm DSCAM 4.33E-08 Plasma Membrane DSE 2.70E-02 Cytoplasm DSG2 1.05E-02 Plasma Membrane DSG3 3.39E-03 Plasma Membrane DST 1.02E-05 Plasma Membrane DSTN 4.58E-03 Cytoplasm DSTYK 1.25E-03 Cytoplasm DTL 1.49E-04 Nucleus DTNA 6.71E-07 Plasma Membrane DTYMK 1.33E-02 Cytoplasm DUOX1 1.26E-03 Plasma Membrane DUS2L 1.71E-03 Cytoplasm DUSP1 1.69E-09 Nucleus DUSP10 1.63E-09 Nucleus DUSP11 1.63E-04 Nucleus DUSP14 5.74E-05 Other DUSP16 2.99E-04 Nucleus DUSP18 3.26E-02 Cytoplasm DUSP2 7.57E-04 Nucleus DUSP27 3.45E-03 Other DUSP3 5.02E-04 Cytoplasm DUSP4 1.05E-08 Nucleus DUSP5 1.35E-02 Nucleus DUSP6 2.20E-07 Cytoplasm DUSP7 1.37E-05 Cytoplasm DUSP8 1.60E-06 Nucleus DUSP9 3.45E-02 Nucleus DVL1 4.13E-02 Cytoplasm DVL2 8.52E-03 Cytoplasm DYRK2 1.35E-05 Cytoplasm DYRK3 1.17E-02 Nucleus E2F3 1.13E-03 Nucleus E2F5 3.38E-02 Nucleus E2F6 4.81E-04 Nucleus E2F7 8.64E-05 Nucleus E4F1 4.88E-04 Nucleus EAPP 3.02E-02 Cytoplasm EARS2 7.24E-04 Cytoplasm EBNA1BP2 2.47E-04 Nucleus EBP 1.91E-02 Cytoplasm ECE1 4.13E-02 Plasma Membrane ECE2 2.17E-02 Plasma Membrane ECHDC1 2.30E-02 Cytoplasm ECI2 2.93E-02 Cytoplasm EDAR 3.90E-03 Plasma Membrane EDIL3 4.62E-02 Extracellular Space EDN1 9.64E-05 Extracellular Space EDN2 1.38E-08 Extracellular Space EDNRA 1.13E-02 Plasma Membrane EDNRB 4.98E-02 Plasma Membrane EED 8.83E-09 Nucleus EEF1D 2.04E-02 Cytoplasm EEF2 3.50E-02 Cytoplasm EFEMP2 1.61E-03 Extracellular Space EFNA1 7.08E-09 Plasma Membrane EFNA3 4.18E-03 Plasma Membrane EFNA4 1.01E-02 Plasma Membrane EFNA5 8.84E-05 Plasma Membrane EFNB1 1.33E-02 Plasma Membrane EGFR 5.16E-04 Plasma Membrane EGLN1 4.37E-10 Cytoplasm EGLN2 4.33E-03 Cytoplasm EGLN3 5.50E-12 Cytoplasm EGR1 2.33E-02 Nucleus EGR3 7.19E-04 Nucleus EGR4 7.14E-03 Nucleus EHD1 1.56E-02 Cytoplasm EHD4 1.30E-05 Plasma Membrane EHF 2.89E-06 Nucleus EHMT1 9.31E-05 Nucleus EI24 4.66E-03 Other EIF1 2.36E-05 Other EIF1AX 3.27E-03 Cytoplasm EIF1AY 2.10E-02 Cytoplasm EIF2AK2 2.25E-02 Cytoplasm EIF2AK3 9.43E-07 Cytoplasm EIF2B1 3.61E-02 Cytoplasm EIF2B2 1.07E-02 Cytoplasm EIF2B3 5.56E-03 Cytoplasm EIF2S1 1.34E-04 Cytoplasm EIF2S2 2.05E-03 Cytoplasm EIF2S3 4.43E-03 Cytoplasm EIF3A 4.95E-03 Cytoplasm EIF3B 7.73E-04 Cytoplasm EIF3C/EIF3CL 1.90E-09 Cytoplasm EIF3D 3.15E-02 Cytoplasm EIF3E 2.86E-02 Cytoplasm EIF3I 1.74E-02 Cytoplasm EIF3J 1.62E-03 Cytoplasm EIF3K 1.03E-02 Cytoplasm EIF3M 1.65E-06 Other EIF4A1 5.06E-04 Cytoplasm EIF4A2 1.83E-03 Cytoplasm EIF4A3 4.30E-05 Nucleus EIF4E 6.85E-05 Cytoplasm EIF4EBP1 5.29E-04 Cytoplasm EIF4EBP2 5.84E-03 Cytoplasm EIF4G1 3.43E-05 Cytoplasm EIF4G2 4.74E-02 Cytoplasm EIF4G3 6.91E-03 Cytoplasm EIF5 1.05E-07 Cytoplasm EIF5A 9.08E-04 Cytoplasm EIF5A2 1.06E-02 Cytoplasm EIF5B 7.20E-03 Cytoplasm ELAC2 2.48E-04 Nucleus ELAVL1 3.91E-02 Cytoplasm ELF1 1.16E-03 Nucleus ELF2 4.19E-03 Nucleus ELF3 5.36E-04 Nucleus ELK1 3.05E-02 Nucleus ELK3 1.22E-02 Nucleus ELK4 3.40E-02 Nucleus ELL 9.89E-03 Nucleus ELMO1 8.62E-04 Cytoplasm ELMO2 7.43E-03 Cytoplasm ELMO3 4.58E-02 Cytoplasm ELN 2.48E-02 Extracellular Space ELOVL1 1.04E-03 Cytoplasm ELOVL6 1.03E-04 Cytoplasm EMP1 3.83E-05 Plasma Membrane EMP2 9.11E-03 Plasma Membrane EMX2 3.74E-03 Nucleus ENAH 9.13E-04 Plasma Membrane ENC1 8.09E-08 Nucleus ENO1 1.39E-02 Cytoplasm ENO2 5.16E-06 Cytoplasm ENOSF1 1.96E-02 Other ENPP2 2.27E-02 Plasma Membrane ENTPD1 2.60E-02 Plasma Membrane ENTPD2 5.48E-03 Cytoplasm ENTPD6 4.46E-04 Cytoplasm ENTPD7 5.42E-05 Cytoplasm EP300 4.87E-02 Nucleus EPAS1 4.14E-03 Nucleus EPB41 1.68E-02 Plasma Membrane EPCAM 2.70E-02 Plasma Membrane EPHA1 8.98E-04 Plasma Membrane EPHA3 8.19E-08 Plasma Membrane EPHA4 4.12E-03 Plasma Membrane EPHA8 9.44E-05 Plasma Membrane EPHB1 2.84E-02 Plasma Membrane EPHB4 4.35E-03 Plasma Membrane EPHB6 2.60E-02 Plasma Membrane EPHX1 1.30E-02 Cytoplasm EPHX2 3.28E-02 Cytoplasm EPM2AIP1 4.10E-05 Cytoplasm EPN1 3.42E-02 Plasma Membrane EPOR 1.49E-03 Plasma Membrane EPRS 6.74E-06 Cytoplasm EPS15L1 1.39E-02 Plasma Membrane EPS8 1.67E-02 Plasma Membrane EPSTI1 4.26E-03 Other EPT1 2.17E-02 Cytoplasm ERAP1 1.10E-03 Extracellular Space ERAP2 2.20E-03 Cytoplasm ERBB2 1.57E-03 Plasma Membrane ERBB3 1.45E-02 Plasma Membrane ERBB4 1.47E-02 Plasma Membrane ERC1 4.79E-03 Cytoplasm ERCC2 1.75E-03 Nucleus ERCC3 1.09E-02 Nucleus ERCC6 4.13E-03 Nucleus EREG 1.05E-05 Extracellular Space ERG 2.46E-02 Nucleus ERN1 6.47E-03 Cytoplasm ERRFI1 2.83E-07 Cytoplasm ESD 3.56E-04 Cytoplasm ESR1 9.59E-05 Nucleus ESRP2 1.27E-04 Nucleus ESRRG 1.15E-04 Nucleus ETFB 4.01E-02 Cytoplasm ETNK1 1.93E-04 Cytoplasm ETS1 9.97E-09 Nucleus ETS2 7.71E-05 Nucleus ETV6 4.43E-05 Nucleus EVI5 2.83E-02 Other EVPL 4.68E-03 Plasma Membrane EWSR1 4.40E-04 Nucleus EXO1 3.23E-02 Nucleus EXOC3 6.45E-03 Plasma Membrane EXOC4 1.69E-03 Cytoplasm EXOC6 1.04E-02 Plasma Membrane EXOC7 9.56E-07 Plasma Membrane EXOC8 1.19E-06 Plasma Membrane EXOSC2 2.67E-04 Nucleus EXOSC4 3.00E-04 Nucleus EXT1 1.07E-03 Cytoplasm EXTL2 3.71E-02 Cytoplasm EXTL3 2.00E-03 Cytoplasm EYA4 2.87E-02 Cytoplasm EZH2 1.05E-02 Nucleus EZR 1.55E-03 Plasma Membrane F11R 2.50E-03 Plasma Membrane F12 7.34E-04 Extracellular Space F13B 3.91E-02 Extracellular Space F2R 3.17E-02 Plasma Membrane F2RL1 3.57E-02 Plasma Membrane F8 3.86E-02 Extracellular Space F9 2.44E-02 Extracellular Space FA2H 3.01E-03 Cytoplasm FAAH2 5.82E-03 Other FABP1 9.76E-03 Cytoplasm FABP3 2.49E-02 Cytoplasm FABP6 1.84E-05 Cytoplasm FABP7 1.18E-02 Cytoplasm FADD 3.93E-06 Cytoplasm FADS1 3.98E-02 Plasma Membrane FADS2 3.98E-03 Plasma Membrane FADS3 3.65E-02 Plasma Membrane FAF1 4.19E-03 Nucleus FAH 3.43E-02 Cytoplasm FAM129A 1.20E-03 Cytoplasm FAM162A 8.35E-07 Cytoplasm FAM175A 1.79E-02 Nucleus FAM208A 3.35E-04 Other FAM20B 9.89E-04 Extracellular Space FANCA 2.64E-07 Nucleus FANCB 7.25E-05 Nucleus FANCC 2.82E-04 Nucleus FANCD2 3.71E-03 Nucleus FANCE 2.46E-06 Nucleus FANCG 5.98E-04 Nucleus FANCL 2.08E-04 Nucleus FANCM 3.32E-05 Nucleus FARS2 1.25E-04 Cytoplasm FARSA 3.19E-04 Cytoplasm FARSB 4.97E-08 Cytoplasm FAS 1.94E-02 Plasma Membrane FASN 2.06E-04 Cytoplasm FASTK 1.88E-02 Cytoplasm FAT4 1.43E-09 Other FBLIM1 1.58E-05 Plasma Membrane FBLN1 1.28E-03 Extracellular Space FBN1 8.43E-04 Extracellular Space FBN2 1.34E-06 Extracellular Space FBXO15 1.58E-03 Other FBXO32 4.12E-08 Cytoplasm FBXO45 1.26E-04 Plasma Membrane FBXO5 4.93E-05 Nucleus FBXW7 3.54E-02 Nucleus FCER1A 2.27E-02 Plasma Membrane FCGR2A 2.56E-02 Plasma Membrane FCGR2C 4.93E-02 Plasma Membrane FDFT1 1.32E-05 Cytoplasm FDXR 1.49E-02 Cytoplasm FEM1B 4.00E-03 Nucleus FEN1 7.30E-04 Nucleus FERMT1 2.67E-05 Plasma Membrane FERMT2 7.25E-05 Cytoplasm FETUB 1.68E-02 Extracellular Space FGD1 2.14E-02 Cytoplasm FGF1 2.54E-04 Extracellular Space FGF2 1.28E-02 Extracellular Space FGF5 5.43E-04 Extracellular Space FGF7 7.59E-03 Extracellular Space FGFBP1 4.71E-04 Extracellular Space FGFR1 3.89E-03 Plasma Membrane FGFR2 9.29E-04 Plasma Membrane FGR 4.63E-02 Nucleus FH 9.83E-03 Cytoplasm FHIT 4.30E-02 Cytoplasm FHL2 2.46E-03 Nucleus FIG4 7.90E-03 Cytoplasm FIP1L1 1.75E-04 Nucleus FKBP11 1.33E-02 Cytoplasm FKBP1A 1.01E-02 Cytoplasm FKBP4 1.10E-03 Nucleus FKBP5 9.61E-03 Nucleus FKTN 2.74E-02 Extracellular Space FLAD1 2.54E-03 Cytoplasm FLNA 1.08E-04 Cytoplasm FLNB 4.07E-05 Cytoplasm FLOT2 3.80E-03 Plasma Membrane FLT1 5.69E-04 Plasma Membrane FMNL2 5.77E-04 Cytoplasm FMNL3 2.36E-02 Cytoplasm FN1 9.95E-05 Extracellular Space FNBP1 2.22E-03 Nucleus FNBP1L 1.43E-04 Cytoplasm FNDC3A 9.23E-03 Cytoplasm FNTB 3.84E-03 Cytoplasm FOLH1 2.19E-02 Plasma Membrane FOLR1 2.04E-06 Plasma Membrane FOS 1.59E-04 Nucleus FOSB 3.49E-04 Nucleus FOSL1 2.85E-05 Nucleus FOSL2 4.93E-04 Nucleus FOXC1 8.29E-04 Nucleus FOXF1 2.71E-02 Nucleus FOXF2 8.22E-03 Nucleus FOXM1 1.10E-02 Nucleus FOXN3 4.82E-06 Nucleus FOXO1 3.75E-04 Nucleus FOXO3 8.83E-04 Nucleus FRAT1 4.32E-03 Cytoplasm FRS2 8.98E-06 Plasma Membrane FRY 1.61E-04 Extracellular Space FRZB 2.31E-09 Extracellular Space FSCN1 1.49E-02 Cytoplasm FST 2.90E-06 Extracellular Space FSTL1 9.73E-07 Extracellular Space FTH1 1.24E-03 Cytoplasm FTSJ1 1.88E-03 Other FTSJ2 1.12E-04 Nucleus FUK 5.96E-03 Other FUT2 1.44E-02 Cytoplasm FUT4 1.56E-05 Cytoplasm FXN 3.70E-02 Cytoplasm FXR1 7.41E-06 Cytoplasm FYB 9.82E-06 Nucleus FYN 2.69E-04 Plasma Membrane FZD10 1.05E-09 Plasma Membrane FZD3 9.69E-05 Plasma Membrane FZD5 3.48E-03 Plasma Membrane FZD6 1.35E-02 Plasma Membrane FZD7 2.48E-08 Plasma Membrane FZD8 2.70E-08 Plasma Membrane FZR1 3.24E-03 Nucleus G0S2 1.64E-05 Cytoplasm G6PD 4.01E-04 Cytoplasm GAA 3.33E-03 Cytoplasm GAB1 2.23E-04 Cytoplasm GABBR1 1.77E-03 Plasma Membrane GABPA 4.84E-02 Nucleus GABPB1 3.70E-02 Nucleus GABRB2 1.14E-02 Plasma Membrane GABRG1 3.03E-02 Plasma Membrane GABRG2 1.64E-02 Plasma Membrane GAD1 2.78E-02 Cytoplasm GADD45A 1.00E-02 Nucleus GADD45B 3.91E-07 Cytoplasm GADD45G 2.28E-05 Nucleus GAK 4.69E-03 Nucleus GALE 3.80E-02 Cytoplasm GALK1 3.87E-02 Cytoplasm GALT 4.05E-02 Cytoplasm GART 9.38E-08 Cytoplasm GAS1 7.46E-07 Plasma Membrane GAS2 2.49E-04 Cytoplasm GAS2L1 6.68E-04 Cytoplasm GAS6 2.27E-04 Extracellular Space GAS7 2.76E-05 Cytoplasm GATA3 6.71E-03 Nucleus GATA6 7.33E-04 Nucleus GATM 2.64E-03 Cytoplasm GBE1 6.57E-08 Cytoplasm GBP1 1.70E-02 Cytoplasm GBP4 8.25E-03 Cytoplasm GCG 1.24E-04 Extracellular Space GCH1 3.78E-03 Cytoplasm GCLC 5.31E-04 Cytoplasm GCLM 9.14E-05 Cytoplasm GCSH 3.34E-02 Cytoplasm GDF11 2.65E-02 Extracellular Space GDF9 2.60E-04 Extracellular Space GDI1 1.79E-02 Cytoplasm GEM 3.83E-08 Plasma Membrane GEN1 9.65E-07 Other GFPT1 2.53E-03 Cytoplasm GGA1 1.87E-02 Cytoplasm GGA3 6.10E-04 Cytoplasm GGCT 5.37E-03 Cytoplasm GGPS1 2.15E-03 Cytoplasm GGT1 1.71E-02 Plasma Membrane GGT5 4.85E-02 Plasma Membrane GHRHR 9.23E-03 Plasma Membrane GIT1 4.99E-02 Nucleus GIT2 4.50E-03 Nucleus GJA5 4.47E-05 Plasma Membrane GJB2 1.96E-02 Plasma Membrane GK5 2.91E-03 Other GLA 2.13E-02 Cytoplasm GLCE 2.59E-05 Cytoplasm GLG1 5.23E-03 Cytoplasm GLIPR1 3.65E-02 Extracellular Space GLIS1 4.51E-02 Nucleus GLRX 1.27E-02 Cytoplasm GLRX2 2.57E-03 Cytoplasm GLRX3 9.98E-04 Cytoplasm GLTSCR2 1.02E-03 Cytoplasm GLUD1 1.76E-03 Cytoplasm GLUD2 1.37E-02 Cytoplasm GLUL 1.04E-02 Cytoplasm GM2A 1.82E-03 Cytoplasm GMFB 6.78E-03 Cytoplasm GMPPB 8.93E-04 Cytoplasm GMPS 2.30E-02 Nucleus GNA11 9.44E-04 Plasma Membrane GNA12 2.12E-02 Plasma Membrane GNA13 4.62E-04 Plasma Membrane GNAI1 1.09E-04 Plasma Membrane GNAI2 8.37E-04 Plasma Membrane GNAL 2.86E-03 Cytoplasm GNAQ 2.37E-02 Plasma Membrane GNAS 7.56E-04 Plasma Membrane GNB2 2.97E-02 Plasma Membrane GNB5 5.27E-03 Plasma Membrane GNE 4.33E-03 Cytoplasm GNG12 1.02E-02 Plasma Membrane GNG4 7.81E-03 Plasma Membrane GNL1 4.83E-03 Other GNL3 1.88E-04 Nucleus GNRH1 1.18E-08 Extracellular Space GNRHR 2.35E-02 Plasma Membrane GOLGA2 5.08E-04 Cytoplasm GOLPH3 3.09E-02 Cytoplasm GOSR1 1.25E-03 Cytoplasm GOSR2 1.55E-06 Cytoplasm GOT1 2.19E-03 Cytoplasm GOT2 1.31E-02 Cytoplasm GPAA1 8.38E-03 Cytoplasm GPAM 8.18E-06 Cytoplasm GPATCH2L 2.40E-03 Other GPD2 1.05E-02 Cytoplasm GPHN 8.31E-03 Plasma Membrane GPLD1 7.01E-03 Cytoplasm GPNMB 8.08E-07 Plasma Membrane GPR126 6.87E-04 Plasma Membrane GPR137B 2.98E-02 Plasma Membrane GPR146 2.12E-04 Plasma Membrane GPR17 3.23E-02 Plasma Membrane GPR176 6.45E-05 Plasma Membrane GPX1 4.30E-02 Cytoplasm GPX3 4.75E-02 Extracellular Space GPX5 1.33E-02 Extracellular Space GPX7 4.72E-03 Other GRB10 5.00E-05 Cytoplasm GRB14 1.16E-07 Plasma Membrane GRB2 6.39E-05 Cytoplasm GRIA3 4.48E-05 Plasma Membrane GRID1 2.76E-02 Plasma Membrane GRIP1 2.37E-06 Plasma Membrane GRK4 1.19E-02 Plasma Membrane GRK5 3.42E-04 Plasma Membrane GRK6 9.26E-04 Plasma Membrane GRM3 1.82E-02 Plasma Membrane GRM5 2.33E-02 Plasma Membrane GRM6 1.51E-02 Plasma Membrane GSN 5.77E-05 Extracellular Space GSPT1 2.18E-09 Cytoplasm GSR 1.78E-03 Cytoplasm GSS 1.26E-02 Cytoplasm GSTA4 1.45E-06 Cytoplasm GSTK1 4.34E-02 Cytoplasm GSTM1 2.74E-02 Cytoplasm GSTM3 4.13E-03 Cytoplasm GSTT1 3.35E-04 Cytoplasm GTF2A1 3.65E-04 Cytoplasm GTF2A2 2.56E-04 Nucleus GTF2B 4.32E-04 Nucleus GTF2E1 2.80E-02 Nucleus GTF2E2 3.07E-02 Nucleus GTF2F1 3.57E-04 Nucleus GTF2H1 3.28E-02 Nucleus GTF2H2 1.37E-05 Nucleus GTF2H3 9.90E-04 Nucleus GTF2H5 1.45E-02 Nucleus GTF2I 5.00E-04 Nucleus GTF3A 1.74E-04 Nucleus GTF3C1 1.61E-02 Nucleus GTF3C2 2.95E-02 Nucleus GTF3C4 5.80E-06 Nucleus GTF3C5 2.15E-02 Nucleus GTPBP1 1.51E-02 Cytoplasm GTPBP4 6.47E-06 Nucleus GUCA1B 1.31E-03 Cytoplasm GUCA1C 3.82E-02 Cytoplasm GUCY1B3 1.09E-04 Cytoplasm GUCY2D 6.06E-03 Plasma Membrane GZMB 6.37E-03 Cytoplasm H1F0 4.27E-04 Nucleus H2AFX 1.76E-06 Nucleus H2AFY 1.13E-09 Nucleus H3F3A/H3F3B 5.23E-05 Other H6PD 1.70E-03 Cytoplasm HABP2 5.55E-03 Extracellular Space HADH 4.29E-02 Cytoplasm HADHA 2.35E-03 Cytoplasm HAGH 5.60E-04 Cytoplasm HAL 3.43E-05 Cytoplasm HAP1 2.10E-02 Cytoplasm HARS 4.05E-03 Cytoplasm HAS3 1.18E-04 Plasma Membrane HAT1 3.63E-02 Nucleus HAVCR2 3.58E-03 Plasma Membrane HBA1/HBA2 1.14E-02 Cytoplasm HBB 1.52E-03 Cytoplasm HBEGF 4.84E-04 Extracellular Space HBS1L 5.71E-05 Cytoplasm HCAR3 2.14E-06 Plasma Membrane HCFC1 1.71E-02 Nucleus HCST 1.25E-02 Plasma Membrane HDAC10 1.67E-03 Nucleus HDAC3 2.52E-03 Nucleus HDAC4 7.52E-03 Nucleus HDAC7 4.79E-03 Nucleus HDAC9 3.49E-09 Nucleus HECW1 2.40E-02 Cytoplasm HECW2 8.64E-05 Extracellular Space HELLS 1.06E-03 Nucleus HELZ2 4.16E-04 Nucleus HERPUD1 9.61E-05 Cytoplasm HES1 2.16E-08 Nucleus HES5 3.31E-08 Nucleus HEXA 1.69E-02 Cytoplasm HEXB 4.26E-02 Cytoplasm HEXIM1 1.71E-04 Nucleus HEY1 2.74E-03 Nucleus HFE 2.68E-05 Plasma Membrane HGS 1.27E-03 Cytoplasm HHAT 1.03E-02 Cytoplasm HHEX 6.49E-03 Nucleus HIBADH 1.15E-02 Cytoplasm HIF1AN 2.08E-04 Nucleus HILPDA 3.17E-12 Cytoplasm HINFP 1.31E-04 Nucleus HIP1 9.60E-03 Cytoplasm HIPK1 1.20E-02 Nucleus HIPK2 2.97E-03 Nucleus HIPK3 4.37E-02 Nucleus HIRA 1.80E-05 Nucleus HIST1H1C 2.95E-02 Nucleus HIST1H1D 3.24E-02 Nucleus HIST1H1T 6.13E-07 Nucleus HIVEP1 1.45E-03 Nucleus HK2 1.97E-07 Cytoplasm HKR1 2.50E-02 Nucleus HLA-B 3.22E-02 Plasma Membrane HLA-C 9.81E-03 Plasma Membrane HLA-DMA 1.21E-03 Plasma Membrane HLA-DOA 1.33E-02 Plasma Membrane HLA-DQB1 1.23E-02 Plasma Membrane HLA-DRB1 4.44E-07 Plasma Membrane HLA-F 1.34E-07 Plasma Membrane HLA-G 3.27E-02 Plasma Membrane HLTF 1.68E-02 Nucleus HMBOX1 5.02E-04 Nucleus HMGA1 3.45E-03 Nucleus HMGA2 1.27E-08 Nucleus HMGB1 1.75E-04 Nucleus HMGB2 7.02E-05 Nucleus HMGCL 1.84E-02 Cytoplasm HMGCR 1.17E-02 Cytoplasm HMGCS1 2.32E-06 Cytoplasm HMGN1 1.89E-02 Nucleus HMGN2 3.73E-02 Nucleus HMMR 1.28E-04 Plasma Membrane HMOX1 1.45E-03 Cytoplasm HMOX2 3.14E-04 Cytoplasm HNF4A 4.79E-02 Nucleus HNMT 1.18E-02 Cytoplasm HNRNPA0 2.36E-04 Nucleus HNRNPA1 2.05E-06 Nucleus HNRNPA2B1 8.47E-05 Nucleus HNRNPA3 2.75E-03 Nucleus HNRNPAB 1.51E-04 Nucleus HNRNPC 5.95E-04 Nucleus HNRNPD 1.76E-03 Nucleus HNRNPF 2.96E-02 Nucleus HNRNPH1 4.07E-04 Nucleus HNRNPK 3.69E-02 Nucleus HNRNPM 5.85E-04 Nucleus HNRNPR 4.57E-05 Nucleus HNRNPU 1.55E-02 Nucleus HNRNPUL1 2.91E-02 Nucleus HNRPDL 1.21E-04 Nucleus HOMER1 1.46E-03 Plasma Membrane HOMER3 4.36E-05 Plasma Membrane HOPX 1.27E-05 Nucleus HOXA1 9.24E-03 Nucleus HOXA5 4.03E-05 Nucleus HOXA7 7.32E-04 Nucleus HOXA9 4.56E-03 Nucleus HOXB6 5.36E-03 Nucleus HOXB7 3.93E-03 Nucleus HOXB9 7.22E-03 Nucleus HOXC10 1.02E-05 Nucleus HOXC6 2.47E-06 Nucleus HPGD 2.21E-04 Cytoplasm HPN 1.03E-02 Plasma Membrane HRAS 7.62E-01 Plasma Membrane HRH1 2.02E-04 Plasma Membrane HRH3 1.99E-02 Plasma Membrane HRH4 1.91E-03 Plasma Membrane HS2ST1 3.02E-05 Cytoplasm HS3ST1 5.10E-04 Cytoplasm HS3ST2 4.19E-07 Cytoplasm HS3ST3A1 2.30E-05 Cytoplasm HS3ST3B1 2.40E-02 Cytoplasm HS6ST3 2.49E-02 Other HSD17B1 4.41E-05 Cytoplasm HSD17B10 1.33E-02 Cytoplasm HSD17B2 8.48E-04 Cytoplasm HSD17B3 4.21E-02 Cytoplasm HSD3B1 7.04E-04 Cytoplasm HSF1 1.18E-03 Nucleus HSP90AA1 1.36E-04 Cytoplasm HSP90AB1 4.45E-05 Cytoplasm HSP90B1 1.26E-02 Cytoplasm HSPA13 5.84E-05 Cytoplasm HSPA14 1.09E-04 Cytoplasm HSPA1A/HSPA1B 9.28E-04 Cytoplasm HSPA4 3.10E-04 Cytoplasm HSPA5 3.03E-03 Cytoplasm HSPA6 1.47E-03 Other HSPA8 3.34E-03 Cytoplasm HSPA9 1.50E-03 Cytoplasm HSPB1 1.05E-02 Cytoplasm HSPB11 1.51E-02 Extracellular Space HSPBAP1 1.40E-02 Other HSPD1 6.54E-04 Cytoplasm HSPE1 2.87E-02 Cytoplasm HSPG2 1.06E-03 Extracellular Space HSPH1 6.49E-06 Cytoplasm HTATIP2 1.79E-03 Nucleus HTR4 2.46E-02 Plasma Membrane HTT 5.63E-04 Cytoplasm HUS1B 3.75E-03 Other HYAL2 1.84E-03 Cytoplasm IARS 2.28E-03 Cytoplasm ICA1 3.47E-03 Cytoplasm ICAM1 6.07E-04 Plasma Membrane ICAM5 5.70E-03 Plasma Membrane ICOSLG 3.20E-03 Plasma Membrane ID1 1.66E-08 Nucleus ID2 1.37E-03 Nucleus ID3 3.46E-08 Nucleus ID4 4.64E-03 Nucleus IDH3A 1.18E-02 Cytoplasm IDH3G 4.22E-02 Cytoplasm IDI1 7.30E-06 Cytoplasm IDS 6.59E-03 Cytoplasm IER2 1.06E-03 Cytoplasm IFI16 8.50E-05 Nucleus IFI6 2.71E-02 Cytoplasm IFIH1 2.65E-03 Nucleus IFIT2 6.54E-06 Cytoplasm IFIT3 2.89E-02 Cytoplasm IFNA1/IFNA13 1.09E-01 Extracellular Space IFNAR1 1.84E-04 Plasma Membrane IFNAR2 1.11E-05 Plasma Membrane IFNB1 1.54E-05 Extracellular Space IFNGR1 1.25E-03 Plasma Membrane IFNGR2 1.86E-02 Plasma Membrane IFNK 5.07E-03 Extracellular Space IFNL1 9.63E-03 Extracellular Space IFT20 1.45E-02 Cytoplasm IFT57 2.51E-03 Cytoplasm IFT88 1.07E-02 Cytoplasm IGBP1 4.06E-03 Cytoplasm IGF1 2.94E-02 Extracellular Space IGF1R 2.60E-03 Plasma Membrane IGF2BP2 5.00E-03 Cytoplasm IGF2BP3 1.03E-04 Cytoplasm IGF2R 3.43E-05 Plasma Membrane IGFBP3 7.37E-12 Extracellular Space IGFBP4 2.31E-02 Extracellular Space IGFBP5 3.47E-05 Extracellular Space IGFBP7 4.03E-05 Extracellular Space IGHA1 3.67E-02 Extracellular Space IGHD 1.02E-02 Extracellular Space IGHG1 5.85E-03 Extracellular Space IGHM 2.43E-02 Plasma Membrane IGLL1/IGLL5 4.71E-02 Plasma Membrane IK 3.90E-02 Extracellular Space IKBIP 4.29E-03 Cytoplasm IKBKB 4.84E-02 Cytoplasm IL11 1.76E-03 Extracellular Space IL12RB2 3.73E-02 Plasma Membrane IL13RA1 1.49E-03 Plasma Membrane IL15 2.78E-07 Extracellular Space IL15RA 4.42E-02 Plasma Membrane IL16 5.60E-03 Extracellular Space IL17RA 2.46E-02 Plasma Membrane IL17RD 1.31E-02 Cytoplasm IL18 6.49E-01 Extracellular Space IL18R1 1.50E-02 Plasma Membrane IL1A 7.36E-05 Extracellular Space IL1B 8.64E-03 Extracellular Space IL1RL1 4.63E-02 Plasma Membrane IL1RN 3.71E-04 Extracellular Space IL20 1.30E-03 Extracellular Space IL23A 1.15E-02 Extracellular Space IL36A 1.52E-02 Extracellular Space IL37 5.49E-05 Extracellular Space IL4 1.07E-01 Extracellular Space IL4R 3.64E-04 Plasma Membrane IL6 6.29E-06 Extracellular Space IL6R 7.30E-03 Plasma Membrane IL6ST 3.75E-04 Plasma Membrane IL8 7.47E-12 Extracellular Space ILF2 4.67E-02 Nucleus ILF3 1.23E-04 Nucleus ILK 4.16E-02 Plasma Membrane IMPA1 4.16E-04 Cytoplasm IMPAD1 5.35E-05 Cytoplasm IMPDH1 1.06E-02 Cytoplasm INADL 6.91E-06 Plasma Membrane ING1 4.70E-08 Nucleus ING2 9.60E-05 Nucleus ING3 3.56E-03 Nucleus ING4 3.41E-03 Nucleus ING5 2.89E-02 Nucleus INHBA 1.12E-09 Extracellular Space INHBB 3.80E-02 Extracellular Space INIP 1.80E-03 Nucleus INPP4A 5.92E-04 Cytoplasm INPP4B 5.67E-05 Cytoplasm INPP5B 1.26E-02 Plasma Membrane INPP5D 2.14E-04 Cytoplasm INPP5F 3.01E-02 Other INPPL1 2.08E-03 Cytoplasm INS 1.56E-02 Extracellular Space INSIG1 3.53E-10 Cytoplasm INSR 3.41E-05 Plasma Membrane IP6K1 1.75E-02 Cytoplasm IP6K2 5.56E-03 Cytoplasm IPMK 1.40E-08 Nucleus IPO5 7.22E-03 Nucleus IQGAP1 4.08E-04 Cytoplasm IRAK1 2.21E-02 Plasma Membrane IRAK2 3.70E-07 Plasma Membrane IRAK3 3.09E-03 Cytoplasm IRF1 1.73E-07 Nucleus IRF2 2.11E-06 Nucleus IRF3 6.95E-04 Nucleus IRF4 4.78E-02 Nucleus IRF5 1.97E-02 Nucleus IRF6 2.04E-02 Nucleus IRS1 1.46E-06 Cytoplasm IRS2 7.55E-08 Cytoplasm ISG15 2.54E-02 Extracellular Space ISG20 1.92E-04 Nucleus ISL1 6.27E-03 Nucleus ISYNA1 3.08E-02 Other ITCH 2.28E-03 Nucleus ITGA2 3.81E-03 Plasma Membrane ITGA2B 5.88E-03 Plasma Membrane ITGA3 1.84E-03 Plasma Membrane ITGA4 2.27E-02 Plasma Membrane ITGA6 3.25E-04 Plasma Membrane ITGA7 2.69E-02 Plasma Membrane ITGAM 1.79E-04 Plasma Membrane ITGB1 2.49E-02 Plasma Membrane ITGB1BP1 2.22E-03 Plasma Membrane ITGB3 4.48E-03 Plasma Membrane ITGB4 4.79E-05 Plasma Membrane ITGB5 4.85E-02 Plasma Membrane ITGB8 4.81E-05 Plasma Membrane ITGBL1 1.44E-02 Other ITPKB 2.64E-03 Cytoplasm ITPR1 1.61E-04 Cytoplasm ITPR2 5.12E-05 Cytoplasm ITPR3 9.62E-04 Cytoplasm ITSN1 2.39E-05 Cytoplasm ITSN2 1.57E-02 Cytoplasm IVD 1.56E-02 Cytoplasm IVL 2.68E-03 Cytoplasm IVNS1ABP 9.40E-04 Nucleus IWS1 1.04E-05 Nucleus JAG1 2.45E-07 Extracellular Space JAG2 1.50E-02 Extracellular Space JAK2 6.19E-04 Cytoplasm JAK3 4.64E-02 Cytoplasm JARID2 8.70E-05 Nucleus JMJD6 6.07E-08 Plasma Membrane JMJD7-PLA2G4B 1.49E-03 Cytoplasm JMY 1.95E-04 Nucleus JUN 2.60E-09 Nucleus JUNB 1.92E-04 Nucleus JUND 1.25E-06 Nucleus KANK1 1.91E-05 Nucleus KARS 1.75E-02 Cytoplasm KAT2B 6.48E-03 Nucleus KAT6A 4.04E-03 Nucleus KAT6B 2.07E-06 Nucleus KCNJ1 1.69E-02 Plasma Membrane KCNJ5 3.24E-04 Plasma Membrane KCNJ8 2.98E-03 Plasma Membrane KCNK2 3.37E-02 Plasma Membrane KCNK3 3.47E-02 Plasma Membrane KCNQ3 9.27E-04 Plasma Membrane KCTD11 2.91E-03 Cytoplasm KDELR1 1.50E-02 Cytoplasm KDM1A 6.56E-07 Nucleus KDM4C 3.33E-07 Nucleus KDM5B 4.45E-04 Nucleus KDSR 2.81E-02 Plasma Membrane KHDRBS1 3.89E-05 Nucleus KIAA0101 1.90E-02 Nucleus KIAA1804 1.08E-02 Other KIAA1967 4.32E-04 Cytoplasm KIDINS220 3.03E-03 Nucleus KIF11 2.34E-02 Nucleus KIF15 3.26E-05 Nucleus KIF20A 1.05E-02 Cytoplasm KIF23 2.48E-06 Cytoplasm KIF2C 2.24E-02 Nucleus KIF3C 5.06E-04 Cytoplasm KIR3DL2 1.49E-02 Plasma Membrane KLC1 1.36E-02 Cytoplasm KLF10 1.09E-02 Nucleus KLF11 2.91E-07 Nucleus KLF4 1.23E-04 Nucleus KLF5 3.92E-02 Nucleus KLF6 3.31E-03 Nucleus KLF9 7.80E-07 Nucleus KLHL20 1.88E-05 Plasma Membrane KLK8 1.01E-02 Extracellular Space KLRC2 4.32E-02 Plasma Membrane KLRC3 9.94E-03 Plasma Membrane KLRD1 7.68E-03 Plasma Membrane KMO 3.40E-02 Cytoplasm KMT2A 5.51E-06 Nucleus KMT2D 7.87E-04 Nucleus KPNA1 1.95E-04 Nucleus KPNA2 3.33E-03 Nucleus KPNA3 1.09E-03 Nucleus KPNA4 3.28E-02 Nucleus KPNA5 1.00E-02 Cytoplasm KPNA6 1.16E-04 Nucleus KPNB1 6.94E-04 Nucleus KRAS 6.27E-04 Cytoplasm KREMEN1 1.82E-04 Plasma Membrane KRT10 3.83E-02 Cytoplasm KRT14 2.58E-02 Cytoplasm KRT2 2.36E-02 Cytoplasm KRT7 9.23E-04 Cytoplasm KRT8 3.57E-04 Cytoplasm KSR1 9.46E-07 Cytoplasm L3MBTL1 4.66E-05 Nucleus LAMA3 9.04E-03 Extracellular Space LAMA4 3.16E-02 Extracellular Space LAMA5 8.49E-06 Extracellular Space LAMB1 2.14E-03 Extracellular Space LAMB3 4.28E-02 Extracellular Space LAMC2 1.71E-05 Extracellular Space LAMP2 1.48E-02 Plasma Membrane LAMP3 5.67E-04 Plasma Membrane LAMTOR5 4.39E-02 Cytoplasm LARS 3.39E-04 Cytoplasm LASP1 3.13E-02 Cytoplasm LATS1 5.82E-03 Nucleus LATS2 6.22E-07 Nucleus LBR 4.91E-04 Nucleus LCMT1 4.69E-04 Other LCT 2.33E-02 Plasma Membrane LDHA 6.24E-03 Cytoplasm LDHAL6B 2.58E-02 Cytoplasm LDHC 1.84E-02 Cytoplasm LDLR 1.56E-03 Plasma Membrane LDLRAP1 6.12E-03 Cytoplasm LDOC1 2.03E-03 Nucleus LEF1 3.80E-04 Nucleus LEP 7.46E-10 Extracellular Space LEPR 1.46E-08 Plasma Membrane LETM1 8.47E-05 Cytoplasm LFNG 3.92E-03 Cytoplasm LGALS1 2.04E-02 Extracellular Space LGALS12 3.77E-02 Extracellular Space LGALS3BP 2.83E-02 Plasma Membrane LGALS8 1.77E-06 Extracellular Space LIAS 5.78E-04 Cytoplasm LIF 2.96E-02 Extracellular Space LIFR 1.04E-02 Plasma Membrane LIG1 1.17E-02 Nucleus LIG4 5.21E-03 Nucleus LIMK2 5.06E-04 Cytoplasm LIMS1 2.03E-03 Plasma Membrane LIN28B 4.95E-02 Other LIPA 3.34E-02 Cytoplasm LIPE 5.11E-03 Cytoplasm LIPT1 2.25E-03 Cytoplasm LITAF 3.15E-03 Nucleus LLGL1 1.17E-04 Cytoplasm LMCD1 1.02E-03 Cytoplasm LMNA 8.73E-04 Nucleus LMNB1 3.53E-05 Nucleus LMNB2 2.58E-04 Nucleus LMO7 4.55E-02 Cytoplasm LOR 6.54E-05 Cytoplasm LOX 6.65E-11 Extracellular Space LPAR1 4.83E-04 Plasma Membrane LPAR2 2.01E-02 Plasma Membrane LPAR3 8.86E-06 Plasma Membrane LPAR5 4.79E-03 Plasma Membrane LPAR6 2.47E-05 Plasma Membrane LPCAT4 3.36E-05 Cytoplasm LPIN2 9.40E-09 Nucleus LPPR4 2.31E-03 Plasma Membrane LRAT 1.68E-02 Cytoplasm LRFN4 6.90E-03 Other LRP1 1.02E-03 Plasma Membrane LRP6 3.33E-04 Plasma Membrane LRP8 3.38E-04 Plasma Membrane LRPAP1 9.27E-04 Plasma Membrane LRRC15 8.54E-03 Plasma Membrane LRRC17 2.40E-04 Extracellular Space LRRC8C 1.25E-04 Cytoplasm LRRK2 5.18E-03 Cytoplasm LSM11 3.39E-03 Nucleus LSM12 1.59E-04 Other LSM14A 4.03E-03 Cytoplasm LSM14B 4.48E-02 Other LSM2 5.00E-03 Nucleus LSM5 1.74E-02 Cytoplasm LSM6 1.75E-02 Nucleus LSS 3.00E-07 Cytoplasm LST1 3.84E-02 Plasma Membrane LTB 5.92E-05 Extracellular Space LTB4R 7.24E-06 Plasma Membrane LTBP1 1.36E-02 Extracellular Space LTBP4 3.01E-02 Extracellular Space LTBR 2.86E-02 Plasma Membrane LUC7L3 1.90E-04 Nucleus LY75 2.50E-05 Plasma Membrane LY9 4.30E-02 Plasma Membrane LYN 6.08E-07 Cytoplasm LYZ 8.26E-03 Extracellular Space LZTS2 1.03E-02 Cytoplasm M6PR 6.74E-04 Cytoplasm MAD1L1 5.32E-04 Nucleus MAD2L1BP 6.56E-05 Nucleus MAF 1.31E-09 Nucleus MAFB 6.88E-08 Nucleus MAFF 6.70E-10 Nucleus MAFK 8.47E-03 Nucleus MAGED4/MAGED4B 1.67E-02 Other MAGEH1 2.53E-02 Cytoplasm MAGI1 5.12E-07 Plasma Membrane MAGI2 2.15E-04 Plasma Membrane MAGOH 5.50E-05 Nucleus MALT1 3.01E-03 Cytoplasm MAML1 1.99E-02 Nucleus MAML2 2.34E-06 Nucleus MAML3 9.64E-08 Nucleus MAN2A1 7.50E-04 Cytoplasm MAOA 1.42E-02 Cytoplasm MAOB 1.34E-02 Cytoplasm MAP1B 1.40E-04 Cytoplasm MAP2K1 1.03E-04 Cytoplasm MAP2K3 2.41E-04 Cytoplasm MAP2K4 5.28E-03 Cytoplasm MAP2K5 8.05E-04 Cytoplasm MAP3K1 1.07E-03 Cytoplasm MAP3K11 2.21E-02 Cytoplasm MAP3K12 2.10E-02 Cytoplasm MAP3K13 1.75E-07 Cytoplasm MAP3K2 8.25E-09 Cytoplasm MAP3K3 4.35E-02 Cytoplasm MAP3K4 3.15E-03 Cytoplasm MAP3K5 6.90E-04 Cytoplasm MAP3K6 1.79E-02 Other MAP3K7 2.59E-03 Cytoplasm MAP3K8 7.95E-10 Cytoplasm MAP3K9 1.43E-02 Cytoplasm MAP4K3 4.12E-03 Other MAP4K4 3.05E-04 Cytoplasm MAPK1 7.96E-04 Cytoplasm MAPK10 2.18E-02 Cytoplasm MAPK13 2.62E-03 Cytoplasm MAPK14 2.84E-03 Cytoplasm MAPK7 3.08E-02 Cytoplasm MAPK8 5.14E-03 Cytoplasm MAPK8IP3 1.84E-04 Cytoplasm MAPK9 5.28E-03 Cytoplasm MAPKAP1 5.05E-05 Cytoplasm MAPKAPK3 2.31E-05 Nucleus MAPRE3 4.26E-02 Cytoplasm MAPT 4.70E-02 Plasma Membrane MARCKS 1.69E-03 Plasma Membrane MARCKSL1 2.20E-04 Cytoplasm MARK1 1.57E-06 Cytoplasm MARK3 7.74E-05 Cytoplasm MARS 1.43E-03 Cytoplasm MARS2 1.50E-06 Cytoplasm MAST2 1.36E-02 Cytoplasm MAT2A 2.50E-10 Cytoplasm MATN1 4.47E-02 Extracellular Space MAVS 1.79E-05 Cytoplasm MAX 3.96E-03 Nucleus MBD1 5.36E-05 Nucleus MBD2 1.51E-02 Nucleus MBD3 4.66E-02 Nucleus MBNL1 3.75E-08 Nucleus MBNL2 9.50E-07 Other MBOAT2 1.05E-02 Cytoplasm MBP 7.32E-05 Extracellular Space MC3R 3.41E-02 Plasma Membrane MCAM 5.08E-04 Plasma Membrane MCC 9.66E-05 Cytoplasm MCCC1 1.63E-02 Cytoplasm MCCC2 4.70E-02 Cytoplasm MCEE 6.94E-03 Cytoplasm MCF2L 5.20E-03 Cytoplasm MCL1 4.04E-03 Cytoplasm MCM2 1.22E-03 Nucleus MCM3 9.23E-03 Nucleus MCM4 1.24E-02 Nucleus MCM5 2.24E-03 Nucleus MCM7 1.24E-02 Nucleus MCMBP 1.62E-03 Nucleus MCOLN3 6.89E-03 Plasma Membrane MCPH1 2.15E-06 Nucleus MDC1 4.37E-02 Nucleus MDH1 6.34E-07 Cytoplasm MDM2 1.80E-05 Nucleus MDM4 3.96E-02 Nucleus ME1 4.37E-02 Cytoplasm MECOM 6.45E-03 Nucleus MECP2 1.87E-03 Nucleus MED1 1.41E-04 Nucleus MED10 3.26E-02 Nucleus MED13 5.20E-03 Nucleus MED13L 2.40E-04 Nucleus MED14 3.20E-04 Nucleus MED16 2.23E-02 Nucleus MED17 3.53E-06 Nucleus MED18 1.15E-06 Nucleus MED20 6.88E-04 Nucleus MED23 5.78E-03 Nucleus MED27 2.98E-03 Nucleus MED30 9.59E-03 Nucleus MED31 3.36E-03 Nucleus MED6 3.79E-03 Nucleus MEF2A 1.36E-07 Nucleus MEIS1 2.76E-05 Nucleus MELK 4.70E-03 Cytoplasm MEN1 4.42E-03 Nucleus MERTK 2.10E-03 Plasma Membrane MET 1.63E-03 Plasma Membrane METAP2 6.30E-04 Cytoplasm METTL22 4.42E-04 Other METTL3 1.50E-05 Nucleus MFHAS1 6.44E-04 Cytoplasm MFN2 3.35E-04 Cytoplasm MGAT4B 2.89E-04 Cytoplasm MGEA5 1.87E-04 Cytoplasm MGMT 4.57E-03 Nucleus MGST1 1.54E-05 Cytoplasm MGST2 1.07E-03 Cytoplasm MIB1 1.03E-04 Cytoplasm MICA 3.67E-05 Plasma Membrane MICAL1 1.25E-02 Cytoplasm MICAL2 4.56E-08 Cytoplasm MICAL3 1.22E-07 Cytoplasm MICB 2.15E-06 Plasma Membrane MIF 4.37E-03 Extracellular Space MINA 8.33E-06 Nucleus mir-21 1.35E-08 Cytoplasm mir-30 2.25E-05 Cytoplasm MITF 4.19E-04 Nucleus MKI67 8.23E-03 Nucleus MKL1 3.57E-02 Nucleus MKNK1 3.68E-05 Cytoplasm MKNK2 7.08E-07 Cytoplasm MLF1 5.86E-05 Nucleus MLF1IP 1.32E-02 Nucleus MLH1 1.55E-03 Nucleus MLLT11 4.63E-02 Cytoplasm MLLT4 1.67E-02 Nucleus MLLT6 3.15E-02 Nucleus MMD 2.54E-06 Plasma Membrane MME 1.43E-03 Plasma Membrane MMP1 1.57E-04 Extracellular Space MMP10 3.41E-02 Extracellular Space MMP14 6.40E-04 Extracellular Space MMP19 7.55E-03 Extracellular Space MMP24 2.92E-02 Plasma Membrane MMP25 7.25E-01 Extracellular Space MMP26 2.36E-02 Extracellular Space MMP28 8.91E-04 Extracellular Space MMP3 3.46E-03 Extracellular Space MNAT1 9.72E-03 Nucleus MNT 4.41E-02 Nucleus MOAP1 1.89E-03 Cytoplasm MOCS2 3.94E-02 Cytoplasm MOCS3 7.49E-05 Cytoplasm MPDZ 8.85E-05 Plasma Membrane MPHOSPH9 4.00E-04 Cytoplasm MPL 3.78E-02 Plasma Membrane MPO 1.89E-02 Cytoplasm MPRIP 5.56E-03 Cytoplasm MR1 4.55E-04 Plasma Membrane MRAS 2.25E-03 Plasma Membrane MRC1 1.47E-02 Plasma Membrane MRE11A 2.74E-03 Nucleus MREG 1.74E-02 Cytoplasm MRPL41 3.19E-02 Cytoplasm MRPS30 3.00E-03 Cytoplasm MS4A1 4.54E-03 Plasma Membrane MSH3 2.11E-02 Nucleus MSMO1 2.89E-05 Cytoplasm MST1 6.17E-04 Extracellular Space MST1R 1.94E-04 Plasma Membrane MSTN 8.89E-05 Extracellular Space MSX1 2.17E-02 Nucleus MSX2 1.14E-05 Nucleus MT1E 9.73E-03 Cytoplasm MT1F 4.21E-04 Other MT1G 1.55E-03 Other MT1H 3.15E-02 Other MT1M 1.43E-06 Other MTA2 3.94E-03 Nucleus MTA3 1.00E-03 Nucleus MTAP 9.56E-07 Nucleus MT-ATP6 3.30E-05 Cytoplasm MT-CO2 6.68E-05 Cytoplasm MT-COI 5.60E-04 Cytoplasm MTCP1 1.13E-04 Cytoplasm MTDH 1.48E-02 Cytoplasm MTF1 1.89E-04 Nucleus MTHFD1L 1.45E-03 Cytoplasm MTHFD2L 3.09E-04 Other MTMR1 4.68E-02 Cytoplasm MTMR14 2.28E-02 Cytoplasm MTMR3 8.34E-03 Cytoplasm MTMR4 3.26E-02 Cytoplasm MTMR9 6.32E-05 Cytoplasm MT-ND2 7.54E-09 Cytoplasm MT-ND4 2.41E-04 Cytoplasm MT-ND5 6.64E-04 Cytoplasm MTR 1.04E-04 Cytoplasm MUC1 2.06E-02 Plasma Membrane MUL1 1.59E-02 Cytoplasm MUT 2.17E-02 Cytoplasm MVK 1.53E-03 Cytoplasm MVP 2.18E-03 Nucleus MXD1 6.74E-09 Nucleus MXD4 1.86E-02 Nucleus MXI1 2.32E-09 Nucleus MYBBP1A 3.57E-04 Nucleus MYBL1 1.64E-02 Nucleus MYC 2.85E-08 Nucleus MYCBP 5.51E-03 Nucleus MYD88 1.82E-04 Plasma Membrane MYH11 1.50E-04 Cytoplasm MYH14 4.05E-02 Extracellular Space MYH3 1.78E-02 Cytoplasm MYH9 2.50E-02 Cytoplasm MYL12A 1.10E-04 Cytoplasm MYL4 4.51E-02 Cytoplasm MYL6 1.36E-02 Cytoplasm MYLIP 1.00E-07 Cytoplasm MYLK 7.20E-03 Cytoplasm MYLK3 4.45E-03 Cytoplasm MYO10 1.88E-07 Cytoplasm MYO1E 4.22E-02 Cytoplasm MYO5A 2.85E-03 Cytoplasm MYO6 1.14E-04 Cytoplasm MYO7A 3.68E-02 Cytoplasm MYOC 8.05E-03 Cytoplasm N4BP1 4.86E-02 Cytoplasm N4BP2L1 1.10E-07 Other N6AMT1 2.77E-02 Other NAA10 9.72E-03 Nucleus NAA38 1.17E-04 Nucleus NAAA 4.01E-03 Cytoplasm NAB1 4.19E-02 Nucleus NAB2 2.54E-06 Nucleus NABP1 1.29E-06 Nucleus NABP2 1.47E-02 Nucleus NACA 2.24E-08 Cytoplasm NADK 3.60E-03 Cytoplasm NAE1 1.08E-02 Cytoplasm NAIP 3.31E-04 Other NAMPT 2.01E-11 Extracellular Space NANOG 5.32E-06 Nucleus NANP 4.27E-03 Other NANS 1.86E-02 Cytoplasm NAP1L1 5.66E-03 Nucleus NAPA 2.37E-02 Cytoplasm NAPEPLD 1.19E-05 Cytoplasm NAPRT1 2.58E-02 Cytoplasm NBN 4.43E-02 Nucleus NCAPG 3.88E-02 Nucleus NCF1 7.10E-03 Cytoplasm NCK2 2.41E-02 Cytoplasm NCKIPSD 2.32E-03 Other NCL 6.91E-03 Nucleus NCOA1 2.26E-03 Nucleus NCOA2 1.44E-02 Nucleus NCOA3 4.63E-04 Nucleus NCOA4 3.51E-02 Nucleus NCOA7 2.25E-03 Nucleus NCOR1 4.38E-04 Nucleus NCOR2 2.43E-02 Nucleus NCR1 2.57E-01 Plasma Membrane NCR3 2.03E-01 Plasma Membrane NCS1 8.96E-04 Cytoplasm NDOR1 4.77E-02 Cytoplasm NDRG1 1.85E-06 Nucleus NDST1 5.58E-05 Cytoplasm NDST2 1.46E-04 Cytoplasm NDST3 1.06E-02 Cytoplasm NDUFA10 5.77E-03 Cytoplasm NDUFA12 4.71E-02 Cytoplasm NDUFA2 4.95E-02 Cytoplasm NDUFA5 3.78E-02 Cytoplasm NDUFAF1 1.03E-02 Cytoplasm NDUFB1 5.65E-05 Cytoplasm NDUFB4 9.32E-09 Cytoplasm NDUFB6 1.23E-02 Cytoplasm NDUFS1 1.32E-03 Cytoplasm NDUFS2 1.63E-03 Cytoplasm NDUFS4 6.29E-04 Cytoplasm NDUFS8 7.52E-04 Cytoplasm NDUFV1 6.45E-03 Cytoplasm NDUFV3 2.47E-04 Cytoplasm NEDD4 7.29E-06 Cytoplasm NEDD4L 7.66E-08 Cytoplasm NEDD8 1.35E-02 Nucleus NEDD9 7.52E-09 Nucleus NEFH 4.62E-02 Cytoplasm NEFL 6.88E-05 Cytoplasm NEFM 3.83E-03 Cytoplasm NEIL1 4.75E-03 Nucleus NEK2 9.33E-04 Cytoplasm NEK6 9.68E-03 Nucleus NEUROD1 4.04E-02 Nucleus NF1 6.88E-07 Cytoplasm NF2 6.40E-06 Plasma Membrane NFAT5 1.57E-03 Nucleus NFATC2 5.37E-04 Nucleus NFATC3 2.54E-03 Nucleus NFATC4 1.39E-05 Nucleus NFE2L1 1.63E-02 Nucleus NFE2L2 4.22E-04 Nucleus NFE2L3 1.79E-02 Nucleus NFIA 6.12E-06 Nucleus NFIB 1.87E-04 Nucleus NFIC 1.27E-02 Nucleus NFIL3 5.11E-08 Nucleus NFKB2 4.37E-03 Nucleus NFKBIA 1.66E-07 Cytoplasm NFKBIE 7.42E-06 Nucleus NFKBIZ 5.77E-04 Nucleus NFS1 2.19E-02 Cytoplasm NFYA 2.61E-05 Nucleus NGF 4.91E-02 Extracellular Space NHP2L1 1.93E-02 Nucleus NID1 8.04E-03 Extracellular Space NINJ1 1.20E-03 Plasma Membrane NIPA2 6.51E-04 Cytoplasm NIPBL 1.08E-03 Nucleus NKIRAS2 1.35E-02 Cytoplasm NKTR 2.34E-06 Plasma Membrane NLK 2.87E-05 Nucleus NLRP1 2.56E-04 Cytoplasm NLRP12 1.95E-02 Cytoplasm NME1 3.78E-02 Cytoplasm NME6 7.87E-04 Cytoplasm NME7 2.68E-03 Cytoplasm NMNAT1 6.52E-03 Nucleus NMNAT2 1.09E-02 Cytoplasm NNT 4.87E-02 Cytoplasm NOC2L 3.72E-04 Nucleus NOD2 3.11E-07 Cytoplasm NODAL 2.95E-02 Extracellular Space NOG 1.90E-04 Extracellular Space NOL3 4.39E-07 Nucleus NOL8 3.40E-02 Nucleus NOLC1 2.24E-04 Nucleus NOP2 8.86E-06 Nucleus NOP58 7.56E-04 Nucleus NOS1 8.17E-04 Cytoplasm NOS3 7.87E-03 Cytoplasm NOTCH2 9.77E-04 Plasma Membrane NOTCH3 3.65E-04 Plasma Membrane NOV 1.57E-04 Extracellular Space NOX4 3.19E-04 Cytoplasm NPAT 1.11E-03 Nucleus NPC2 8.77E-05 Extracellular Space NPIP (includes others)2.61E-02 Nucleus NPM1 2.84E-02 Nucleus NPPB 6.51E-05 Extracellular Space NPR2 1.80E-02 Plasma Membrane NPR3 1.63E-04 Plasma Membrane NQO1 1.82E-02 Cytoplasm NQO2 4.72E-02 Cytoplasm NR0B2 3.09E-02 Nucleus NR1D1 2.08E-04 Nucleus NR1H2 7.60E-03 Nucleus NR1H4 2.17E-02 Nucleus NR2C1 3.92E-05 Nucleus NR2C2 4.02E-03 Nucleus NR2F2 2.14E-06 Nucleus NR2F6 3.45E-03 Nucleus NR3C1 3.32E-07 Nucleus NR3C2 4.05E-04 Nucleus NR4A1 4.24E-02 Nucleus NR4A2 6.49E-09 Nucleus NR4A3 1.60E-04 Nucleus NR5A2 2.71E-02 Nucleus NR6A1 1.90E-02 Nucleus NRCAM 4.66E-04 Plasma Membrane NRF1 1.24E-05 Nucleus NRG1 1.37E-05 Other NRIP1 6.19E-05 Nucleus NRP1 1.95E-02 Plasma Membrane NRP2 2.49E-04 Plasma Membrane NSD1 5.61E-06 Nucleus NSMAF 1.23E-05 Cytoplasm NSUN5P2 3.93E-02 Other NT5C2 2.69E-04 Cytoplasm NTF4 1.13E-02 Extracellular Space NTNG1 7.35E-04 Extracellular Space NTRK2 2.41E-03 Plasma Membrane NTRK3 4.93E-02 Plasma Membrane NUAK1 4.05E-05 Other NUDC 1.55E-02 Cytoplasm NUDT11 3.72E-03 Cytoplasm NUDT12 7.28E-05 Cytoplasm NUDT15 1.16E-02 Other NUDT3 8.19E-04 Cytoplasm NUDT4 1.24E-02 Cytoplasm NUMA1 1.77E-03 Nucleus NUMB 9.00E-05 Plasma Membrane NUP153 1.79E-04 Nucleus NUP62 2.85E-05 Nucleus NUP98 1.76E-03 Nucleus NUSAP1 2.87E-03 Nucleus OAS1 7.76E-03 Cytoplasm OAS2 7.73E-03 Cytoplasm OAS3 3.55E-02 Cytoplasm OASL 3.01E-02 Cytoplasm OAZ2 3.75E-03 Cytoplasm OCLN 1.50E-02 Plasma Membrane ODC1 2.32E-05 Cytoplasm ODF1 1.70E-02 Cytoplasm OGDH 6.87E-03 Cytoplasm OGFR 3.25E-02 Plasma Membrane OLR1 1.46E-07 Plasma Membrane OPA1 6.47E-03 Cytoplasm OPN1SW 3.68E-03 Plasma Membrane OPRK1 4.76E-02 Plasma Membrane OPRL1 4.26E-02 Plasma Membrane OPTN 1.43E-03 Cytoplasm ORC1 2.69E-02 Nucleus ORC2 1.08E-03 Nucleus ORC5 2.84E-06 Nucleus ORM1 2.05E-02 Extracellular Space OSM 3.44E-02 Extracellular Space OSMR 1.39E-05 Plasma Membrane OTUB1 1.92E-02 Cytoplasm OVOL2 2.18E-02 Nucleus OXSM 2.24E-05 Cytoplasm OXTR 2.59E-07 Plasma Membrane P2RY1 4.21E-04 Plasma Membrane P2RY14 5.26E-03 Plasma Membrane P4HB 1.36E-03 Cytoplasm PA2G4 6.54E-03 Nucleus PAFAH1B1 3.00E-05 Cytoplasm PAFAH1B2 1.28E-03 Cytoplasm PAG1 3.25E-07 Plasma Membrane PAIP1 1.33E-02 Cytoplasm PAIP2 4.56E-04 Cytoplasm PAK1 1.29E-02 Cytoplasm PAK1IP1 9.24E-06 Nucleus PAK2 1.03E-02 Cytoplasm PAK6 5.95E-07 Cytoplasm PALB2 7.55E-04 Nucleus PALD1 2.93E-02 Cytoplasm PAOX 7.04E-03 Cytoplasm PAPOLA 7.43E-04 Nucleus PAPPA 4.26E-05 Extracellular Space PARD6B 5.99E-08 Plasma Membrane PARD6G 1.71E-02 Plasma Membrane PARK7 3.53E-03 Nucleus PARP1 4.21E-04 Nucleus PARP10 2.79E-02 Nucleus PARP11 6.30E-03 Other PARP16 1.76E-04 Cytoplasm PARP2 4.41E-02 Nucleus PARP6 5.56E-04 Other PARP8 8.35E-06 Other PARP9 2.45E-03 Nucleus PARS2 3.92E-06 Cytoplasm PARVA 2.78E-03 Cytoplasm PAWR 2.99E-04 Nucleus PAX2 2.93E-02 Nucleus PAX3 2.76E-02 Nucleus PAX6 1.60E-02 Nucleus PBK 4.58E-02 Cytoplasm PBRM1 3.50E-05 Nucleus PBX1 2.43E-05 Nucleus PBX2 8.90E-03 Nucleus PBXIP1 1.66E-02 Nucleus PCBP2 2.34E-07 Nucleus PCCA 2.72E-05 Cytoplasm PCCB 2.57E-02 Cytoplasm PCDH1 2.08E-03 Plasma Membrane PCGF6 4.34E-03 Nucleus PCNA 1.20E-03 Nucleus PCNP 9.40E-08 Nucleus PCOLCE 1.54E-02 Extracellular Space PCYOX1 6.09E-03 Cytoplasm PCYT1A 4.98E-05 Cytoplasm PCYT1B 3.73E-03 Cytoplasm PCYT2 8.88E-04 Cytoplasm PDAP1 5.33E-06 Cytoplasm PDCD1LG2 2.69E-03 Plasma Membrane PDCD5 1.25E-08 Nucleus PDCD6IP 1.21E-03 Cytoplasm PDCD7 3.54E-04 Nucleus PDE11A 2.44E-02 Cytoplasm PDE12 1.53E-03 Cytoplasm PDE1A 1.74E-02 Cytoplasm PDE1C 3.82E-02 Cytoplasm PDE2A 5.00E-03 Cytoplasm PDE3A 3.54E-02 Cytoplasm PDE3B 2.33E-02 Cytoplasm PDE4B 3.80E-03 Cytoplasm PDE4D 1.27E-07 Cytoplasm PDE4DIP 7.71E-05 Cytoplasm PDE5A 9.33E-05 Cytoplasm PDE6B 1.85E-02 Cytoplasm PDE6D 2.48E-03 Cytoplasm PDE7A 1.47E-07 Cytoplasm PDE7B 2.45E-02 Cytoplasm PDE8A 7.01E-05 Cytoplasm PDGFA 3.42E-04 Extracellular Space PDGFB 1.04E-03 Extracellular Space PDGFC 6.30E-04 Extracellular Space PDGFD 2.61E-03 Extracellular Space PDGFRA 3.71E-02 Plasma Membrane PDHA2 4.73E-02 Cytoplasm PDHB 2.61E-04 Cytoplasm PDIA2 3.26E-02 Cytoplasm PDIA3 8.47E-04 Cytoplasm PDK1 1.16E-09 Cytoplasm PDLIM7 6.03E-05 Cytoplasm PDPK1 6.85E-03 Cytoplasm PDS5B 4.33E-02 Nucleus PDSS1 4.94E-04 Cytoplasm PDSS2 1.07E-03 Cytoplasm PDXK 2.19E-03 Cytoplasm PDZK1 1.93E-05 Plasma Membrane PDZK1IP1 2.45E-02 Extracellular Space PDZRN3 4.03E-06 Extracellular Space PEA15 3.09E-04 Cytoplasm PEBP1 5.33E-03 Cytoplasm PEG10 2.63E-02 Nucleus PELP1 1.10E-02 Nucleus PENK 3.03E-03 Extracellular Space PER2 3.90E-02 Nucleus PES1 5.98E-03 Nucleus PET112 1.91E-06 Cytoplasm PEX2 1.55E-03 Cytoplasm PFAS 7.89E-05 Cytoplasm PFDN6 6.51E-03 Cytoplasm PFKFB2 1.84E-03 Cytoplasm PFKFB3 2.14E-10 Cytoplasm PFKFB4 2.85E-07 Cytoplasm PFKL 4.82E-02 Cytoplasm PGAM1 4.50E-02 Cytoplasm PGD 2.79E-02 Cytoplasm PGF 1.79E-07 Extracellular Space PGK1 1.96E-04 Cytoplasm PGLS 2.36E-03 Cytoplasm PGM1 1.33E-02 Cytoplasm PGM2 1.04E-04 Cytoplasm PGM3 4.58E-04 Cytoplasm PGM5 4.89E-02 Cytoplasm PHACTR2 8.91E-05 Other PHB 2.87E-02 Nucleus PHC1 7.16E-04 Nucleus PHC3 6.13E-04 Nucleus PHKA2 2.09E-02 Cytoplasm PHKB 4.35E-08 Cytoplasm PHKG2 6.08E-04 Cytoplasm PHLDA1 4.58E-08 Cytoplasm PI4K2A 4.34E-04 Cytoplasm PI4KA 1.73E-02 Cytoplasm PI4KB 1.49E-02 Cytoplasm PIAS1 1.15E-03 Nucleus PIAS2 4.75E-08 Nucleus PIAS4 1.10E-03 Nucleus PIBF1 2.99E-03 Nucleus PICALM 3.49E-02 Cytoplasm PICK1 3.43E-02 Cytoplasm PIGA 5.94E-10 Cytoplasm PIGF 4.81E-04 Cytoplasm PIK3AP1 1.14E-02 Cytoplasm PIK3C2A 2.97E-07 Cytoplasm PIK3C2G 2.26E-04 Cytoplasm PIK3C3 9.01E-04 Cytoplasm PIK3CA 4.75E-02 Cytoplasm PIK3CB 9.00E-07 Cytoplasm PIK3CD 1.87E-02 Cytoplasm PIK3IP1 1.01E-07 Other PIK3R1 2.73E-06 Cytoplasm PIK3R2 1.09E-02 Cytoplasm PIK3R3 6.58E-05 Cytoplasm PIK3R4 1.77E-04 Cytoplasm PIKFYVE 2.74E-03 Cytoplasm PILRB 3.66E-07 Plasma Membrane PIM3 4.78E-04 Other PIN1 2.66E-02 Nucleus PINK1 3.80E-02 Cytoplasm PINX1 4.60E-04 Nucleus PIP4K2B 1.43E-02 Cytoplasm PIP5K1A 6.08E-04 Cytoplasm PIP5K1B 4.94E-04 Cytoplasm PIP5K1C 4.04E-02 Plasma Membrane PITPNC1 2.47E-03 Cytoplasm PITPNM1 5.40E-04 Cytoplasm PITX2 7.08E-03 Nucleus PKD1 2.07E-02 Plasma Membrane PKD1P1 2.73E-02 Other PKIA 1.09E-04 Cytoplasm PKIB 1.66E-04 Other PKIG 6.70E-05 Other PKM 3.91E-03 Cytoplasm PKN2 1.47E-02 Cytoplasm PKP1 4.53E-03 Plasma Membrane PKP3 1.37E-05 Plasma Membrane PLA2G12A 7.95E-03 Other PLA2G4C 1.24E-03 Plasma Membrane PLA2G4D 2.48E-02 Cytoplasm PLA2G6 3.13E-02 Cytoplasm PLA2R1 6.78E-06 Plasma Membrane PLAGL1 8.31E-07 Nucleus PLAGL2 3.18E-02 Nucleus PLAT 4.55E-03 Extracellular Space PLAU 1.33E-03 Extracellular Space PLAUR 3.91E-06 Plasma Membrane PLCB1 8.40E-03 Cytoplasm PLCB4 3.80E-04 Cytoplasm PLCE1 5.00E-02 Cytoplasm PLCG2 1.97E-02 Cytoplasm PLCL2 4.52E-03 Cytoplasm PLCXD1 3.89E-02 Other PLCZ1 3.55E-02 Other PLD1 5.77E-05 Cytoplasm PLEC 6.29E-05 Cytoplasm PLEKHA2 7.14E-06 Cytoplasm PLEKHF2 1.64E-02 Cytoplasm PLIN2 1.65E-09 Plasma Membrane PLIN3 6.39E-03 Cytoplasm PLK1 3.01E-02 Nucleus PLK2 9.86E-06 Nucleus PLK3 2.63E-06 Nucleus PLK4 4.85E-05 Cytoplasm PLN 2.43E-02 Cytoplasm PLOD2 5.42E-09 Cytoplasm PLRG1 1.01E-03 Nucleus PLSCR1 1.03E-03 Plasma Membrane PLSCR4 3.47E-02 Plasma Membrane PLTP 2.69E-02 Extracellular Space PLXNA1 2.04E-04 Plasma Membrane PLXNA2 7.44E-05 Plasma Membrane PLXNB1 2.26E-04 Plasma Membrane PLXNB2 1.06E-02 Plasma Membrane PLXNC1 3.18E-04 Plasma Membrane PMAIP1 4.76E-08 Cytoplasm PML 2.40E-05 Nucleus PMM2 8.96E-04 Cytoplasm PMP22 4.33E-03 Plasma Membrane PMPCA 4.99E-04 Cytoplasm PMS2 2.26E-03 Nucleus PMVK 1.39E-03 Cytoplasm PNKD 3.25E-04 Nucleus PNKP 3.79E-02 Nucleus PNLIPRP3 3.43E-03 Other PNOC 2.20E-02 Extracellular Space PNP 4.29E-04 Nucleus PNPLA3 1.02E-03 Cytoplasm PNPLA6 3.06E-05 Cytoplasm PNPLA8 6.43E-07 Cytoplasm PNPO 4.40E-02 Cytoplasm PNRC1 1.59E-06 Nucleus PNRC2 4.63E-02 Nucleus POLD1 1.05E-03 Nucleus POLG 1.26E-03 Cytoplasm POLM 4.85E-03 Nucleus POLR1A 3.15E-04 Nucleus POLR1B 2.05E-08 Nucleus POLR1C 1.22E-06 Nucleus POLR1D 6.72E-05 Nucleus POLR2A 7.23E-03 Nucleus POLR2B 9.97E-03 Nucleus POLR2C 1.28E-03 Nucleus POLR2D 3.01E-06 Nucleus POLR2E 8.19E-03 Nucleus POLR2J2/POLR2J3 1.51E-06 Other POLR2K 6.39E-03 Nucleus POLR2L 4.69E-03 Nucleus POLR2M 1.49E-04 Nucleus PON3 2.72E-02 Extracellular Space POR 3.72E-02 Cytoplasm POSTN 3.67E-04 Extracellular Space POT1 4.22E-02 Nucleus POU2F1 2.82E-03 Nucleus POU2F2 5.15E-04 Nucleus POU4F1 1.87E-02 Nucleus POU5F1 1.95E-03 Nucleus PPAP2A 1.71E-03 Plasma Membrane PPAP2B 2.97E-03 Plasma Membrane PPAPDC1A 1.09E-02 Other PPAPDC1B 4.76E-03 Other PPAPDC2 2.10E-03 Other PPARA 1.60E-05 Nucleus PPARD 2.18E-06 Nucleus PPARGC1B 1.06E-05 Nucleus PPAT 7.30E-05 Cytoplasm PPBP 1.36E-04 Extracellular Space PPFIA1 3.33E-04 Plasma Membrane PPFIBP2 3.81E-03 Nucleus PPIA 4.25E-06 Cytoplasm PPIB 1.21E-02 Cytoplasm PPID 1.43E-03 Cytoplasm PPIP5K1 1.87E-02 Nucleus PPM1A 1.50E-04 Cytoplasm PPM1B 2.94E-06 Cytoplasm PPM1D 2.63E-04 Cytoplasm PPM1F 3.87E-02 Cytoplasm PPM1K 2.19E-03 Cytoplasm PPP1CB 2.44E-02 Cytoplasm PPP1R10 1.52E-02 Nucleus PPP1R11 1.28E-02 Cytoplasm PPP1R12A 4.12E-02 Cytoplasm PPP1R12B 1.45E-02 Cytoplasm PPP1R12C 2.83E-02 Cytoplasm PPP1R13B 1.43E-02 Cytoplasm PPP1R15A 1.21E-04 Cytoplasm PPP1R1C 3.66E-04 Cytoplasm PPP1R3C 1.33E-10 Cytoplasm PPP1R3D 3.82E-06 Cytoplasm PPP1R8 1.35E-03 Nucleus PPP2CA 1.96E-02 Cytoplasm PPP2CB 1.94E-02 Cytoplasm PPP2R1A 4.25E-02 Cytoplasm PPP2R1B 2.51E-04 Other PPP2R2A 2.21E-03 Cytoplasm PPP2R2B 7.60E-09 Cytoplasm PPP2R2D 8.82E-08 Nucleus PPP2R3A 7.76E-06 Nucleus PPP2R4 2.83E-04 Cytoplasm PPP2R5A 3.48E-02 Cytoplasm PPP2R5C 2.74E-09 Nucleus PPP2R5E 4.03E-04 Cytoplasm PPP3CC 8.28E-07 Cytoplasm PPP3R1 3.72E-03 Cytoplasm PPP5C 4.15E-02 Nucleus PPRC1 2.56E-06 Nucleus PPTC7 5.49E-05 Cytoplasm PRDM1 1.14E-06 Nucleus PRDM4 6.47E-04 Nucleus PRDX2 3.74E-03 Cytoplasm PREB 7.98E-05 Nucleus PRELID1 9.99E-03 Cytoplasm PRKAA1 3.22E-07 Cytoplasm PRKAA2 7.18E-08 Cytoplasm PRKAB1 2.86E-02 Nucleus PRKAB2 4.54E-03 Cytoplasm PRKACG 4.41E-02 Cytoplasm PRKAG1 1.49E-02 Nucleus PRKAG2 5.32E-03 Other PRKAR1A 2.49E-03 Cytoplasm PRKAR2A 2.33E-05 Cytoplasm PRKCA 9.46E-08 Cytoplasm PRKCD 1.88E-02 Cytoplasm PRKCE 2.25E-05 Cytoplasm PRKCH 5.03E-05 Cytoplasm PRKCI 9.65E-05 Cytoplasm PRKD2 6.13E-04 Cytoplasm PRKD3 6.86E-04 Nucleus PRKDC 2.56E-05 Nucleus PRKRIR 4.48E-02 Nucleus PRLR 3.81E-02 Plasma Membrane PRMT1 2.18E-02 Nucleus PRMT2 4.36E-05 Nucleus PRMT5 2.24E-02 Cytoplasm PRNP 2.09E-02 Plasma Membrane PROS1 1.16E-03 Extracellular Space PRPF18 5.45E-03 Nucleus PRPF19 1.22E-04 Nucleus PRPF3 2.88E-02 Nucleus PRPF31 1.97E-02 Nucleus PRPF38A 2.68E-02 Nucleus PRPF38B 1.46E-09 Other PRPF4 3.26E-07 Nucleus PRPF40A 5.33E-05 Nucleus PRPF4B 1.72E-04 Nucleus PRPF6 1.88E-04 Nucleus PRPF8 3.83E-03 Nucleus PRPS1L1 4.61E-02 Other PRR5L 9.91E-06 Cytoplasm PRRC2C 6.83E-04 Cytoplasm PRRX1 1.22E-02 Nucleus PRRX2 4.72E-02 Nucleus PRSS23 1.98E-03 Extracellular Space PSAP 1.20E-02 Extracellular Space PSCA 2.22E-03 Plasma Membrane PSEN1 2.34E-02 Plasma Membrane PSIP1 4.05E-02 Nucleus PSMA3 1.15E-05 Cytoplasm PSMA5 2.34E-03 Cytoplasm PSMA7 3.54E-02 Cytoplasm PSMA8 3.86E-02 Cytoplasm PSMB2 6.23E-04 Cytoplasm PSMB7 7.63E-05 Cytoplasm PSMB9 1.54E-02 Cytoplasm PSMC2 3.35E-04 Nucleus PSMC3 7.46E-03 Nucleus PSMD1 4.94E-03 Cytoplasm PSMD11 1.84E-03 Cytoplasm PSMD12 1.70E-03 Cytoplasm PSMD13 4.31E-02 Cytoplasm PSMD3 3.63E-03 Cytoplasm PSMD4 3.90E-02 Cytoplasm PSMD5 3.53E-02 Other PSMD6 2.52E-03 Cytoplasm PSMD7 9.05E-04 Cytoplasm PSME1 7.53E-03 Cytoplasm PSME3 3.07E-04 Cytoplasm PSME4 2.49E-02 Cytoplasm PSMF1 2.11E-02 Cytoplasm PSPH 9.99E-03 Cytoplasm PTAFR 4.68E-04 Plasma Membrane PTBP1 9.51E-04 Nucleus PTCD2 5.49E-05 Cytoplasm PTCH1 5.11E-07 Plasma Membrane PTEN 1.81E-05 Cytoplasm PTGER3 2.56E-02 Plasma Membrane PTGER4 8.24E-03 Plasma Membrane PTGES2 1.10E-03 Cytoplasm PTGFR 2.93E-02 Plasma Membrane PTGIS 5.06E-03 Cytoplasm PTGR1 7.01E-04 Cytoplasm PTGS1 1.11E-06 Cytoplasm PTGS2 1.17E-02 Cytoplasm PTHLH 2.81E-07 Extracellular Space PTK2 7.70E-05 Cytoplasm PTP4A1 5.57E-04 Cytoplasm PTPDC1 1.38E-04 Extracellular Space PTPLA 1.13E-02 Other PTPLB 3.73E-03 Cytoplasm PTPN1 3.18E-04 Cytoplasm PTPN11 2.27E-05 Cytoplasm PTPN12 1.51E-04 Cytoplasm PTPN13 3.48E-03 Cytoplasm PTPN14 3.08E-05 Cytoplasm PTPN2 3.41E-05 Cytoplasm PTPN20A/PTPN20B 1.64E-02 Extracellular Space PTPN22 4.86E-04 Cytoplasm PTPN3 7.23E-03 Cytoplasm PTPN4 2.00E-02 Cytoplasm PTPN9 5.82E-05 Cytoplasm PTPRA 1.53E-03 Plasma Membrane PTPRB 5.14E-03 Plasma Membrane PTPRD 4.74E-02 Plasma Membrane PTPRE 3.12E-07 Plasma Membrane PTPRF 1.35E-07 Plasma Membrane PTPRG 8.05E-03 Plasma Membrane PTPRJ 1.13E-02 Plasma Membrane PTPRK 4.85E-04 Plasma Membrane PTPRM 3.88E-04 Plasma Membrane PTPRO 3.39E-03 Plasma Membrane PTPRS 2.41E-04 Plasma Membrane PTPRZ1 2.18E-03 Plasma Membrane PTRF 2.03E-03 Nucleus PTTG1 7.00E-05 Nucleus PTTG1IP 2.05E-02 Nucleus PTX3 1.25E-10 Extracellular Space PVR 2.75E-04 Plasma Membrane PVRL1 4.64E-03 Plasma Membrane PVRL3 1.23E-02 Plasma Membrane PYCARD 7.20E-04 Cytoplasm PYCR2 9.24E-07 Cytoplasm PYGB 3.45E-04 Cytoplasm PYGO1 4.24E-02 Nucleus QDPR 7.92E-03 Cytoplasm QRSL1 2.97E-02 Cytoplasm QSOX1 4.43E-05 Cytoplasm RAB1A 1.73E-02 Cytoplasm RAB2A 3.71E-03 Cytoplasm RAB32 2.23E-04 Cytoplasm RAB3A 2.01E-02 Cytoplasm RAB3B 7.24E-04 Cytoplasm RAB4B 4.07E-02 Plasma Membrane RAB5A 4.88E-04 Cytoplasm RAB5C 4.66E-03 Cytoplasm RAB8B 1.69E-05 Cytoplasm RAC2 1.88E-02 Cytoplasm RAD1 6.80E-05 Nucleus RAD21 2.12E-03 Nucleus RAD23B 5.92E-05 Nucleus RAF1 8.53E-06 Cytoplasm RAG1 7.32E-03 Nucleus RALBP1 6.18E-03 Cytoplasm RALGDS 1.33E-05 Cytoplasm RALGPS2 4.91E-03 Other RAN 1.64E-02 Nucleus RANBP1 1.54E-02 Nucleus RANBP9 8.27E-03 Nucleus RANGAP1 4.58E-04 Nucleus RAP1A 1.52E-02 Cytoplasm RAP2B 5.35E-03 Plasma Membrane RAPGEF1 2.59E-02 Cytoplasm RAPGEF2 9.12E-06 Cytoplasm RAPGEF4 8.93E-05 Cytoplasm RAPGEF5 5.60E-09 Nucleus RAPH1 1.04E-04 Plasma Membrane RARA 1.04E-02 Nucleus RARS 3.18E-02 Cytoplasm RARS2 4.39E-03 Cytoplasm RASA2 5.73E-04 Cytoplasm RASGRP1 3.66E-06 Cytoplasm RASGRP2 3.40E-03 Cytoplasm RASSF1 5.73E-03 Nucleus RASSF2 6.00E-03 Nucleus RASSF5 1.36E-03 Plasma Membrane RB1 7.18E-04 Nucleus RB1CC1 2.65E-03 Nucleus RBBP4 8.88E-05 Nucleus RBBP5 1.59E-04 Nucleus RBBP6 4.69E-03 Nucleus RBBP8 2.80E-03 Nucleus RBCK1 2.86E-04 Cytoplasm RBFOX2 6.72E-04 Nucleus RBL2 2.17E-03 Nucleus RBM25 1.45E-08 Nucleus RBM38 2.92E-03 Other RBM4 5.65E-08 Nucleus RBM5 2.46E-04 Nucleus RBM6 1.73E-05 Nucleus RBM8A 5.42E-05 Nucleus RBMS1 5.62E-03 Nucleus RBMS3 2.66E-04 Other RBP1 2.93E-04 Extracellular Space RBPJ 6.36E-06 Nucleus RBPMS 1.47E-04 Other RBX1 3.56E-02 Cytoplasm RCAN1 2.96E-04 Nucleus RCC1 6.50E-03 Cytoplasm RCOR1 3.28E-02 Nucleus RCOR2 7.21E-03 Nucleus RDH10 2.33E-02 Nucleus RDH11 1.85E-03 Cytoplasm RDH12 1.25E-04 Cytoplasm RDH13 1.98E-07 Cytoplasm RDH5 3.78E-03 Cytoplasm RDX 5.28E-05 Cytoplasm RECK 2.57E-06 Plasma Membrane REG3A 2.55E-02 Extracellular Space REL 8.85E-03 Nucleus RELA 1.45E-02 Nucleus RELB 2.78E-02 Nucleus RELN 1.66E-02 Extracellular Space RELT 2.60E-02 Plasma Membrane REST 1.97E-04 Nucleus RET 5.50E-06 Plasma Membrane REV3L 1.66E-07 Nucleus RFC1 1.96E-03 Nucleus RFC2 4.09E-02 Nucleus RFC3 2.71E-04 Nucleus RFC5 2.52E-04 Nucleus RFK 9.59E-03 Cytoplasm RFTN1 3.49E-03 Plasma Membrane RFWD2 2.63E-02 Cytoplasm RFX3 8.17E-08 Nucleus RFX4 1.30E-02 Nucleus RGCC 1.44E-02 Cytoplasm RGPD3 (includes others)1.68E-02 Nucleus RGS10 2.48E-04 Cytoplasm RGS12 1.26E-05 Nucleus RGS14 9.39E-04 Cytoplasm RGS2 1.82E-07 Nucleus RGS20 3.82E-04 Cytoplasm RGS3 8.18E-04 Nucleus RGS5 6.68E-05 Plasma Membrane RHBDD1 5.53E-04 Extracellular Space RHEB 1.52E-07 Cytoplasm RHOA 4.93E-02 Cytoplasm RHOB 4.96E-02 Cytoplasm RHOBTB1 3.07E-02 Other RHOBTB3 5.83E-03 Other RHOC 1.90E-04 Plasma Membrane RHOF 1.01E-02 Cytoplasm RHOG 1.09E-02 Cytoplasm RHOJ 2.35E-03 Cytoplasm RHOQ 1.01E-04 Plasma Membrane RHOT1 4.70E-05 Cytoplasm RHOT2 1.24E-03 Cytoplasm RHOU 3.54E-09 Cytoplasm RHPN2 2.97E-05 Cytoplasm RICTOR 2.09E-04 Cytoplasm RIF1 7.27E-05 Nucleus RIN2 2.02E-04 Cytoplasm RIN3 1.16E-02 Cytoplasm RIPK2 3.68E-03 Plasma Membrane RIPK3 2.19E-02 Plasma Membrane RLN1 9.76E-03 Extracellular Space RLN2 1.64E-04 Extracellular Space RNASEH1 2.46E-04 Nucleus RNASEL 1.09E-04 Cytoplasm RND1 9.94E-03 Cytoplasm RNF130 9.34E-03 Cytoplasm RNF139 2.48E-02 Cytoplasm RNF167 2.22E-03 Cytoplasm RNF19A 4.78E-07 Nucleus RNF41 3.57E-03 Cytoplasm RNF7 2.92E-02 Nucleus RNGTT 1.20E-03 Nucleus RNLS 1.01E-02 Extracellular Space RNPC3 4.60E-04 Nucleus ROBO1 1.70E-02 Plasma Membrane ROBO2 2.49E-03 Plasma Membrane ROCK1 2.30E-03 Cytoplasm ROCK2 1.95E-02 Cytoplasm ROMO1 2.36E-02 Cytoplasm ROR2 4.67E-02 Plasma Membrane RORA 2.28E-06 Nucleus RPA2 2.30E-04 Nucleus RPE65 1.28E-02 Cytoplasm RPGRIP1L 8.49E-03 Cytoplasm RPIA 9.47E-04 Cytoplasm RPL10 1.20E-02 Cytoplasm RPL10A 6.20E-04 Other RPL12 2.91E-03 Cytoplasm RPL13 3.46E-04 Cytoplasm RPL13A 9.97E-03 Cytoplasm RPL14 2.00E-02 Cytoplasm RPL15 5.13E-04 Cytoplasm RPL17 5.81E-07 Cytoplasm RPL18 2.69E-04 Cytoplasm RPL19 8.57E-03 Cytoplasm RPL21 2.02E-02 Cytoplasm RPL22L1 1.34E-02 Other RPL23 1.80E-02 Cytoplasm RPL24 3.35E-02 Cytoplasm RPL27 1.84E-02 Cytoplasm RPL27A 1.95E-02 Nucleus RPL28 1.54E-05 Cytoplasm RPL31 2.57E-07 Other RPL34 3.31E-03 Cytoplasm RPL35 7.03E-03 Cytoplasm RPL35A 2.69E-02 Cytoplasm RPL36AL 3.95E-02 Cytoplasm RPL37 2.06E-06 Cytoplasm RPL37A 3.34E-09 Cytoplasm RPL39L 1.87E-02 Other RPL5 6.63E-04 Cytoplasm RPL7 3.02E-03 Nucleus RPL7L1 3.90E-05 Cytoplasm RPLP2 1.73E-02 Cytoplasm RPS10 3.95E-03 Cytoplasm RPS11 5.00E-02 Cytoplasm RPS12 4.04E-02 Cytoplasm RPS14 7.43E-04 Cytoplasm RPS15A 9.44E-04 Cytoplasm RPS16 9.56E-04 Cytoplasm RPS18 3.82E-02 Cytoplasm RPS19 1.48E-04 Cytoplasm RPS20 2.44E-02 Cytoplasm RPS21 3.37E-04 Cytoplasm RPS24 1.42E-09 Cytoplasm RPS25 3.11E-02 Cytoplasm RPS26 2.25E-03 Cytoplasm RPS27 2.08E-05 Cytoplasm RPS27A 1.48E-02 Cytoplasm RPS27L 1.53E-02 Nucleus RPS28 4.87E-03 Cytoplasm RPS3 4.99E-02 Cytoplasm RPS3A 5.19E-03 Cytoplasm RPS5 2.58E-03 Other RPS6KA1 2.73E-02 Cytoplasm RPS6KA3 6.26E-03 Cytoplasm RPS6KA4 4.26E-06 Cytoplasm RPS6KA5 1.06E-03 Nucleus RPS6KA6 2.06E-02 Cytoplasm RPS6KB1 4.94E-03 Cytoplasm RPS6KB2 2.88E-03 Cytoplasm RPS8 3.44E-02 Cytoplasm RPS9 2.71E-02 Cytoplasm RPTOR 7.04E-03 Cytoplasm RRAD 6.71E-04 Cytoplasm RRAS2 2.28E-02 Plasma Membrane RRBP1 1.36E-03 Cytoplasm RRM1 2.01E-02 Nucleus RRM2 2.05E-03 Nucleus RSAD2 7.95E-05 Cytoplasm RSL1D1 9.64E-05 Nucleus RSU1 9.60E-05 Cytoplasm RTN1 4.97E-02 Cytoplasm RTN4 5.52E-03 Cytoplasm RUNX2 7.75E-08 Nucleus RUVBL1 1.87E-03 Nucleus RXRA 5.65E-03 Nucleus RYBP 2.87E-08 Nucleus RYK 4.01E-02 Plasma Membrane S100A10 2.37E-08 Cytoplasm S100A11 5.18E-03 Cytoplasm S100A4 3.84E-04 Cytoplasm S100A6 2.23E-02 Cytoplasm S100A7 3.63E-03 Cytoplasm S1PR1 1.45E-02 Plasma Membrane S1PR3 5.73E-05 Plasma Membrane S1PR5 7.43E-03 Plasma Membrane SAA2 8.66E-06 Extracellular Space SACM1L 1.65E-03 Cytoplasm SAE1 2.17E-03 Cytoplasm SAFB 1.18E-02 Nucleus SAG 3.18E-02 Cytoplasm SAMD4A 5.06E-07 Cytoplasm SAP30 1.04E-07 Nucleus SAP30BP 2.82E-04 Nucleus SARS 1.50E-03 Cytoplasm SART1 1.75E-02 Nucleus SART3 8.89E-07 Nucleus SAT1 6.85E-03 Cytoplasm SAT2 1.18E-03 Plasma Membrane SATB1 5.08E-07 Nucleus SBDS 2.52E-02 Nucleus SC5D 6.51E-04 Cytoplasm SCAF11 2.07E-08 Nucleus SCAMP1 2.14E-03 Cytoplasm SCAMP2 3.68E-04 Cytoplasm SCAMP4 1.83E-02 Cytoplasm SCD 1.61E-04 Cytoplasm SCD5 1.74E-05 Cytoplasm SCEL 4.00E-02 Cytoplasm SCG2 2.67E-02 Extracellular Space SCG5 4.52E-05 Extracellular Space SCGB1A1 3.29E-02 Extracellular Space SCNN1G 5.81E-05 Plasma Membrane SCO2 1.33E-02 Cytoplasm SCRIB 4.05E-05 Cytoplasm SDC1 3.35E-03 Plasma Membrane SDC2 6.58E-03 Plasma Membrane SDC3 2.04E-02 Plasma Membrane SDCBP 2.40E-04 Plasma Membrane SDHA 6.60E-05 Cytoplasm SDHC 3.38E-03 Cytoplasm SDHD 8.28E-05 Cytoplasm SDR9C7 4.14E-03 Other SDSL 4.67E-02 Cytoplasm SEC16A 1.06E-03 Cytoplasm SEC22B 1.63E-04 Other SEC61A1 3.97E-02 Cytoplasm SEL1L 6.62E-04 Cytoplasm SEL1L3 7.71E-04 Other SELP 2.92E-02 Plasma Membrane SELPLG 3.91E-05 Plasma Membrane SEMA3C 8.80E-03 Extracellular Space SEMA3F 1.27E-02 Extracellular Space SEMA3G 4.97E-02 Other SEMA4A 4.66E-07 Plasma Membrane SEMA4B 6.90E-03 Plasma Membrane SEMA4C 2.23E-02 Plasma Membrane SEMA4D 2.82E-05 Plasma Membrane SEMA4F 4.69E-02 Plasma Membrane SEMA4G 1.76E-02 Plasma Membrane SEMA5A 4.74E-03 Plasma Membrane SEMA6A 8.49E-09 Plasma Membrane SEMA6D 6.73E-08 Other SENP3 2.48E-05 Nucleus SEPHS2 4.07E-05 Other SEPP1 7.13E-03 Extracellular Space SEPSECS 1.84E-04 Cytoplasm SEPT2 6.78E-05 Cytoplasm SEPT6 2.00E-06 Cytoplasm SEPT7 1.73E-02 Cytoplasm SEPT9 6.38E-03 Cytoplasm SERPINA3 6.09E-04 Extracellular Space SERPINB1 3.70E-04 Cytoplasm SERPINB5 3.14E-05 Extracellular Space SERPINB9 3.84E-02 Cytoplasm SERPINC1 1.09E-02 Extracellular Space SERPINE1 9.49E-05 Extracellular Space SERPINE2 1.48E-02 Extracellular Space SERPINF1 9.14E-04 Extracellular Space SERPING1 8.84E-04 Extracellular Space SERPINH1 1.39E-02 Extracellular Space SERTAD1 6.03E-04 Nucleus SERTAD2 4.58E-08 Cytoplasm SET 8.74E-04 Nucleus SETDB1 3.04E-01 Nucleus SETMAR 4.58E-06 Other SF1 5.00E-06 Nucleus SF3A1 9.85E-03 Nucleus SF3A2 1.39E-02 Nucleus SF3A3 4.28E-02 Nucleus SF3B1 4.87E-03 Nucleus SF3B2 1.15E-02 Nucleus SF3B3 1.55E-02 Nucleus SFPQ 1.21E-04 Nucleus SFRP1 1.33E-05 Plasma Membrane SGCA 4.96E-02 Plasma Membrane SGCB 3.19E-03 Plasma Membrane SGK1 9.33E-07 Cytoplasm SGMS2 3.09E-04 Plasma Membrane SGPL1 3.39E-02 Cytoplasm SGPP2 1.26E-02 Cytoplasm SGSM3 4.05E-03 Plasma Membrane SH2B2 1.33E-02 Cytoplasm SH2B3 7.79E-03 Plasma Membrane SH2D1B 4.00E-03 Cytoplasm SH3BGRL3 1.85E-03 Nucleus SH3BP2 7.16E-07 Cytoplasm SH3BP4 7.60E-06 Cytoplasm SH3BP5 2.46E-06 Cytoplasm SH3GL3 6.10E-05 Cytoplasm SH3GLB1 4.71E-03 Cytoplasm SH3KBP1 4.02E-08 Cytoplasm SH3PXD2A 3.55E-03 Cytoplasm SH3RF1 2.04E-02 Cytoplasm SHANK2 4.00E-08 Plasma Membrane SHC1 6.78E-05 Cytoplasm SHC3 2.31E-02 Cytoplasm SHMT1 6.92E-03 Cytoplasm SHMT2 8.20E-03 Cytoplasm SIAH1 2.48E-04 Nucleus SIK3 3.71E-03 Other SIKE1 2.19E-06 Cytoplasm SIN3A 4.45E-03 Nucleus SIN3B 3.50E-03 Nucleus SIPA1L2 1.99E-02 Other SIRPG 4.58E-02 Plasma Membrane SIRT1 7.17E-05 Nucleus SIRT2 1.59E-04 Nucleus SIRT4 1.82E-05 Cytoplasm SIRT5 1.25E-02 Cytoplasm SIRT7 6.52E-05 Nucleus SIVA1 9.07E-04 Cytoplasm SKI 1.82E-02 Nucleus SKIL 8.36E-03 Nucleus SKP1 3.12E-02 Nucleus SKP2 4.89E-05 Nucleus SLAMF1 1.78E-02 Plasma Membrane SLAMF7 2.25E-05 Plasma Membrane SLBP 1.64E-03 Nucleus SLC12A2 6.70E-03 Plasma Membrane SLC12A4 1.20E-03 Plasma Membrane SLC12A6 1.23E-03 Plasma Membrane SLC16A1 1.35E-04 Plasma Membrane SLC16A3 3.79E-02 Plasma Membrane SLC16A7 1.26E-04 Plasma Membrane SLC17A8 3.74E-04 Plasma Membrane SLC1A1 3.17E-04 Plasma Membrane SLC1A2 2.45E-03 Plasma Membrane SLC1A4 1.76E-02 Plasma Membrane SLC1A5 1.15E-02 Plasma Membrane SLC1A6 2.67E-09 Plasma Membrane SLC20A1 1.14E-07 Plasma Membrane SLC22A18 1.86E-04 Plasma Membrane SLC22A3 1.80E-02 Plasma Membrane SLC23A2 8.52E-03 Plasma Membrane SLC24A6 2.89E-02 Plasma Membrane SLC25A10 6.90E-03 Cytoplasm SLC25A11 5.05E-04 Cytoplasm SLC25A13 2.54E-03 Cytoplasm SLC25A14 1.05E-02 Cytoplasm SLC25A27 4.03E-05 Cytoplasm SLC25A4 1.67E-05 Cytoplasm SLC25A6 2.25E-02 Cytoplasm SLC27A2 1.56E-03 Cytoplasm SLC27A4 1.77E-02 Plasma Membrane SLC27A5 2.33E-03 Cytoplasm SLC29A2 8.73E-03 Cytoplasm SLC2A1 1.10E-05 Plasma Membrane SLC2A3 1.71E-11 Plasma Membrane SLC2A5 7.70E-04 Plasma Membrane SLC35A2 5.90E-04 Cytoplasm SLC35F6 1.67E-04 Cytoplasm SLC38A1 4.94E-03 Plasma Membrane SLC4A11 3.36E-02 Plasma Membrane SLC4A2 6.34E-03 Plasma Membrane SLC5A3 4.65E-06 Plasma Membrane SLC6A11 1.95E-06 Plasma Membrane SLC6A13 6.08E-05 Plasma Membrane SLC7A1 5.99E-05 Plasma Membrane SLC7A11 3.25E-02 Plasma Membrane SLC7A5 8.33E-03 Plasma Membrane SLC7A7 1.08E-02 Plasma Membrane SLC7A8 6.66E-03 Plasma Membrane SLC8A1 4.39E-07 Plasma Membrane SLC8A3 4.35E-02 Plasma Membrane SLC9A1 8.11E-06 Plasma Membrane SLCO3A1 4.64E-06 Plasma Membrane SLCO4A1 1.20E-03 Plasma Membrane SLCO4C1 2.84E-02 Plasma Membrane SLIT2 3.39E-02 Extracellular Space SLIT3 9.61E-04 Extracellular Space SLK 4.58E-03 Nucleus SMAD1 1.74E-08 Nucleus SMAD2 3.24E-04 Nucleus SMAD3 1.92E-05 Nucleus SMAD4 1.30E-04 Nucleus SMAD5 1.07E-02 Nucleus SMAD6 2.75E-02 Nucleus SMAD7 6.28E-07 Nucleus SMAD9 9.62E-11 Nucleus SMARCA2 4.47E-05 Nucleus SMARCA4 3.29E-02 Nucleus SMARCD2 4.22E-03 Nucleus SMC2 2.11E-03 Nucleus SMC3 3.90E-03 Nucleus SMPD3 1.01E-04 Cytoplasm SMPD4 6.88E-03 Cytoplasm SMS 4.94E-03 Cytoplasm SMTN 1.78E-03 Extracellular Space SMU1 5.19E-03 Nucleus SMURF1 7.31E-03 Cytoplasm SMURF2 1.58E-05 Cytoplasm SNAI2 5.97E-03 Nucleus SNAP23 7.81E-07 Plasma Membrane SNAPC1 7.56E-08 Nucleus SNCA 2.37E-02 Cytoplasm SNRNP200 5.20E-03 Nucleus SNRNP27 3.97E-03 Nucleus SNRNP35 4.68E-05 Nucleus SNRNP40 6.70E-04 Nucleus SNRPA 8.96E-03 Nucleus SNRPB2 1.20E-04 Nucleus SNRPD1 2.02E-04 Nucleus SNRPE 1.89E-03 Nucleus SNRPF 4.77E-02 Nucleus SNRPN 1.58E-05 Nucleus SNTA1 4.82E-03 Plasma Membrane SNTB1 5.45E-03 Plasma Membrane SNTB2 2.21E-02 Plasma Membrane SNW1 2.80E-02 Nucleus SNX9 2.94E-02 Cytoplasm SOCS1 2.41E-04 Cytoplasm SOCS3 8.37E-06 Cytoplasm SOCS5 1.07E-04 Extracellular Space SOCS6 1.32E-02 Cytoplasm SOCS7 5.73E-04 Cytoplasm SOD2 3.07E-08 Cytoplasm SON 1.52E-04 Nucleus SORBS1 2.97E-03 Plasma Membrane SORBS2 2.99E-04 Plasma Membrane SORD 2.97E-05 Cytoplasm SOS1 9.46E-04 Cytoplasm SOS2 9.77E-04 Cytoplasm SOST 5.54E-03 Extracellular Space SOSTDC1 2.86E-02 Extracellular Space SOX11 3.85E-02 Nucleus SOX12 6.10E-03 Nucleus SOX15 4.39E-04 Nucleus SOX2 3.53E-03 Nucleus SOX4 4.83E-06 Nucleus SOX5 4.00E-03 Nucleus SOX6 1.75E-04 Nucleus SOX7 6.33E-07 Nucleus SOX9 7.66E-08 Nucleus SP1 5.00E-03 Nucleus SP100 1.14E-06 Nucleus SP3 3.90E-05 Nucleus SP4 1.69E-02 Nucleus SPARC 1.62E-03 Extracellular Space SPDYA 2.09E-10 Nucleus SPEG 1.30E-02 Nucleus SPEN 5.72E-04 Nucleus SPHK1 5.77E-04 Cytoplasm SPHK2 3.63E-02 Cytoplasm SPIB 2.63E-02 Nucleus SPN 2.08E-03 Plasma Membrane SPP1 8.43E-03 Extracellular Space SPRY1 8.59E-08 Other SPRY2 6.94E-06 Plasma Membrane SPTAN1 2.60E-05 Plasma Membrane SPTB 1.40E-02 Plasma Membrane SPTBN1 7.99E-03 Plasma Membrane SPTLC2 1.40E-02 Cytoplasm SPTSSA 3.47E-03 Cytoplasm SQLE 4.02E-09 Cytoplasm SQSTM1 5.63E-06 Cytoplasm SRA1 1.05E-03 Nucleus SRC 7.77E-03 Cytoplasm SRD5A3 3.07E-04 Cytoplasm SREBF1 2.62E-03 Nucleus SREBF2 3.98E-03 Nucleus SRF 3.47E-04 Nucleus SRGAP1 3.87E-04 Cytoplasm SRGAP2 1.23E-03 Cytoplasm SRGAP3 5.21E-04 Cytoplasm SRM 1.53E-03 Cytoplasm SRPX 8.11E-04 Cytoplasm SRR 2.14E-03 Cytoplasm SRSF1 6.96E-05 Nucleus SRSF2 2.91E-03 Nucleus SRSF3 5.41E-03 Nucleus SRSF7 2.10E-10 Nucleus SRXN1 1.27E-02 Cytoplasm SSFA2 3.78E-02 Plasma Membrane SSH1 5.19E-03 Cytoplasm SSH2 1.58E-02 Cytoplasm SSH3 1.39E-02 Cytoplasm SSR1 1.59E-03 Cytoplasm SSR4 3.34E-03 Cytoplasm SSRP1 9.78E-05 Nucleus SSTR2 3.49E-02 Plasma Membrane ST3GAL2 5.76E-03 Cytoplasm ST6GALNAC4 8.31E-03 Cytoplasm ST8SIA1 9.08E-04 Cytoplasm STAG1 4.11E-06 Nucleus STAG2 1.21E-02 Nucleus STAM 2.18E-03 Cytoplasm STAMBP 3.39E-02 Nucleus STARD4 7.93E-04 Cytoplasm STAT1 4.43E-02 Nucleus STAT2 3.98E-02 Nucleus STAT3 8.36E-04 Nucleus STAT4 3.92E-04 Nucleus STAT5A 3.06E-02 Nucleus STAT6 4.59E-02 Nucleus STAU1 3.37E-02 Cytoplasm STC1 3.18E-08 Extracellular Space STIP1 1.67E-05 Cytoplasm STK11 1.64E-03 Cytoplasm STK17A 6.25E-04 Nucleus STK24 6.82E-08 Cytoplasm STK25 1.64E-02 Cytoplasm STK3 1.90E-03 Cytoplasm STK36 3.02E-02 Cytoplasm STK4 1.69E-05 Cytoplasm STOML2 2.55E-02 Plasma Membrane STON2 2.18E-05 Cytoplasm STRADA 1.71E-03 Nucleus STRAP 4.63E-07 Plasma Membrane STRBP 1.28E-04 Cytoplasm STRN 5.83E-07 Cytoplasm STUB1 4.20E-02 Cytoplasm STX11 4.18E-02 Plasma Membrane STX1A 9.53E-04 Cytoplasm STX4 1.20E-04 Plasma Membrane STX6 1.54E-07 Cytoplasm STXBP3 8.01E-03 Plasma Membrane STYX 2.68E-02 Cytoplasm SUB1 2.55E-02 Nucleus SUCLA2 3.29E-06 Cytoplasm SUCLG1 1.29E-03 Cytoplasm SUCLG2 8.30E-03 Cytoplasm SUDS3 4.77E-02 Nucleus SULF1 5.45E-06 Cytoplasm SULT1E1 7.18E-06 Cytoplasm SUPV3L1 3.83E-04 Cytoplasm SURF1 1.56E-02 Cytoplasm SURF4 1.88E-02 Cytoplasm SURF6 3.03E-05 Nucleus SUV39H1 1.27E-03 Nucleus SUV39H2 2.11E-03 Nucleus SVIL 8.58E-04 Other SYK 4.40E-03 Cytoplasm SYMPK 1.23E-04 Cytoplasm SYNCRIP 4.37E-06 Nucleus SYNJ1 1.18E-02 Cytoplasm SYT12 2.44E-02 Plasma Membrane TAB2 1.33E-03 Cytoplasm TAB3 2.37E-05 Cytoplasm TACC1 1.27E-02 Nucleus TACC3 3.63E-04 Nucleus TACSTD2 8.76E-03 Plasma Membrane TAF1 2.96E-03 Nucleus TAF11 9.92E-05 Nucleus TAF13 1.13E-05 Nucleus TAF15 2.15E-06 Nucleus TAF1A 1.47E-05 Nucleus TAF1D 5.55E-05 Nucleus TAF3 4.49E-03 Nucleus TAF4B 1.93E-04 Nucleus TAF5 1.63E-03 Nucleus TAF5L 4.55E-05 Nucleus TAF6L 6.48E-04 Nucleus TAF9B 3.31E-11 Nucleus TAGLN 4.46E-02 Cytoplasm TANK 8.44E-03 Cytoplasm TAOK1 9.88E-05 Cytoplasm TAP2 2.25E-02 Cytoplasm TARP 5.80E-03 Cytoplasm TARS 3.04E-02 Nucleus TARS2 7.00E-04 Cytoplasm TARSL2 9.98E-04 Other TAT 3.14E-02 Cytoplasm TBC1D3F (includes others)1.25E-05 Extracellular Space TBC1D8 7.74E-03 Plasma Membrane TBK1 2.20E-03 Cytoplasm TBL1XR1 1.47E-05 Nucleus TBRG1 7.96E-04 Nucleus TBRG4 2.99E-04 Cytoplasm TBX1 3.69E-02 Nucleus TBX3 3.44E-03 Nucleus TCEA1 4.34E-02 Nucleus TCEB2 1.23E-03 Nucleus TCERG1 7.10E-06 Nucleus TCF12 1.94E-05 Nucleus TCF19 2.87E-03 Nucleus TCF21 2.40E-02 Nucleus TCF3 3.04E-02 Nucleus TCF4 1.55E-05 Nucleus TCF7L2 1.40E-04 Nucleus TDO2 4.86E-02 Cytoplasm TDP1 3.63E-02 Nucleus TDP2 1.27E-02 Cytoplasm TEK 9.37E-07 Plasma Membrane TERF1 4.61E-04 Nucleus TERF2 5.69E-06 Nucleus TERF2IP 3.77E-03 Nucleus TES 2.98E-03 Plasma Membrane TET2 1.73E-04 Other TFAM 3.96E-05 Cytoplasm TFAP2A 1.96E-03 Nucleus TFCP2 6.21E-03 Nucleus TFDP2 1.08E-02 Nucleus TFPI 5.83E-05 Extracellular Space TFPI2 1.33E-03 Extracellular Space TFRC 1.49E-05 Plasma Membrane TGFA 1.77E-02 Extracellular Space TGFB1 5.82E-01 Extracellular Space TGFB1I1 8.41E-04 Nucleus TGFB2 5.53E-05 Extracellular Space TGFBR1 1.86E-03 Plasma Membrane TGFBR2 9.41E-05 Plasma Membrane TGFBR3 1.63E-05 Plasma Membrane TGIF1 2.44E-05 Nucleus TGM1 1.34E-02 Plasma Membrane TGM2 8.94E-03 Cytoplasm TGS1 7.86E-07 Nucleus THBD 7.15E-07 Plasma Membrane THBS1 1.67E-05 Extracellular Space THEM4 2.58E-05 Plasma Membrane THOP1 2.47E-05 Cytoplasm THRAP3 1.47E-06 Nucleus THRB 3.70E-05 Nucleus THTPA 1.02E-02 Cytoplasm TIA1 1.97E-07 Nucleus TIAF1 3.61E-04 Cytoplasm TICAM2 9.16E-06 Plasma Membrane TIFA 7.40E-06 Other TIMM17A 2.05E-03 Cytoplasm TIMM50 2.48E-03 Cytoplasm TIMP3 9.86E-04 Extracellular Space TINF2 5.79E-03 Nucleus TIPARP 1.30E-02 Other TIPIN 1.25E-02 Nucleus TIRAP 1.34E-02 Cytoplasm TJP1 1.05E-04 Plasma Membrane TJP2 8.55E-07 Plasma Membrane TK2 1.50E-02 Cytoplasm TKT 1.22E-03 Cytoplasm TLE1 7.50E-06 Nucleus TLE3 1.22E-04 Nucleus TLE4 9.50E-04 Nucleus TLK1 1.44E-04 Nucleus TLK2 8.67E-03 Cytoplasm TLN1 1.27E-03 Plasma Membrane TLN2 9.86E-05 Nucleus TLR10 1.63E-03 Plasma Membrane TLR2 1.31E-08 Plasma Membrane TLR3 6.42E-06 Plasma Membrane TM4SF1 4.99E-02 Plasma Membrane TM7SF2 3.59E-04 Cytoplasm TMBIM4 2.37E-02 Nucleus TMBIM6 1.43E-02 Nucleus TMEFF2 2.29E-03 Cytoplasm TMEM117 3.30E-04 Cytoplasm TMEM123 4.19E-02 Plasma Membrane TMEM158 9.87E-05 Plasma Membrane TMEM161A 2.44E-02 Other TMEM2 6.21E-03 Other TMEM67 2.15E-05 Plasma Membrane TMEM9B 4.03E-04 Other TMLHE 2.95E-02 Cytoplasm TMPO 4.38E-04 Nucleus TMPRSS11D 7.89E-04 Extracellular Space TMX1 4.85E-05 Cytoplasm TNC 1.03E-07 Extracellular Space TNF 7.12E-10 Extracellular Space TNFAIP1 3.94E-03 Plasma Membrane TNFAIP2 3.39E-04 Extracellular Space TNFAIP3 1.74E-09 Nucleus TNFAIP6 3.34E-11 Extracellular Space TNFAIP8 2.81E-07 Cytoplasm TNFRSF10A 2.07E-08 Plasma Membrane TNFRSF10B 3.20E-02 Plasma Membrane TNFRSF10D 7.48E-04 Plasma Membrane TNFRSF11A 1.55E-05 Plasma Membrane TNFRSF12A 5.98E-03 Plasma Membrane TNFRSF18 2.57E-02 Plasma Membrane TNFRSF19 1.54E-06 Plasma Membrane TNFRSF1A 1.27E-03 Plasma Membrane TNFRSF21 2.77E-05 Plasma Membrane TNFRSF25 3.59E-03 Plasma Membrane TNFSF10 1.90E-05 Extracellular Space TNFSF11 1.04E-04 Extracellular Space TNFSF13B 7.61E-07 Extracellular Space TNFSF15 1.58E-05 Extracellular Space TNFSF4 1.07E-03 Extracellular Space TNFSF8 1.08E-02 Plasma Membrane TNFSF9 2.32E-02 Plasma Membrane TNIK 5.62E-06 Cytoplasm TNIP2 4.83E-03 Cytoplasm TNIP3 3.12E-02 Other TNK2 4.17E-03 Cytoplasm TNKS 1.21E-05 Nucleus TNKS2 1.77E-03 Nucleus TNNI2 1.74E-03 Cytoplasm TNNI3 1.63E-02 Cytoplasm TNPO1 1.72E-08 Nucleus TNS1 1.81E-06 Plasma Membrane TNS3 3.01E-04 Other TOB2 1.86E-05 Nucleus TOE1 6.47E-04 Nucleus TOLLIP 2.59E-04 Cytoplasm TOM1 1.67E-02 Cytoplasm TONSL 5.46E-05 Cytoplasm TOP1 3.27E-06 Nucleus TOP2A 1.50E-04 Nucleus TOPORS 2.83E-06 Nucleus TOR1A 3.59E-03 Cytoplasm TOR2A 2.66E-02 Extracellular Space TOR4A 3.54E-03 Other TP53AIP1 1.51E-06 Cytoplasm TP53BP1 3.40E-03 Nucleus TP53BP2 1.57E-06 Nucleus TP63 1.38E-04 Nucleus TPD52L2 1.49E-03 Cytoplasm TPI1 5.23E-03 Cytoplasm TPK1 3.83E-02 Cytoplasm TPM1 7.38E-06 Cytoplasm TPM2 2.54E-03 Cytoplasm TPM3 2.85E-03 Cytoplasm TPM4 1.42E-05 Cytoplasm TPP1 1.23E-04 Cytoplasm TPP2 5.84E-04 Cytoplasm TPR 3.99E-04 Nucleus TPSAB1/TPSB2 2.12E-02 Extracellular Space TRAF1 3.09E-03 Cytoplasm TRAF3 1.84E-03 Cytoplasm TRAF3IP2 4.64E-02 Cytoplasm TRAF4 3.23E-05 Cytoplasm TRAF5 3.38E-05 Cytoplasm TRAK1 3.57E-04 Nucleus TRAP1 1.79E-02 Cytoplasm TREM1 3.70E-01 Plasma Membrane TREX1 3.17E-02 Nucleus TRH 3.54E-02 Extracellular Space TRIAP1 1.14E-02 Cytoplasm TRIB1 1.12E-05 Cytoplasm TRIB2 2.69E-04 Plasma Membrane TRIB3 5.87E-04 Nucleus TRIM16 3.94E-03 Cytoplasm TRIM21 3.41E-03 Nucleus TRIM22 2.87E-03 Cytoplasm TRIM25 2.90E-05 Cytoplasm TRIM27 4.18E-05 Nucleus TRIM28 8.90E-05 Nucleus TRIM32 5.43E-05 Nucleus TRIM35 3.62E-04 Cytoplasm TRIM39 2.48E-02 Cytoplasm TRIM69 9.05E-04 Nucleus TRIO 5.70E-04 Cytoplasm TRIOBP 1.60E-04 Nucleus TRIP11 1.05E-03 Cytoplasm TRNT1 5.35E-04 Cytoplasm TRPC1 9.07E-05 Plasma Membrane TRPC6 1.73E-02 Plasma Membrane TRPS1 8.59E-06 Nucleus TRPV1 3.20E-03 Plasma Membrane TSC1 3.67E-04 Cytoplasm TSC2 6.99E-03 Cytoplasm TSC22D1 8.01E-03 Nucleus TSC22D3 2.96E-08 Nucleus TSEN2 8.18E-06 Cytoplasm TSEN54 1.96E-03 Nucleus TSLP 7.49E-06 Extracellular Space TSPAN17 5.27E-03 Other TSPAN2 7.53E-03 Extracellular Space TSPAN5 3.40E-04 Plasma Membrane TSPAN6 1.86E-02 Plasma Membrane TSTA3 3.72E-03 Plasma Membrane TTF1 3.33E-05 Nucleus TTK 2.38E-06 Nucleus TTLL4 5.05E-04 Other TTN 3.80E-02 Other TUBA1A 3.91E-03 Cytoplasm TUBA1B 6.01E-03 Cytoplasm TUBA4A 1.18E-02 Cytoplasm TUBB 4.64E-03 Cytoplasm TUBB1 4.49E-02 Cytoplasm TUBB2A 1.82E-03 Cytoplasm TUBB2B 1.54E-03 Cytoplasm TUBB3 4.44E-04 Cytoplasm TUBB4B 2.03E-04 Cytoplasm TUBB6 2.81E-02 Cytoplasm TUBG1 2.35E-03 Cytoplasm TUSC2 5.99E-05 Nucleus TWF1 3.92E-04 Cytoplasm TWIST1 5.88E-03 Nucleus TWIST2 1.02E-02 Nucleus TWSG1 1.67E-02 Extracellular Space TXLNA 3.98E-03 Extracellular Space TXN2 5.64E-04 Cytoplasm TXNDC12 6.07E-08 Cytoplasm TXNDC5 9.68E-03 Cytoplasm TXNIP 4.64E-08 Cytoplasm TXNL4B 1.79E-02 Nucleus TXNRD1 2.29E-02 Cytoplasm TYK2 7.38E-03 Plasma Membrane TYMP 2.32E-03 Extracellular Space U2AF1 1.39E-04 Nucleus U2AF2 1.55E-05 Nucleus U2SURP 5.55E-05 Nucleus UBA1 4.02E-03 Cytoplasm UBA6 1.18E-03 Cytoplasm UBE2B 1.30E-02 Cytoplasm UBE2D2 3.03E-02 Cytoplasm UBE2D3 5.67E-04 Other UBE2D4 2.38E-02 Other UBE2E2 3.01E-02 Cytoplasm UBE2G1 2.99E-04 Cytoplasm UBE2G2 2.71E-02 Cytoplasm UBE2H 6.38E-04 Other UBE2I 4.75E-03 Nucleus UBE2J1 1.05E-04 Cytoplasm UBE2J2 2.52E-03 Cytoplasm UBE2K 9.37E-03 Cytoplasm UBE2L3 1.42E-02 Nucleus UBE2M 1.01E-02 Cytoplasm UBE2Q1 7.91E-03 Other UBE2R2 7.77E-03 Other UBE2S 3.12E-02 Nucleus UBE2V2 3.86E-03 Cytoplasm UBE3A 1.81E-05 Nucleus UBE3B 8.00E-03 Extracellular Space UBE4B 2.17E-03 Cytoplasm UBLCP1 2.46E-02 Nucleus UBR1 1.59E-04 Cytoplasm UBR2 3.05E-03 Nucleus UBR5 1.42E-02 Nucleus UBTF 4.85E-04 Nucleus UCHL1 1.16E-02 Cytoplasm UCHL5 2.04E-04 Cytoplasm UCK2 3.83E-02 Cytoplasm UCN2 3.95E-02 Extracellular Space UCP3 1.69E-02 Cytoplasm UGCG 1.90E-03 Cytoplasm UGP2 3.10E-03 Cytoplasm UGT1A1 2.82E-04 Cytoplasm UGT1A9 (includes others)5.37E-04 Cytoplasm UHMK1 3.10E-06 Nucleus UHRF2 3.81E-02 Nucleus UIMC1 4.33E-02 Nucleus UMPS 4.19E-07 Cytoplasm UNC119 2.11E-03 Cytoplasm UNC5CL 1.09E-02 Cytoplasm UNC93B1 4.07E-02 Cytoplasm UPB1 2.25E-03 Cytoplasm UQCR11 1.05E-02 Cytoplasm UQCRC2 7.42E-04 Cytoplasm URI1 3.85E-07 Nucleus UROD 7.58E-04 Cytoplasm UROS 1.52E-02 Cytoplasm USO1 3.35E-04 Cytoplasm USP1 3.39E-04 Cytoplasm USP10 1.51E-06 Cytoplasm USP12 3.80E-04 Cytoplasm USP14 2.29E-03 Cytoplasm USP15 1.54E-03 Cytoplasm USP19 4.36E-03 Cytoplasm USP2 1.27E-04 Cytoplasm USP20 2.60E-04 Cytoplasm USP22 1.23E-03 Nucleus USP24 2.10E-02 Other USP25 1.21E-05 Other USP27X 4.46E-04 Other USP28 6.32E-04 Nucleus USP29 3.09E-02 Other USP3 1.57E-03 Cytoplasm USP31 3.20E-04 Other USP32 1.00E-03 Cytoplasm USP34 3.78E-06 Extracellular Space USP36 6.58E-05 Nucleus USP37 1.64E-07 Nucleus USP38 1.11E-03 Other USP4 4.30E-02 Nucleus USP40 5.12E-04 Other USP42 3.82E-03 Other USP43 7.71E-03 Other USP46 3.66E-04 Other USP47 3.13E-04 Cytoplasm USP48 1.08E-03 Plasma Membrane USP53 2.18E-04 Extracellular Space USP54 2.24E-02 Other USP6 5.77E-03 Cytoplasm USP7 9.33E-05 Nucleus USP9X 1.04E-02 Plasma Membrane USP9Y 6.13E-03 Cytoplasm UST 4.19E-06 Cytoplasm UTP11L 1.44E-04 Nucleus UTP20 2.62E-03 Nucleus UTP6 2.74E-04 Nucleus UTRN 1.65E-02 Plasma Membrane UTS2 2.40E-02 Extracellular Space UTS2R 2.74E-02 Plasma Membrane UXT 1.89E-02 Cytoplasm VAMP3 7.27E-06 Plasma Membrane VAMP8 4.87E-02 Plasma Membrane VAPA 4.54E-02 Plasma Membrane VAPB 1.10E-03 Plasma Membrane VARS 7.11E-05 Cytoplasm VASP 9.63E-06 Plasma Membrane VAV3 2.88E-06 Extracellular Space VCAN 5.34E-03 Extracellular Space VCL 7.48E-03 Plasma Membrane VCP 3.13E-05 Cytoplasm VDR 1.15E-05 Nucleus VEGFA 1.21E-07 Extracellular Space VEGFB 3.58E-02 Extracellular Space VHL 2.09E-04 Nucleus VIM 2.00E-07 Cytoplasm VIMP 3.12E-02 Cytoplasm VIPR2 3.66E-02 Plasma Membrane VLDLR 2.88E-08 Plasma Membrane VNN1 8.33E-05 Plasma Membrane VPS13A 5.80E-04 Cytoplasm VPS25 3.24E-02 Cytoplasm VPS33B 3.08E-02 Cytoplasm VPS37B 9.28E-05 Cytoplasm VPS4A 5.71E-03 Cytoplasm VRK2 1.41E-03 Nucleus VRK3 1.75E-04 Nucleus VTA1 9.02E-04 Cytoplasm VTI1A 1.23E-02 Plasma Membrane VTI1B 1.32E-02 Plasma Membrane WAPAL 4.84E-02 Nucleus WARS2 2.21E-02 Cytoplasm WASF1 2.64E-03 Nucleus WASF3 1.55E-02 Cytoplasm WBP5 7.71E-03 Other WDFY3 3.75E-03 Cytoplasm WDR12 2.79E-04 Cytoplasm WDR33 2.84E-03 Nucleus WDR6 1.25E-04 Cytoplasm WDR91 1.26E-02 Other WEE1 1.04E-05 Nucleus WIF1 8.47E-03 Extracellular Space WIPF1 1.32E-03 Cytoplasm WISP1 1.52E-03 Extracellular Space WISP2 1.17E-02 Extracellular Space WLS 3.94E-05 Cytoplasm WNK1 5.14E-05 Cytoplasm WNK2 1.12E-02 Cytoplasm WNK3 9.46E-05 Plasma Membrane WNK4 7.70E-03 Plasma Membrane WNT10A 3.88E-05 Extracellular Space WNT2B 8.60E-03 Extracellular Space WNT3 1.38E-04 Extracellular Space WNT4 1.12E-02 Extracellular Space WNT5A 3.25E-02 Extracellular Space WNT5B 3.26E-02 Extracellular Space WNT7A 8.48E-03 Extracellular Space WRN 2.38E-03 Nucleus WTAP 8.23E-07 Nucleus WWTR1 2.22E-04 Nucleus XAF1 9.61E-06 Nucleus XBP1 4.51E-05 Nucleus XCL1 2.42E-02 Extracellular Space XCL2 3.62E-02 Extracellular Space XPC 4.31E-03 Nucleus XPO1 5.88E-08 Nucleus XRCC1 1.09E-02 Nucleus XRCC4 6.86E-03 Nucleus XRCC5 3.39E-07 Nucleus XYLT1 1.62E-05 Cytoplasm YAP1 6.61E-03 Nucleus YARS 1.62E-04 Cytoplasm YBX1 1.73E-02 Nucleus YES1 1.27E-02 Cytoplasm YKT6 2.83E-02 Cytoplasm YME1L1 6.08E-03 Cytoplasm YRDC 1.18E-03 Other YTHDF2 3.08E-07 Other YWHAB 2.21E-03 Cytoplasm YWHAE 2.51E-04 Cytoplasm YWHAH 3.35E-06 Cytoplasm YWHAZ 2.53E-02 Cytoplasm YY1 1.59E-03 Nucleus YY1AP1 3.06E-03 Nucleus ZAK 1.73E-04 Cytoplasm ZBED1 2.02E-05 Nucleus ZBED2 7.13E-05 Other ZBTB11 5.87E-03 Nucleus ZBTB16 4.93E-05 Nucleus ZBTB40 1.77E-03 Nucleus ZBTB5 3.25E-04 Nucleus ZC3H12A 1.91E-02 Cytoplasm ZC3H12D 2.19E-02 Cytoplasm ZC3HAV1 2.66E-06 Plasma Membrane ZCCHC2 2.99E-03 Cytoplasm ZDHHC17 2.96E-03 Cytoplasm ZEB1 2.01E-03 Nucleus ZFHX3 7.95E-03 Nucleus ZFP36 1.28E-05 Nucleus ZFP36L1 1.42E-06 Nucleus ZFP36L2 1.70E-06 Nucleus ZFYVE9 2.36E-02 Cytoplasm ZHX2 2.00E-04 Nucleus ZMAT3 1.34E-05 Nucleus ZMIZ1 1.12E-06 Nucleus ZMYM2 3.08E-05 Nucleus ZMYND11 6.94E-04 Nucleus ZNF24 1.09E-03 Nucleus ZNF25 6.18E-03 Nucleus ZNF259 3.04E-02 Nucleus ZNF267 6.09E-04 Nucleus ZNF280C 1.26E-03 Nucleus ZNF318 9.65E-07 Nucleus ZNF330 1.81E-02 Nucleus ZNF346 3.24E-04 Nucleus ZNF365 2.14E-02 Cytoplasm ZNF443 9.39E-05 Nucleus ZNF512B 3.55E-06 Nucleus ZNF622 4.12E-03 Nucleus ZNF639 8.63E-03 Nucleus ZNF668 2.32E-03 Cytoplasm ZNF678 3.16E-02 Nucleus ZNFX1 1.03E-05 Nucleus ZW10 2.31E-02 Nucleus ZWINT 1.62E-02 Nucleus ZYX 5.73E-04 Plasma Membrane Genes are associated with proliferation Genes are associated with migration Genes in dataset Fold Change Genes in dataset Fold Change A2M 1.228 ABI3 1.070 AATF -1.211 ABL1 -1.208 ABI1 1.915 ACKR1 1.135 ABL1 -1.208 ACKR2 1.388 ABTB1 -1.096 ACKR3 3.021 ACE2 1.228 ACTN4 -1.410 ACHE 1.196 ACVRL1 1.168 ACPP 1.165 ADAM10 1.606 ACSL4 -1.523 ADAM17 -1.366 ACTB -1.085 ADARB1 1.134 ACTN1 -3.037 ADORA1 -1.138 ACTN4 -1.410 ADORA2A 1.637 ACVR1B 1.243 ADRA2A 1.073 ACVRL1 1.168 AGER 1.087 ADAM10 1.606 AIMP1 -1.141 ADAM12 1.187 AKT1 -1.482 ADAM15 -1.548 ALB 1.163 ADAM17 -1.366 ALCAM 1.832 ADAMTS8 -1.146 ALOX15B -1.543 ADAR -1.029 AMICA1 1.131 ADARB1 1.134 AMOT 1.029 ADCY3 -1.899 ANXA1 -5.807 ADD2 1.237 APC -3.473 ADIPOQ -1.248 APP -1.407 ADM 3.655 ASAP2 1.141 ADRA1A -1.165 BCAR1 -1.971 ADRA1B -1.066 BCR -1.627 ADRA2A 1.073 BMP10 1.084 AES -1.375 C1QBP -1.275 AGO2 -1.557 C3 1.535 AHCY -1.321 C5AR2 1.020 AIF1 1.091 CAMP 1.165 AK2 -1.434 CAST -1.558 AK4 1.509 CAV1 -1.175 AKAP13 1.907 CBL -1.438 AKR1C1/AKR1C21.597 CCL1 1.217 AKR1C3 1.542 CCL11 1.098 AKT1 -1.482 CCL16 -1.054 ALDH1A2 1.285 CCL17 1.142 ALK 1.200 CCL18 1.122 ALOX15B -1.543 CCL19 -1.080 ANAPC5 -3.888 CCL2 -1.035 ANG 4.200 CCL20 4.505 ANXA1 -5.807 CCL21 1.028 ANXA11 1.196 CCL22 -1.113 ANXA2 -1.257 CCL23 1.097 APC -3.473 CCL24 1.071 APCDD1 1.293 CCL25 -1.103 APOA1 -1.058 CCL26 1.099 APOE 1.503 CCL28 2.070 APPL1 -1.342 CCL3 1.029 APPL2 -1.616 CCL4 1.116 AR 1.199 CCL5 3.746 ARAF 1.269 CCL8 -1.082 AREG -14.959 CCR1 1.105 ARF6 -1.849 CCR3 1.069 ARHGEF1 -1.084 CCR4 1.157 ARHGEF2 -1.845 CCR5 1.060 ARL1 -1.210 CCR6 1.129 ARL3 1.191 CCR7 1.141 ARL6IP5 -1.705 CD151 1.017 ARRB2 1.022 CD2 1.196 ASB2 4.813 CD226 -1.089 ASGR1 1.477 CD28 1.261 ASH2L -1.176 CD4 1.212 ATF3 15.394 CD40 -1.293 ATF4 1.322 CD40LG 1.049 ATG7 -1.326 CD44 -2.722 ATM -1.319 CD47 2.614 ATP5A1 -1.175 CD48 1.159 ATP5B -1.225 CD58 1.221 ATP5F1 -1.218 CD81 -1.025 ATP5G1 -1.181 CD9 -2.398 ATP5G2 1.204 CD97 -1.545 ATP6V0D1 -1.651 CDH13 1.370 ATP6V0E1 -1.170 CDH5 -1.040 ATP6V0E2 -1.032 CDK1 1.143 ATP8A2 1.288 CEACAM1 -1.408 AXIN2 1.855 CHRD 1.030 B4GALT2 -1.373 CITED2 2.254 BAG1 1.627 CKLF 1.336 BAI1 -1.081 CLEC11A 1.459 BAMBI 1.249 CLIC4 1.589 BAP1 -2.378 CMKLR1 1.130 BATF 1.018 COL18A1 -1.303 BCAP31 -1.327 CORO1A -1.216 BCAR1 -1.971 CREB3 -1.312 BCAT1 -1.141 CRK -2.003 BCL2 1.419 CSF1 1.279 BCL2L1 -1.445 CSF2 4.348 BCL3 1.612 CSF2RA -1.098 BCL6 2.025 CSF3 1.042 BCR -1.627 CSF3R 1.061 BECN1 -2.300 CTNNB1 -2.310 BIK 1.865 CTSB -1.401 BIN1 1.247 CTSG -1.028 BIN3 -2.045 CTSZ -1.588 BIRC2 1.556 CTTN/Cortactin 3.00 BIRC5 -1.241 CX3CL1 1.112 BIRC6 -1.102 CXADR 2.186 BLM 1.091 CXCL10 4.427 BLOC1S2 -1.131 CXCL11 2.895 BLZF1 -1.341 CXCL12 1.110 BMI1 1.141 CXCL13 1.002 BMP1 -1.580 CXCL16 1.066 BMP10 1.084 CXCL2 19.899 BMP2 9.415 CXCL3 3.73 BMP4 -1.003 CXCL5 3.409 BMP5 1.127 CXCL6 2.750 BMPR2 -1.646 CXCL8 35.879 BNC1 -1.194 CXCR1 1.079 BRAF 2.039 CXCR2 10.18 BRAP 1.113 CXCR3 1.100 BRCA1 -1.174 CXCR4 1.991 BRCA2 -1.575 CYR61 -2.267 BRF1 -1.149 DAG1 -1.502 BRF2 -1.415 DDR2 7.217 BSG 1.499 DDX58 -1.601 BTC -1.192 DEFB1 -1.038 BTG1 2.935 DEFB4A 1.317 BTG3 2.596 DEK 1.207 BTLA 1.086 DLC1 2.072 BTN3A1 1.134 DOCK1 1.760 BUB1B 1.169 DPP4 1.567 C1QBP -1.275 DPYSL2 1.621 C9orf78 -1.141 EDN1 -1.716 CACNB3 1.196 EDN2 5.698 CACUL1 -2.177 EDN3 1.092 CALM1 -2.031 EDNRB 1.204 CALR -1.645 EGF 1.096 CAMKK2 1.413 EGFR 1.597 CAMP 1.165 ENG -1.099 CAPN1 -2.369 ENPP2 1.248 CAPNS1 -2.116 EPHA1 -1.697 CAPRIN2 -1.080 EPHA2 -1.290 CAPS -1.234 ERBB2 -1.466 CAPZA1 -2.106 EZR -1.484 CASC3 -1.429 F10 -1.018 CASP1 -1.456 F11R -1.339 CASP3 -1.243 F2 1.050 CASP9 -1.076 F2R -1.394 CAT -2.359 F2RL1 -1.168 CAV1 -1.175 F3 1.191 CBFA2T3 1.321 F7 1.091 CBX1 1.258 FAS 1.227 CBX7 1.039 FASLG 1.077 CCKAR -1.054 FCGR3A 1.214 CCKBR 1.129 FERMT3 1.149 CCL2 -1.035 FGFR1OP 1.292 CCL23 1.097 FIGF 1.171 CCL4 1.116 FN1 2.102 CCL5 3.746 FPR1 1.058 CCNA2 -1.113 FPR2 -1.072 CCND1 -2.258 FYN -1.590 CCND3 1.052 GDNF -1.090 CCNE1 -1.453 GH1 1.138 CCNG1 1.266 GNA11 -1.629 CCNI -1.268 GNA12 -1.144 CCNL2 -1.456 GNA13 1.523 CCR5 1.060 GNAI2 -1.464 CCT2 -3.221 GNAO1 1.115 CCT3 -1.060 GNAS -1.635 CCT5 -1.273 GNAZ 1.013 CCT7 -1.494 GNB2L1 1.290 CD14 1.252 GRP 1.096 CD160 1.031 GTPBP4 -2.138 CD164 1.131 HARS -1.581 CD19 -1.100 HBEGF -1.563 CD1D 1.352 HGF 1.101 CD2 1.196 HLA-G 1.275 CD209 1.089 HMMR 1.559 CD24 1.105 HMOX1 -1.574 CD244 -1.117 HOXA7 -1.413 CD247 1.274 HRAS -1.026 CD248 1.191 HSPB1 -1.307 CD27 -1.155 HSPD1 -4.066 CD274 1.162 ICAM1 2.623 CD276 -1.243 ICAM2 -1.119 CD28 1.261 ICOS 1.118 CD33 1.125 ID2 -1.560 CD37 -1.049 IFNG 1.048 CD38 1.227 IGF1 1.213 CD3E 1.205 IGF1R 1.296 CD4 1.212 IGF2 1.221 CD40 -1.293 IGFBP3 4.55 CD40LG 1.049 IGHG1 1.495 CD44 -2.722 IL10 1.059 CD46 1.183 IL11 1.327 CD47 2.614 IL13 -1.123 CD55 2.896 IL16 1.307 CD6 1.238 IL18 -1.034 CD63 -1.173 IL1B -1.290 CD70 1.190 IL2 -1.045 CD74 1.206 IL21R -1.116 CD80 1.137 IL3 -1.097 CD81 -1.025 IL33 1.133 CD82 -2.050 IL4 1.138 CD83 1.549 IL5 -1.054 CD84 1.180 IL6R -1.626 CD86 2.053 IL7 1.070 CD8A 1.216 IL9 1.039 CDA -1.309 ILK -1.188 CDC123 -1.540 INPP5D -1.607 CDC14A -1.415 INS -1.320 CDC16 1.219 INSR 1.736 CDC25A -3.518 IQGAP1 -1.531 CDC25B -1.595 ITGA2 -1.416 CDC25C 1.413 ITGA3 -2.133 CDC27 -1.611 ITGA4 -1.211 CDC45 -1.158 ITGA5 1.236 CDC6 -1.453 ITGA6 -2.342 CDC7 1.114 ITGAL 1.101 CDC73 1.186 ITGAM 1.580 CDCA7L 1.065 ITGB1 -1.744 CDH1 -1.601 ITGB2 1.192 CDH13 1.370 ITGB3 1.290 CDH23 1.090 ITGB8 1.616 CDH5 -1.040 JAK2 1.812 CDHR2 1.043 JAM3 -1.108 CDK10 -1.993 KANK1 1.818 CDK13 -1.590 KCNMA1 1.189 CDK2 1.228 KDR -1.004 CDK3 1.283 KITLG -1.172 CDK4 -1.466 KRAS 1.382 CDK5 1.045 L1CAM 1.092 CDK5R1 1.122 LAMA3 1.261 CDK6 -2.171 LAMB1 -1.327 CDK7 -1.104 LASP1 -1.275 CDK9 -2.264 LEF1 1.857 CDKN1A -1.047 LEP 9.667 CDKN1B 4.629 LGALS3 1.113 CDKN2A 1.218 LGMN 1.102 CDKN2AIP -1.300 LSP1 1.174 CDKN2B 1.322 LYN 2.232 CDKN2C 1.729 MAP3K13 3.193 CDKN2D 1.220 MET 1.368 CDKN3 -1.097 MGAT3 1.076 CEACAM1 -1.408 MIA3 1.216 CEBPA -1.567 MINK1 1.063 CEBPB 1.069 MMP14 -2.068 CER1 -1.051 MMP2 1.317 CGRRF1 -1.082 MMP7 1.136 CHEK1 1.454 MMP9 1.161 CHERP -1.580 MST1R -1.742 CHMP1A -1.855 MYLK -1.306 CHRD 1.030 NARS -1.109 CHRM3 1.265 NCKAP1L 1.074 CHRM5 -1.038 NEDD9 4.979 CHRNA10 1.105 NEXN 1.362 CHRNA7 1.161 NF1 -3.270 CIAO1 -1.363 NF2 -2.840 CIRBP -2.628 NKX2-1 -1.208 CITED2 2.254 NPM1 1.180 CKLF 1.336 NRTN 1.005 CKS1B 1.233 PAK1 1.305 CKS2 1.633 PAK4 -1.084 CLCF1 -1.272 PARD6B 4.022 CLCN7 -1.211 PARP9 -1.307 CLEC11A 1.459 PCSK4 1.160 CLECL1 1.237 PDGFA -1.544 CLK1 1.614 PDGFB 1.394 CLK2 1.033 PDGFRA 1.218 CLOCK -1.382 PDGFRB 1.141 CLSPN -1.204 PECAM1 -1.087 CNKSR1 -1.074 PENK 1.535 CNOT7 -2.269 PF4 -1.001 CNOT8 3.758 PGF 4.404 CNTFR -1.077 PIGR 1.185 COL18A1 -1.303 PIK3C2B 1.148 COL2A1 1.186 PIK3CD -1.207 COL4A3 1.219 PIK3CG -1.042 COL6A1 1.160 PIP5K1C -1.191 COL6A2 -1.101 PLAU -2.044 COPE -1.761 PLAUR 2.111 CORO1A -1.216 PLCB2 -1.185 COX17 1.323 PLXND1 1.139 CPSF4 -1.170 PPAP2B -1.400 CR1 1.244 PPARA 1.732 CR2 1.040 PPARG 1.173 CREB1 1.758 PRKCA 2.558 CREB3 -1.312 PRKCB 1.264 CREG1 -1.090 PRKCE 2.085 CREM 1.263 PRKCZ -1.049 CRH 1.105 PRKX -1.087 CRIP1 -1.151 PROC -1.065 CRLF3 -1.005 PTEN 1.809 CRTC2 -1.024 PTGDR 1.181 CSE1L -1.294 PTGDR2 1.151 CSF1 1.279 PTK2 1.720 CSF1R 1.087 PTK2B 1.109 CSF2 4.348 PTPN1 -1.813 CSF2RA -1.098 PTPRJ 1.270 CSF2RB 1.356 PTPRK 2.054 CSF3 1.042 PTPRU -1.102 CSF3R 1.061 PTPRZ1 1.402 CSK -1.516 RAC1 -2.288 CSNK1D -2.032 RAC2 1.190 CSNK1E 1.212 RALGDS 1.915 CSNK2B -1.091 RARRES2 1.073 CSPG4 -1.065 RASGRP1 1.878 CTBP1 -1.315 RET 2.412 CTBP2 -1.185 RGS1 1.101 CTLA4 -1.220 RHOA -1.790 CTNNB1 -2.310 RNF20 -1.273 CTNNBIP1 1.028 RNF41 -1.324 CTSB -1.401 ROCK1 -1.324 CTSC -1.459 ROR2 1.217 CTSD -1.378 RTN4 1.270 CTSL -1.050 S100A12 1.012 CTSZ -1.588 S100A8 -1.080 CUL1 -1.666 S100A9 1.032 CUL2 1.152 S1PR1 -1.311 CUL3 -3.046 S1PR2 1.052 CUL4A -1.229 S1PR3 1.782 CUL5 -3.276 SBDS -1.182 CXCL1 4.871 SCAI 1.402 CXCL10 4.427 SCG2 1.357 CXCL12 1.110 SDCBP 1.451 CXCL16 1.066 SELE 1.115 CXCL2 19.899 SELL 1.128 CXCL5 3.409 SELP 1.235 CXCL8 35.879 SELPLG 1.810 CXCR9 -1.176 SEMA4A 3.015 CXCR3 1.100 SEMA4D 1.677 CYP19A1 1.127 SERPINB3 -1.250 CYP20A1 -1.074 SERPINB5 -1.990 CYP27B1 -1.226 SERPINC1 1.514 CYR61 -2.267 SERPINE1 2.287 CYSLTR1 1.187 SFRP1 -2.067 CYSLTR2 1.098 SFRP2 1.054 DAB2 1.112 SGPP1 1.159 DACT3 1.090 SIRPA -1.321 DAP -1.227 SLIT2 1.233 DAP3 -1.393 SLPI 1.091 DAXX -1.278 SMAD7 2.117 DBF4 -1.289 SMPD1 -1.134 DBF4B -1.288 SMPD2 1.052 DBN1 -1.618 SNAI2 -1.270 DCBLD2 -1.899 SOCS3 3.143 DCN 1.338 SPAG9 1.203 DCTN2 -1.136 SPATA13 1.928 DCUN1D3 -1.221 SPHK1 -1.784 DDB1 -1.472 SPP1 1.350 DDIT3 2.518 SPRY2 -2.349 DDR1 -1.048 SRC -1.721 DDX11 -1.830 SRCIN1 -1.052 DDX3X -2.230 STAB1 1.152 DDX56 -1.519 STK24 3.582 DERL2 -1.118 STK25 -1.345 DES 1.154 SYK -1.617 DFNA5 -1.021 TAC1 1.163 DGKA -2.184 TBX5 1.180 DHPS 1.191 TDP2 -1.249 DIRAS1 1.093 TGFB1 -1.137 DKC1 -2.794 TGFB2 1.855 DKK3 1.218 TGFBR1 1.570 DLC1 2.072 TGM2 1.314 DLG5 -2.592 THBS1 -2.556 DLK1 1.230 THY1 1.185 DLL4 -1.020 TIMP1 1.027 DNAJA1 -1.481 TIMP2 -1.069 DNAJA2 -1.495 TLR4 1.209 DNAJA3 -1.212 TNF 10.058 DNAJB1 1.898 TNFRSF1A -1.331 DNAJB2 -1.089 TNFRSF1B 1.025 DNMT3B 1.074 TNFSF18 5.802 DPAGT1 1.063 TNFSF14 1.101 DPP4 1.567 TNS1 3.205 DSP 1.046 TPM1 2.250 DTNB 1.083 TPM3 -1.560 DTYMK -1.220 TRIM32 -1.864 DUS2 -1.438 TRIP6 -1.616 DUSP22 1.170 TSC1 1.416 DUSP6 -2.561 VCAM1 1.139 DUSP9 1.163 VCAN 1.411 E2F1 -1.036 VCL -1.436 E2F4 -1.075 VEGFA 3.05 E4F1 -1.462 VEGFB 1.236 EAPP -1.291 VEGFC 1.151 EBI3 -1.080 VHL -2.020 EBNA1BP2 -1.443 VIL1 1.130 ECM1 1.217 WARS -1.205 EDN1 -1.716 WAS 1.064 EDN2 5.698 WNT5A 1.195 EED 2.836 WNT5B -1.569 EFNA1 1.763 XCL1 1.320 EGF 1.096 XCL2 1.253 EGFR 1.597 ZAP70 -1.074 EGR1 1.582 ZBTB16 3.072 EGR2 1.014 EGR4 1.340 EHF 2.333 EI24 -1.457 EID2 -1.072 EIF2AK2 -1.359 EIF2AK4 -1.108 EIF2B2 -1.274 EIF3I -1.239 EIF4A1 -1.956 EIF4B -1.329 EIF4EBP1 1.425 EIF4G1 -3.537 EIF5A -4.058 EIF5A2 -1.256 ELF1 -1.520 ELF3 1.644 ELF4 -1.123 ELK1 1.190 ELL -1.509 ELN -1.201 EMP1 -2.005 EMP2 1.298 EMP3 1.047 ENG -1.099 ENO1 -1.974 ENPEP 1.180 ENPP1 1.184 ENPP7 1.252 EPCAM 1.235 EPHA5 1.067 EPHB3 -1.050 EPHB4 -1.445 EPHB6 -1.271 EPO 1.063 EPS15 1.144 EPS15L1 -1.314 EPS8 1.275 ERBB2 -1.466 ERBB3 1.316 ERBB4 1.422 ERCC3 -1.601 EREG -2.026 ERG 1.330 ESR1 1.699 ETFB -1.231 ETFDH -1.216 ETS1 3.548 EVI5 -1.645 EXOSC2 -1.647 EXOSC4 -1.473 EZH2 1.276 EZR -1.484 F2 1.050 F2R -1.394 F2RL1 -1.168 FABP3 1.256 FABP6 1.658 FABP7 1.273 FADD -2.336 FADS1 -1.526 FADS2 -1.287 FADS3 -1.423 FAS 1.227 FASLG 1.077 FBN1 1.465 FBN2 -2.981 FBP1 1.051 FBXW11 1.118 FCER2 -1.033 FES 1.082 FGF1 2.027 FGF10 1.014 FGF11 1.382 FGF19 1.054 FGF2 1.240 FGF3 1.113 FGF4 1.110 FGF5 -1.437 FGF7 10.898 FGF8 1.127 FGF9 1.189 FGFBP1 -1.431 FGFR1 1.704 FGFR1OP 1.292 FGFR2 9.842 FGFR3 -1.132 FGFR4 1.191 FHIT 1.241 FKBP4 -1.469 FKTN 1.246 FLOT1 1.152 FLOT2 -1.583 FLT3 -1.009 FLT4 1.105 FN1 2.102 FOSL1 -1.804 FOXM1 -1.768 FOXO3 1.412 FOXO4 -1.178 FOXP3 -1.089 FRZB 19.924 FSCN1 -2.007 FST -2.049 FTH1 1.365 FTL -1.246 FURIN -1.312 FXN -1.182 GAB1 1.666 GAB2 -1.029 GABARAP -1.014 GADD45A 1.222 GAK -1.478 GAS2L1 -1.489 GAS6 -1.990 GATA1 1.074 GATA2 1.135 GDF15 -1.245 GDF2 -1.131 GDF9 1.639 GDNF -1.090 GFI1B 1.072 GFRA1 -1.029 GFRA2 -1.212 GGA1 -1.224 GGA2 -1.237 GGT5 1.186 GH1 1.138 GHRH 1.084 GHRHR 1.264 GHRL -1.017 GJA1 1.047 GJA5 -1.872 GJB2 1.189 GLA 1.232 GLDC -1.000 GLI1 -1.057 GLI2 -1.094 GLMN -1.138 GLP2R -1.040 GLTSCR2 1.355 GLUL -1.329 GNB2L1 1.290 GNG4 1.345 GNL3 -1.470 GNPAT 1.058 GNRH1 4.373 GOLGA2 -1.878 GOLPH3 -1.178 GPC3 -1.026 GPC4 1.172 GPER1 1.114 GPLD1 -1.259 GPNMB 4.047 GPR56 -1.117 GPX1 -1.342 GPX3 -1.550 GPX4 1.109 GRB2 -1.525 GREM1 1.061 GRIN2C 1.137 GRN -1.090 GRPR 1.043 GSK3B 1.085 GSN -1.940 GSS -1.446 GSTP1 -1.177 GTF2B -1.455 GTPBP1 -1.320 GTPBP3 -1.191 GTPBP4 -2.138 HADHA -1.446 HADHB 1.025 HAVCR2 1.370 HBEGF -1.563 HCK -1.084 HDAC1 -1.183 HDAC10 -1.346 HDAC2 1.062 HDAC3 1.313 HDAC4 1.274 HDAC5 -1.200 HDAC6 -1.065 HDGF -1.095 HES1 4.455 HES5 -4.109 HEXB 1.183 HFE -1.778 HGF 1.101 HGFAC 1.033 HGS -1.833 HHEX 1.400 HIF1A -1.035 HILPDA 20.863 HIPK2 -1.439 HK1 1.051 HLCS 1.081 HMGA1 -1.496 HMOX1 -1.574 HNF4A 1.154 HNRNPA0 -1.573 HNRNPA2B1 -3.092 HNRNPC -3.024 HNRNPD -1.625 HNRNPF -1.501 HNRNPK -1.220 HNRNPM -1.942 HNRNPR -1.751 HNRNPU -1.462 HOXA9 -1.274 HOXC10 1.754 HPN 1.242 HRAS -1.026 HRH2 -1.046 HSD11B2 1.093 HSPA5 -1.403 HSPD1 -4.066 HSPG2 -1.642 HTR1A -1.153 HTR2B 1.113 HTR4 1.281 HYAL1 1.113 HYAL2 -1.316 ICAM1 2.623 ICAM3 1.142 ICOS 1.118 ICOSLG 1.466 ID1 -4.928 ID2 -1.560 ID3 -5.454 IDO1 1.015 IFI30 1.057 IFIT3 -1.297 IFNG 1.048 IFNL1 1.331 IGF1 1.213 IGF1R 1.296 IGF2 1.221 IGF2R -3.546 IGFBP2 -1.075 IGFBP3 3.418 IGFBP4 1.216 IGFBP5 2.208 IGFBP6 1.161 IGFBP7 1.674 IGHG1 1.495 IGHM 1.267 IKBKB -1.175 IKBKG -1.158 IL10 1.059 IL10RA -1.145 IL11 1.327 IL12B 1.171 IL12RB1 1.075 IL12RB2 -1.238 IL13 -1.123 IL13RA2 1.058 IL15 3.726 IL15RA 1.201 IL17C -1.042 IL17RA -1.256 IL18 -1.034 IL1A 2.315 IL1B -1.290 IL1R1 1.141 IL1RL1 1.208 IL2 -1.045 IL21 1.102 IL22 1.152 IL23A 1.318 IL23R 1.169 IL24 -1.067 IL27 1.032 IL2RA -1.076 IL2RB 1.076 IL3 -1.097 IL31RA 1.213 IL4 1.138 IL4R -1.854 IL5 -1.054 IL5RA -1.146 IL6 2.154 IL6R -1.626 IL6ST 1.647 IL7 1.070 IL7R 1.130 IL9 1.039 IL9R -1.159 ILF3 -2.575 ILK -1.188 ING1 3.104 ING2 1.509 ING4 1.333 ING5 -1.217 INHBA 5.510 INHBB 1.284 INS -1.320 INSIG1 7.474 INSR 1.736 IP6K2 1.442 IRF1 3.274 IRF2 2.444 IRF3 -1.447 IRF6 -1.482 IRF8 1.149 IRS1 -2.248 ISG20 1.616 ITGA2B -1.339 ITGA3 -2.133 ITGA5 1.236 ITGA6 -2.342 ITGA7 1.173 ITGAV 1.060 ITGB1 -1.744 ITGB1BP1 1.349 ITGB3 1.290 ITGB4 -2.413 ITPKC 1.214 JAG1 -4.280 JAG2 -1.358 JUN 4.707 JUNB 1.538 JUND 2.292 KAT2B 1.305 KCNA3 1.097 KCNK3 1.236 KCNMA1 1.189 KCNN4 1.001 KCTD11 2.504 KDM4C 4.011 KDR -1.004 KHDRBS1 3.080 KIF11 1.198 KIF15 1.856 KIF2C -1.250 KIR2DS4 -1.119 KIT -1.012 KITLG -1.172 KLC2 -1.103 KLF10 -1.337 KLF11 3.446 KLF4 1.480 KLF5 1.172 KLF6 -2.184 KLK3 1.174 KLK8 1.310 KMT2D -1.497 KRAS 1.382 LAIR1 1.120 LAMA2 1.074 LAMA3 1.261 LAMB1 -1.327 LAMC1 -1.189 LAMP3 1.422 LCK -1.047 LDOC1 -1.476 LEF1 1.857 LEP 9.567 LGALS1 1.208 LGALS12 1.216 LGALS3 1.113 LGALS7 1.088 LGI1 1.130 LIF 1.207 LIFR 1.478 LILRB1 1.075 LILRB2 1.063 LILRB4 1.050 LIN28B 1.238 LITAF -1.440 LPAR2 -1.282 LRP5 1.152 LTA -1.019 LTBP3 -1.261 LTBP4 -1.211 LTBR -1.259 LTK 1.108 LY75 2.412 LY86 1.191 LYN 2.232 LZTS1 1.184 LZTS2 1.259 MAD2L2 -1.053 MAFF 5.506 MAGEA4 1.040 MAGED1 -1.100 MAGED2 1.090 MAP2K1 1.651 MAP2K2 -1.786 MAP2K7 -1.211 MAP3K1 -1.767 MAP3K11 -1.264 MAP4K4 -1.460 MAPK1 1.570 MAPK14 -1.411 MAPK3 -1.127 MAPRE1 -1.176 MATK 1.098 MBD1 -2.213 MBD3 -1.210 MBP -1.887 MCAM -1.505 MCL1 -1.432 MCM2 -1.488 MCM3 -1.369 MCM5 -1.509 MCM7 -1.312 MCTS1 1.234 MDM2 -2.130 MDM4 1.223 MECOM -1.413 MELK 1.269 MEN1 -1.627 MEST 1.090 MET 1.368 METAP2 -1.996 METTL3 -2.484 MGAT1 -1.102 MGAT3 1.076 MGAT4B -2.624 MGMT 1.280 MIA 1.079 MICA 2.032 MIF 1.289 MINA -2.162 mir-21 -3.656 MKI67 1.270 MLXIPL -1.002 MMP11 1.190 MMP15 -1.134 MMP2 1.317 MMP24 -1.259 MMP7 1.136 MMP9 1.161 MNAT1 1.282 MNT 1.186 MOG 1.122 MPL 1.207 MS4A1 1.576 MSH2 -1.079 MST1 1.383 MST1R -1.742 MSX1 1.309 MT2A 1.058 MT-ATP6 -3.184 MTCH1 1.076 MTCP1 1.609 MTHFD1 -1.129 MTOR -1.242 MTPN -1.212 MUL1 -1.204 MVP -1.773 MX1 -1.042 MXD1 4.526 MXD4 -1.310 MXI1 4.784 MYBBP1A -1.636 MYBL2 -1.094 MYC -3.753 MYCBP -1.260 MYD88 -1.463 MYOCD 1.087 N6AMT1 -1.219 NAB2 2.231 NACC1 -1.113 NAGA -1.096 NAMPT 12.139 NANOG 2.334 NAP1L1 1.260 NCK1 -1.215 NCK2 1.190 NCKAP1L 1.074 NCOR2 -1.359 NDN 1.129 NDUFA13 1.190 NDUFS3 -1.078 NDUFV1 -1.623 NEU1 -1.330 NEU4 1.071 NEURL1 1.082 NF2 -2.840 NFATC2 1.405 NFKB2 1.752 NFKBIA 6.428 NGF 1.179 NGFR -1.128 NKIRAS2 -1.448 NKX2-5 1.107 NKX3-1 -1.064 NME1 -1.204 NME6 -1.524 NOG 2.264 NOL8 -1.175 NOP2 -2.088 NOP58 -1.518 NOS2 -1.113 NOS3 1.313 NOTCH1 -1.123 NOTCH2 -1.413 NOX4 1.743 NPM1 1.180 NPY 1.113 NQO1 -1.281 NR1H2 -1.380 NR4A2 4.958 NR6A1 1.255 NRAS 1.084 NRD1 1.176 NRG1 -2.467 NUBP1 1.057 NUDC -1.332 NUMB 1.657 NUP62 -2.746 NUP98 -1.535 ODC1 -2.129 OGFR -1.178 OPTN 1.405 OSM 1.200 OSMR 1.874 PA2G4 -3.249 PARP1 -1.923 PAWR -1.508 PCDH1 -1.333 PCNA -1.476 PDAP1 -2.420 PDCD1 -1.034 PDCD10 -1.101 PDCD1LG2 -1.372 PDGFA -1.544 PDGFB 1.394 PDGFRA 1.218 PDGFRB 1.141 PDK1 8.738 PDS5B 1.174 PDXK -2.084 PDZK1 1.934 PECAM1 -1.087 PEG10 -1.248 PEMT 1.149 PER1 1.163 PES1 -1.373 PF4 -1.001 PFN2 1.037 PGF 4.404 PGK1 4.135 PHB -1.628 PHLDA1 -4.111 PHLDA2 -1.153 PIK3CA 1.200 PIK3CD -1.207 PIK3CG -1.042 PIM1 1.147 PIM2 1.012 PKD2 1.102 PKM -2.068 PKN1 -1.021 PKP1 -1.851 PKP3 -2.269 PLAT -1.277 PLAU -2.044 PLAUR 2.111 PLCD1 -1.130 PLCE1 1.169 PLD1 -2.823 PLEC -2.698 PLIN2 6.588 PLIN3 -1.317 PLK1 -1.329 PLK4 1.703 PML -1.743 PNP -2.223 POLA1 1.022 POLM -1.382 POLR2J -1.019 POR -1.784 POU3F2 1.013 PPARD 1.954 PPAT -2.002 PPM1D 1.852 PPM1G -1.371 PPP1CA -1.197 PPP1R1C 1.759 PPP1R9B -1.159 PPP2CA -1.521 PPP2R1A -1.600 PPP2R5C -6.552 PPP5C -1.445 PPT1 -1.152 PRAME -1.059 PRDM4 -1.406 PRDX3 1.125 PREB -1.616 PRG2 1.032 PRKAB1 -1.241 PRKCA 2.558 PRKCB 1.264 PRKCD -1.286 PRKCE 2.085 PRKCH 1.724 PRKCI -1.629 PRKCSH -1.180 PRKCZ -1.049 PRKD1 1.203 PRKRA 1.096 PRKRIR -1.164 PRL 1.094 PRMT5 -1.469 PROK2 -1.103 PROX1 1.248 PRPF19 -1.914 PRPF8 -1.584 PRSS2 1.076 PRTN3 1.123 PSAP -1.907 PSEN1 -1.360 PSMB10 1.146 PSMB2 1.579 PSMC1 -1.102 PSMC3 -2.520 PSMC4 -1.127 PSMC5 -1.150 PSMD10 -1.190 PSMD2 -1.395 PSME2 1.038 PSMF1 -1.397 PTEN 1.809 PTGS1 3.071 PTGS2 1.246 PTHLH -2.938 PTK2 1.720 PTK2B 1.109 PTN 1.191 PTPN11 -2.868 PTPN6 1.190 PTPRA 1.369 PTPRC 1.126 PTPRJ 1.270 PTPRK 2.054 PTPRU -1.102 PTTG1 1.574 PXDN 1.133 PYY 1.104 QSOX1 1.917 RAC1 -2.288 RAC2 1.190 RAD17 -1.176 RAF1 -1.800 RAP1GAP 1.049 RAPGEF3 -1.060 RARA -1.249 RARB -1.179 RARRES3 1.055 RASGRP4 -1.031 RB1 -1.553 RBBP4 -1.682 RBBP7 -1.115 RBFOX2 -1.893 RBL2 1.306 RBM3 -1.073 RBM38 -1.389 RBM5 -1.917 RBM6 -1.954 RBP3 -1.085 REG1B -1.007 REG3A -1.215 REL 1.348 RELA -1.264 RELB 1.358 RELT 1.234 RET 2.412 RGCC 1.222 RGL2 -1.009 RHBDF1 1.140 RHOA -1.790 RHOB 1.203 RHOG -1.450 RNF139 -1.244 RNF41 -1.324 ROMO1 1.295 ROR2 1.217 ROS1 1.047 RPS15A 1.436 RPS19 1.812 RPS6KA1 1.208 RPS6KA2 -1.157 RPS6KA3 1.268 RPS9 1.210 RRAD 3.318 RSL1D1 -2.019 RUVBL1 -2.326 RXRA -1.426 RXRB -1.100 S100B 1.202 S1PR2 1.052 S1PR3 1.782 SAE1 -1.401 SART1 -1.326 SART3 -2.219 SBDS -1.182 SCAF11 -6.876 SCAMP2 -1.557 SCAMP3 -1.054 SCAMP4 -1.271 SCIN 1.172 SCN5A 1.211 SCRIB -1.667 SDC2 1.341 SEC61A1 -1.399 SEMA6A 4.941 SERPINE1 2.287 SERPINE2 1.233 SERPINF1 1.467 SERPINF2 1.117 SERPINH1 -1.399 SERTAD1 1.499 SERTAD2 2.902 SERTAD3 -1.040 SESN1 -1.119 SET -3.920 SF3A3 -1.282 SF3B2 -1.472 SF3B3 -1.233 SFRP1 -2.067 SFRP2 1.054 SFRP4 1.196 SFTPD 1.046 SGMS1 1.194 SHC1 -1.749 SHH -1.046 SIPA1 -1.055 SIRPA -1.321 SIRPG 1.321 SIRT1 1.805 SKI -1.253 SKP1 1.237 SKP2 -1.850 SLAMF1 1.229 SLAMF7 -1.783 SLC12A4 -1.708 SLC22A1 1.177 SLC22A18 1.456 SLC25A5 -1.066 SLC26A3 -1.055 SLC2A8 -1.151 SLC35F6 -1.817 SLC3A2 1.076 SLC4A1 -1.058 SLC7A7 1.289 SLC9A3R1 -1.095 SLC9A3R2 1.090 SLIT2 1.233 SLIT3 1.606 SMAD2 1.521 SMAD3 2.610 SMAD4 -2.081 SMARCA2 -1.883 SMARCA4 -1.667 SMARCB1 1.217 SMO -1.046 SOCS1 1.518 SOCS3 3.143 SOD1 -1.034 SOD2 14.966 SOX11 1.270 SOX17 1.175 SOX4 1.857 SOX7 -5.028 SP1 1.255 SPARC -1.398 SPDYA 9.574 SPEG -1.449 SPHK1 -1.784 SPINT2 -1.116 SPN -1.490 SPP1 1.350 SPRY2 -2.349 SRA1 -1.473 SRC -1.721 SRF -1.524 SRPK2 1.525 SRSF2 -1.318 SSR1 -1.562 SST 1.172 SSTR2 1.236 SSTR3 1.112 SSTR4 -1.083 SSTR5 -1.203 ST3GAL2 -1.315 STAMBP 1.176 STAT3 -1.771 STAT5A 1.217 STAU1 1.188 STIL 1.168 STK11 -1.459 STRAP -3.066 STRN 2.546 SUPV3L1 -1.432 SURF1 -1.267 SURF4 -1.486 SURF6 -1.946 SYK -1.617 SYMPK -1.799 TACSTD2 1.324 TAF1D 1.830 TAF6 -1.006 TAF9 -1.149 TAF9B 14.297 TBC1D8 1.318 TBP 1.016 TBRG1 -1.494 TBRG4 -1.910 TBX3 1.283 TBX5 1.180 TCF19 1.432 TCF3 1.249 TCF7L2 1.502 TCHP -1.095 TCIRG1 1.064 TCL1A 1.131 TCN2 1.130 TERF2 -2.439 TERT -1.055 TES 1.341 TESC 1.044 TF -1.132 TFAP2A -1.616 TFAP2B 1.145 TFAP4 1.043 TFDP1 -1.207 TFF3 1.060 TFG -1.219 TFPI2 -1.385 TGFA -1.208 TGFB1 -1.137 TGFB1I1 1.344 TGFB2 1.855 TGFB3 1.050 TGFBI -1.118 TGFBR1 1.570 TGFBR2 -1.783 TGFBR3 1.934 THBS1 -2.556 THEM4 -1.752 THPO 1.104 TIGIT 1.036 TIMELESS -1.123 TIMP1 1.027 TIMP2 -1.069 TIMP3 1.378 TJAP1 1.032 TJP1 1.799 TJP3 -1.125 TLR9 -1.047 TMSB10/TMSB4X1.131 TNF 16.028 TNFRSF10B 1.168 TNFRSF11A 1.999 TNFRSF12A -1.611 TNFRSF14 1.008 TNFRSF17 -1.046 TNFRSF1A -1.331 TNFRSF1B 1.025 TNFRSF21 -3.723 TNFRSF25 -1.545 TNFRSF4 1.082 TNFRSF6B -1.268 TNFRSF8 1.087 TNFRSF9 1.099 TNFSF10 1.917 TNFSF12 1.180 TNFSF13 1.094 TNFSF13B 2.440 TNFSF14 1.101 TNFSF4 1.472 TNFSF8 1.263 TNFSF9 1.291 TNKS 1.731 TOB1 -1.928 TOB2 -2.058 TOE1 -1.719 TOP3B -1.194 TP53 -1.021 TP53BP1 -1.488 TP53I11 1.145 TP63 -2.208 TP73 1.080 TPD52L2 -2.079 TPX2 -1.091 TRAF2 1.058 TRAIP 1.023 TRAP1 -1.311 TRIM27 -2.370 TRIM28 -1.933 TRIM32 -1.864 TSC1 1.416 TSHR -1.116 TSLP 2.749 TSPAN31 1.090 TSPO -1.071 TSPY1 (includes others)1.135 TSPYL2 1.065 TTK 1.976 TUSC2 -1.664 TXLNA -1.828 TXN -1.090 TXNL1 1.112 UBA1 -2.132 UBC 1.059 UBE2A 1.099 UBE2E3 1.143 UBE2I -1.379 UBE2L3 -1.782 UBE2L6 -1.199 UBE2V2 1.396 UHMK1 -2.580 ULBP1 1.038 ULBP2 -1.389 UMOD 1.072 UNC119 1.322 USP28 1.555 USP3 1.398 USP4 -1.366 USP47 -1.521 USP7 -1.494 USP8 -1.187 USP9X -1.224 UTP20 -1.442 UTP6 -1.440 VAMP8 -1.244 VAV3 2.314 VCAM1 1.139 VCAN 1.411 VDR -2.421 VEGFA 3.05 VEGFB 1.236 VEGFC 1.151 VHL -2.020 VIP 1.116 VIPR1 1.002 VPS28 1.041 VPS33B -1.194 VRK3 -1.623 VSIG4 1.028 VTI1B 1.276 VTN 1.234 WARS -1.205 WAS 1.064 WDR12 -1.607 WDR6 -1.889 WEE1 1.831 WNK2 1.232 WNT1 1.140 WNT2 1.052 WNT5A 1.195 WNT9A -1.102 WT1 1.095 WWTR1 -1.527 XBP1 -1.948 XCL1 1.320 XIAP -1.250 XPC -1.320 XRCC1 -1.244 XRCC5 -4.802 YAP1 1.259 YME1L1 1.366 YTHDF2 -2.526 ZAP70 -1.074 ZBED1 -2.911 ZBTB17 1.100 ZC3H12D 1.279 ZEB1 1.681 ZFHX3 -1.229 ZFP36L2 -3.370 ZMAT3 -2.417 ZMYM2 1.647 ZMYND11 -1.574 ZNF148 1.379 ZNF639 -1.227 ZP4 1.108 ZPR1 -1.574 ZYX -1.759