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The Pennsylvania State University

The Graduate School

Physiology Graduate Program

IMMUNOMODULATORY EFFECTS OF ISOTRETINOIN IN VIVO

A Dissertation in

Physiology

Melanie Claire Dispenza

2011 Melanie Claire Dispenza

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2011

The dissertation of Melanie Claire Dispenza was reviewed and approved* by the following:

Diane M. Thiboutot Professor of Dermatology Dissertation Advisor Chair of Committee

Robert H. Bonneau Professor of Microbiology and Immunology and Pediatrics

Charles H. Lang Distinguished Professor of Cellular and Molecular Physiology Program Director for Physiology

Gary A. Clawson Professor of Pathology

Stephen R. Rannels Associate Professor of Cellular and Molecular Physiology

*Signatures are on file in the Graduate School

ii ABSTRACT

Acne is a common human ailment that can cause significant physical and psychological morbidity. Though it is thought to be partially caused by Propionibacterium acnes colonization, studies suggest that patients‟ specific immune responses to P.acnes may play a larger role in acne than the pathogenicity of P.acnes itself. Isotretinoin, a pro-drug for retinoic acid, is the only agent that induces a permanent remission of acne, but the mechanism underlying its long-term efficacy is unknown. We hypothesized that modulation of the immune response to P.acnes is key to isotretinoin‟s ability to induce long-term or permanent remissions of acne. In this Thesis, we show that acne patients‟ monocytes express higher levels of TLR-2 and secrete more IL-1β, IL-6, IL-8, IL-10, and IL-12 in response to P. acnes than monocytes from normal volunteers. Isotretinoin therapy significantly decreases TLR-2 expression and inflammatory cytokine secretion by acne patients‟ monocytes compared to baseline. These changes continue through 20 weeks of isotretinoin therapy and persist for at least six months after the cessation of therapy, indicating that modification of patients‟ immune responses to P. acnes may represent a long-term mechanism by which isotretinoin can cure acne. Because the exogenous administration of retinoids causes severe side-effects including birth defects, studies characterizing the action of isotretinoin in humans in vivo are lacking. This is the first study to show that isotretinoin has immunomodulatory effects in acne patients in vivo. Findings from this study extend to other diseases for which isotretinoin is used as a therapy as well as inflammatory disorders characterized by TLR-2 over-expression.

iii TABLE OF CONTENTS

List of Figures ...... vii

List of Tables ...... xiv

List of Abbreviations ...... xv

Acknowledgements ...... xvii

Chapter 1: Literature Review ...... 1

1.1 Introduction ...... 1 1.2 Skin ...... 2 1.2.1 Structure of the skin ...... 2 1.2.1.1 Epidermis ...... 2 1.2.1.2 Dermis and hypodermis ...... 3 1.2.1.3 Sebaceous glands ...... 4 1.2.2 Immune function of the skin ...... 5 1.2.2.1 Innate immunity in the skin ...... 6 1.2.2.2 Antigen presenting and phagocytic cells...... 10 1.2.2.3 T cells ...... 12 1.2.2.4 Regulatory T cells ...... 14 1.3 Acne ...... 16 1.3.1 Epidemiology ...... 16 1.3.2 Pathophysiology...... 17 1.3.2.1 Follicular hyperkeratinization ...... 17 1.3.2.2 Excess sebum production ...... 18 1.3.2.3 P.acnes and inflammation ...... 19 1.3.3 Current treatments for acne ...... 22 1.3.3.1 Topical treatments ...... 22 1.3.3.2 Systemic treatments ...... 23 1.3.3.3 Isotretinoin ...... 24 1.3.4 Model systems for acne research ...... 25 1.3.4.1 Cell lines ...... 25 1.3.4.2 Animal models ...... 26 1.3.4.3 Human subjects ...... 27 1.4 Retinoids ...... 27 1.4.1 Retinoid biology, function, and metabolism ...... 27 1.4.2 Retinoid effects on the immune system ...... 33 1.4.2 Mechansims of retinoids in acne therapy...... 37 1.5 Significance of research project ...... 40

Chapter 2: Systemic Isotretinoin Treatment Modulates Acne Patients’ Immune Response to P. acnes ...... 42

2.1 Chapter abstract ...... 42 2.2 Introduction ...... 42 2.3 Results ...... 43 2.3.1 Subject demographics ...... 43

iv 2.3.2 All enrolled patients responded clinically to isotretinoin therapy ...... 45 2.3.3 Serum concentrations of retinoids do not differ between acne patients and normal volunteers ...... 46 2.3.4 Acne patients have higher serum concentrations of IL-10, but not inflammatory cytokines, compared to healthy volunteers ...... 46 2.3.5 Isotretinoin therapy down-regulates surface expression of TLR-2 on acne patients‟ monocytes in vivo ...... 49 2.3.6 TLR-2 expression on peripheral blood monocytes does not correlate with age ...... 51 2.3.7 Acne patients‟ monocytes express lower levels of TLR-4 than normal volunteers‟...... 53 2.3.8 Isotretinoin therapy decreases inflammatory cytokine production by acne patients‟ monocytes in response to P. acnes...... 54 2.3.9 Isotretinoin therapy does not affect TLR-2 expression in the epidermis of acne patients as determined by immunohistochemistry ...... 56 2.3.10 Isotretinoin therapy does not affect proportions of Treg in peripheral blood ...... 59 2.3.11 Lymphocyte proliferation in response to P. acnes is blunted in acne patients compared to normal volunteers ...... 61 2.3.12 Isotretinoin therapy does not affect peripheral blood lymphocyte cytokine secretion ...... 66 2.4 Discussion ...... 66 2.5 Methods ...... 71 2.5.1 Human subjects ...... 71 2.5.2 P. acnes cultures ...... 72 2.5.3 Cell culture ...... 72 2.5.4 Antibodies and flow cytometry ...... 73 2.5.5 Cytokine detection using bead arrays ...... 74 2.5.6 Lymphocyte proliferation assay ...... 74 2.5.7 High-performance liquid chromatography and mass spectrometry ...... 75 2.5.8 Human skin biopsies and immunohistochemistry ...... 76 2.5.9 Statistical analysis of the data ...... 77

Chapter 3: Development and Characterization of a Primary Sebocyte Cell Line (pSEB) ...... 78

3.1 Chapter abstract ...... 78 3.2 Introduction ...... 78 3.3 Results ...... 80 3.3.1 Methods for growing pSEB from human sebaceous glands ...... 80 3.3.2 pSEB can be successfully expanded with Y-27632 for over 30 passages ...... 80 3.3.3 pSEB grown with Y-27632 show characterisitics of primary sebocytes ...... 83 3.3.4 Gene array analysis of Y-27632‟s effects in pSEB ...... 85 3.4 Discussion ...... 89 3.5 Methods ...... 92 3.5.1 Sebaceous gland dissestion and cell culture ...... 92 3.5.2 Oil Red O staining ...... 92 3.5.3 Lipogenesis assays ...... 93 3.5.4 Gene chips ...... 93 3.5.5 BrdU incorporation assays ...... 94

Chapter 4: Discussion ...... 95

4.1 Introduction ...... 95

v 4.2 Rationale, hypothesis, and results of this work ...... 95 4.3 Explanation of model ...... 96 4.4 Future directions ...... 96 4.4.1 What factors are responsible for the higher TLR-2 expression in acne patients‟ monocytes? ...... 97 4.4.2 How do retinoids work to down-regulate TLR-2 expression? ...... 97 4.4.3 Does isotretinoin also affect leukocytes in the skin or other microenvironments? ... 98 4.4.4 What are the downstream consequences of TLR-2 down-regulation on monocytes? ...... 99 4.4.5 What factors determine if an acne patient will stay in remission with isotretinoin therapy? ...... 99 4.4.6 Could isotretinoin effectively treat other disorders characterized by TLR-2 over- expression? ...... 100 4.5 Conclusions ...... 101

Appendix A: Mouse Studies ...... 102

A.1 Chapter abstract ...... 102 A.2 Introduction...... 102 A.3 Results ...... 103 A.3.1 Live P. acnes subcutaneous injection successfully induces a cutaneous inflammatory response in C57BL/6 mice ...... 103 A.3.2 Prior exposure to P. acnes does not alter ear swelling in response to P. acnes challenge in C57BL/6 mice ...... 104 A.3.3 Prior intradermal injection with P. acnes does not alter the proportions of CD4+ or CD8+ T cells in ears or draining lymph nodes in response to P. acnes challenge in C57BL/6 mice ...... 104 A.3.4 Prior intradermal injection with P. acnes does not alter the proportions of T cell subsets or activation markers in ears or draining lymph nodes in response to P. acnes challenge in C57BL/6 mice ...... 108 A.4 Discussion ...... 108 A.5 Methods ...... 111 A.5.1 Mice ...... 111 A.5.2 Sensitization and challenge with P. acnes ...... 111 A.5.3 Tissue harvest and processing ...... 112 A.5.4 Cell culture ...... 113 A.5.5 Antibodies and flow cytometry ...... 113 A.5.6 Statistical analysis of the data ...... 114

Appendix B: The Function of NGAL’s Glycan...... 115

B.1 Chapter abstract ...... 115 B.2 Introduction ...... 115 B.3 Results ...... 119 B.3.1 NGAL runs as a double band in western blots ...... 119 B.3.2 Mammalian-expressed NGAL is glycosylated, while bacterially-expressed NGAL is not ...... 120 B.3.3 NGAL‟s glycan is successfully removed by PNGase F ...... 122 B.3.4 PNGase F removal of NGAL‟s glycan is confirmed by mass spectrometry ...... 123 B.3.5 Deglycosylated NGAL can be detected by western blot ...... 123 B.3.6 Deglycosylated NGAL cannot be purified from solutions using MagneHis beads. 124

vi B.3.7 NGAL can be successfully isolated from solution using Dynabeads ...... 125 B.3.8 NGAL cannot be detected with glycan staining of total cell lysates ...... 127 B.3.9 Tunicamycin can inhibit glycosylation of NGAL produced in SEB-1 ...... 127 B.3.10 NGAL‟s N-linked glycan is not essential for its secretion from SEB-1 ...... 129 B.3.11 Apoptosis studies with deglycosylated NGAL were inconclusive ...... 129 B.4 Discussion ...... 133 B.5 Methods ...... 137 B.5.1 SEB-1 culture and treatments ...... 137 B.5.2 Western blots ...... 137 B.5.3 Glycan staining of protein gels ...... 138 B.5.4 PNGase F treatment of NGAL ...... 138 B.5.5 Immunoprecipitation ...... 139 B.5.6 Cell death assays ...... 140 B.5.7 Mass spectrometry ...... 140 B.5.8 Annexin V/PI staining and flow cytometry ...... 140 B.5.9 TUNEL assays ...... 141

Appendix C: NF-κB Studies ...... 142

C.1 Chapter abstract ...... 142 C.2 Introduction ...... 142 C.3 Results ...... 144 C.3.1 The NF-κB pathway is intact in SEB-1 ...... 144 C.3.2 13-cis RA activates NF-κB in a delayed time-course in SEB-1 ...... 146 C.3.3 NF-κB stimuli induce NGAL expression in SEB-1 ...... 147 C.4 Discussion ...... 148 C.5 Methods ...... 149 C.5.1 SEB-1 cell culture and treatments ...... 149 C.5.2 p65 translocation assays ...... 150 C.5.3 Western blots ...... 150

References ...... 152

vii

LIST OF FIGURES

Chapter 1:

Figure 1.1: Layers of the skin. Skin has three main layers: 1) the epidermis, which primarily consists of keratinocytes and functions in physical protection; 2) the dermis, which contains the pilosebaceous unit, nerve endings, blood vessels, and sweat glands surrounded by collagen-rich connective tissue; and 3) the hypodermis, which is composed primarily of adipose tissue ...... 3

Figure 1.2: TLR signaling in cells. TLRs recognize antigen patterns that induce the transcription of antimicrobial peptides and inflammatory cytokines. Most TLRs signal through MyD88, converging on NF-κB and IRF nuclear transcription factors...... 8

Figure 1.3: Acne vulgaris. Acne usually affects areas of high sebum production such as the face, chest, and back ...... 17

Figure 1.4: Types of acne lesions. Acne lesions are formed from blockages of the hair follicle called microcomedones. Microcomedones can develop into (a) inflammatory pustules, (b) non- inflammatory open comedones, or (c) closed comedones ...... 18

Figure 1.5: Retinoid metabolism. Retinol is absorbed from food in the intestine and transported to the liver, where RDH enzymes convert it to retinal. Circulating retinal bound to RBP is taken up by cells and converted to ATRA by Raldh enzymes. ATRA then binds to nuclear receptors to initiate the transcription of target genes. CYP26 enzymes hydroxylate ATRA and its isomers to promote their excretion from the body. TTR, transthyretin; SDRs; short-chain dehydrogenase/reductase ...... 29

Figure 1.6: Retinoid isomers and metabolites. Retinol is reversibly converted to retinal by RDH enzymes. Raldh enzymes in cells irreversibly convert retinal to ATRA, which can then isomerize to 13-cis RA or 9-cis RA. The three isomers are metabolized into their 4-oxo metabolites by hydroxylation...... 30

Figure 1.7: Retinoid receptors and their binding partners. ATRA is the main ligand for RAR receptors, which dimerize with RXRs to bind to bind to RARE or RXRE elements in gene promoters. Alternatively, RXR can dimerize with several other nuclear receptors such as PPAR and VDR, thereby regulating gene transcription in many different pathways ...... 31

Figure 1.8: Retinoid effects on T cell differentiation and mucosal immunity. a) ATRA promotes naïve T cell differentiation into Foxp3+ Treg while suppressing differentiation of TH17 cells. In some circumstances ATRA can induce gut-homing molecules on T cells, though there is now evidence that in vivo it can also promote skin-homing in T cells activated in skin-draining lymph nodes. b) ATRA plays an important role in maintaining gut mucosal immunity by promoting B cell class switching to IgA. GALT, gut-associated lymphoid tissue; MALT, mucosal-associated lymphoid tissue...... 34

Figure 1.9: Retinoid effects on lymphocyte homing. a) ATRA treatment of naïve T cells in vitro promotes gut-homing receptor expression and suppresses skin-homing receptor expression. However, new evidence suggests that DC in skin-draining lymph nodes can also produce ATRA

viii to induce a skin-homing phenotype in T cells. b) ATRA treatment also promotes a gut-homing phenotype in B cells ...... 36

Figure 1.10: Study hypothesis. Studies have shown that patients‟ immune response to P. acnes is more pathogenic in acne than the pathogenicity of P. acnes itself. This study hypothesized that isotretinoin induces an immune tolerance to P. acnes as part of its long-term mechanism of action...... 40

Chapter 2:

Figure 2.1: Lesion counts of acne patients on isotretinoin therapy. Mean (± SEM) inflammatory and non-inflammatory lesion counts are displayed for acne patients prior to (n = 25) and during isotretinoin therapy (1 week, n = 19; 4 weeks, n = 19; 8 weeks, n = 17; 20 weeks, n = 10). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with patients‟ baseline counts ...... 45

Figure 2.2: Serum concentrations of retinoids in acne patients and normal volunteers. Levels of the three retinoic acid isomers and their corresponding 4-oxo metabolites in the serum of normal volunteers (n = 19) and acne patients at baseline (n = 25), 1 week (n = 19), 4 weeks (n = 19), 8 weeks (n = 17), and 20 weeks (n = 10) of isotretinoin therapy were analyzed by HPLC/ mass spectrometry. Mean (± SEM) serum concentrations are displayed in ng/mL for the retinoic acid isomers and as relative peak areas (counts) for the 4-oxo metabolites. *P < 0.05; **P < 0.01, and ***P < 0.001 compared with patients‟ baseline counts ...... 47

Figure 2.3: Serum concentrations of inflammatory cytokines in acne patients and normal volunteers. Mean (± SEM) serum concentrations of cytokines are displayed for normal volunteers (Vols) (n = 13) and acne patients at baseline (n = 26), 1 week (n = 20), 4 weeks (n = 18), 8 weeks (n = 16), and 20 weeks (n = 9) of isotretinoin therapy as well as six months after the cessation of therapy (n = 8). +P < 0.05 and ++P < 0.01 compared with normal volunteers ..... 48

Figure 2.4: Analysis of patients’ monocytes by flow cytometry. Flow cytometry contour plots of CD14 versus TLR-2 for unstimulated monocytes and monocytes stimulated with P. acnes sonicate are shown from a representative patient over the course of isotretinoin therapy ...... 50

Figure 2.5: Analysis of TLR expression on patients’ monocytes. Histogram plots of TLR-2 MFI is displayed below for unstimulated monocytes and P. acnes-stimulated monocytes from one representative patient over the course of isotretinoin therapy ...... 51

Figure 2.6: TLR-2 expression in acne patients’ peripheral monocytes during isotretinoin therapy. Blood samples were taken from normal volunteers (Vols) (n = 22) and acne patients at baseline (n = 25), 1 week (n = 19), 4 weeks (n = 19), 8 weeks (n = 17), and 20 weeks (n = 10) of isotretinoin therapy as well as six months after the cessation of therapy (n = 8). Isolated peripheral monocytes were treated with no antigen (unstimulated; blue bars) or 1 μg/mL P. acnes sonicate (red bars) for 20 hours and analyzed by flow cytometry. a) Mean (± SEM) relative TLR-2 MFI values are displayed for both normal volunteers and acne patients. b) Mean (± SEM) cumulative percent changes in TLR-2 MFI are displayed for acne patients‟ monocytes over the course of isotretinoin therapy. * denotes significance compared to acne patients‟ baseline, P < 0.05; ** P < 0.01; *** P < 0.001. + denotes significance compared to normal volunteers, P < 0.05; ++ P < 0.01; +++ P < 0.001 ...... 52

ix Figure 2.7: TLR-2 expression versus age in the study population. TLR-2 MFI values in unstimulated (left) and P. acnes-treated (right) monocytes are plotted against age for both normal volunteers (Vols) and acne patients at baseline ...... 53

Figure 2.8: TLR-4 expression in monocytes of acne patients and normal volunteers. Mean (± SEM) TLR-4 MFI values are graphed for unstimulated (blue bars) and P. acnes-treated (red bars) monocytes from both volunteers (Vols) and patients during isotretinoin therapy. + denotes significance compared to normal volunteers, P < 0.05...... 54

Figure 2.9: Inflammatory cytokine production by monocytes from acne patients treated with isotretinoin. Mean (± SEM) concentrations of inflammatory cytokines are displayed for unstimulated (blue bars) and P. acnes-treated (red bars) monocytes from normal volunteers (Vols) and acne patients during isotretinoin therapy. * denotes significance compared to acne patients‟ baseline, P < 0.05; ** P < 0.01; *** P < 0.001. + denotes significance compared to normal volunteers, P < 0.05; ++ P < 0.01; +++ P < 0.001 ...... 55

Figure 2.10: TLR-2 expression in the epidermis of acne patients on isotretinoin therapy. IHC for TLR-2 was performed on skin biopsies from acne patients at baseline and 8 weeks of isotretinoin therapy. TLR-2 expression (brown staining) is observed in the epidermal keratinocytes...... 57

Figure 2.11: Analysis of peripheral Treg proportions in acne patients and normal volunteers. a) Dot plots of CD25 versus Foxp3 are shown for one representative patient‟s lymphocytes at baseline and during isotretinoin therapy. Treg populations are gated by a box gate. b) The mean (± SEM) percent of CD4+ T cells that expressed CD25 and Foxp3 are displayed for normal volunteers (Vols) and acne patients at different points during isotretinoin therapy ...... 60

Figure 2.12: Lymphocyte proliferation in acne patients and normal volunteers. Lymphocyte proliferation in response to P. acnes and anti-CD3/anti-CD28 treatment for acne patients and normal volunteers (Vols) is displayed as mean (± SEM) counts per minute (CPM). + denotes significance compared to normal volunteers, P < 0.05; ++ P < 0.01 ...... 61

Figure 2.13: Cytokine secretion by acne patients’ lymphocytes after 20 hours stimulation. Displayed below are mean (± SEM) media concentrations of secreted cytokines from acne patients‟ and normal volunteers‟ (Vols) lymphocytes after 20 hours of stimulation with a) anti- CD3 and anti-CD28 antibodies or b) P. acnes sonicate ...... 62

Figure 2.14: Cytokine secretion by acne patients’ lymphocytes after five days stimulation. Displayed below are mean (± SEM) media concentrations of secreted cytokines from acne patients‟ and normal volunteers‟ (Vols) lymphocytes after five days of stimulation with a) anti- CD3 and anti-CD28 antibodies or b) P. acnes sonicate ...... 64

Chapter 3:

Figure 3.1: Y-27632 drug structure and target. a) The chemical structure of Y-27632 is shown. b) Y-27632 acts on the integrin-Rho pathway. ROCK = Rho-associated protein kinase; Myosin PPTase = myosin phosphatase; MLCP = myosin light chain phosphatase ...... 79

x Figure 3.2: pSEB in culture. pSEB grown in the presence of Y-27632 are shown at 20x magnification with (a) and without (b) 3T3 fibroblasts present. At 40x magnification (c), lipid droplets are visible (yellow dots) in pSEB colonies ...... 81

Figure 3.3: pSEB growth curves. pSEB were continuously passaged for over 200 days in the presence of Y-27632 and 3T3 fibroblast feeder layers. Periodically, some cultures were deprived of Y-27632, 3T3, or both until complete growth arrest or death ...... 82

Figure 3.4: BrdU incorporation assays. BrdU incorporation was used as a measure for cell proliferation in pSEB continuously treated with Y-27632 and pSEB that had been deprived of Y- 27632 for 11 days. Y = Y-27632 drug ...... 82

Figure 3.5: Oil Red O staining for lipids in pSEB. Oil Red O staining shows lipid droplets (red) in SEB-1 cells and pSEB that had grown without Y-26732 for two days ...... 83

Figure 3.6: Effect of Y-26732 on total lipogenesis in pSEB. Total lipogenesis assays were performed on SEB-1 and on pSEB that were cultured in the continued presence of Y-27632 or were deprived of Y-27632 for five days. Mean (± SEM) counts per minute (CPM) are displayed. ** P < 0.01...... 84

Figure 3.7: Effect of synthetic androgen on total lipogenesis. Total lipogenesis assays were performed on pSEB were treated with 10 nM R1881 or vehicle (P.S. Medium) for 24 and 48 hours in the presence or absence of 5 μM Y-27632. CPM = counts per minute ...... 84

Appendix A:

Figure A.1: Ear thickness measurements in response to a single P. acnes injection. Mice were given a one-time subcutaneous injection in one ear with live P. acnes and vehicle in the opposite ear. Ear thickness was measured using micrometers at baseline (before challenge) and then immediately following sacrifice. Fold-changes in ear thickness for each group were compared using Student‟s t-test. * denotes significance compared to baseline, P < 0.05; ** P < 0.01; *** P < 0.001 ...... 103

Figure A.2: P. acnes sensitization time-line. Mice were sensitized with three intradermal injections of heat-killed P. acnes in the skin of the back every three days (days -27, -24, and -21). Three weeks after the last sensitization injection (day 0), mice were injected subcutaneously once in one ear with live P. acnes and saline vehicle in the opposite ear. Mice were sacrificed 2, 4, and 7 days after the challenge injection ...... 105

Figure A.3: Ear thickness measurements in P. acnes-sensitized mice in response to P. acnes challenge. Mice were sensitized with intradermal injections of 450 μg heat-killed P. acnes every three days for three times total. Two weeks after the last sensitization injection, mice were challenged with 20 μg subcutaneous injections of live P. acnes in one ear and vehicle in the opposite ear. Ear thickness measurements were taken and analyzed as described. * denotes significance compared to baseline, P < 0.05 ...... 105

xi Chal.‟) or saline vehicle („Saline Chal.‟) as described. Mean (± SEM) percentages of CD4+ and CD8+ T cells observed in each of the P. acnes-sensitized (dark-blue bars) and vehicle-sensitized (light-blue bars) groups are shown for the ears and their corresponding draining cervical lymph nodes (CerLN) ...... 106

Figure A.5: Proportions of Treg in ears and draining lymph nodes in P. acnes-sensitized mice in response to P. acnes challenge. Mean (± SEM) percentages of CD4+ T cells that expressed Foxp3+ in each of the P. acnes-sensitized (dark-green bars) and vehicle-sensitized (light-green bars) groups are shown for ears and their corresponding draining cervical lymph nodes (CerLN) ...... 107

Appendix B:

Figure B.1: Model for cellular iron-trafficking by NGAL. Siderophore-free NGAL (apo-NGAL) binds to intracellular siderophores to transport iron to the extracellular space. Siderophore- associated NGAL (holo-NGAL) binds iron in the extracellular space and delivers it into cells by means of receptor-mediated endocytosis. In a parallel pathway, iron can be transported into cells by binding to transferrin, which is then endocytosed using an iron transferrin receptor (FeTfR). Sid, siderophore; DMT, divalent metal ion transporter; FPN, ferroportin (iron efflux channel) ...... 117

Figure B.2: NGAL’s protein structure. The diagram below shows the complete protein structure of NGAL with its N-linked glycan on N65 as well as the cysteine residue at which NGAL forms disulphide bonds in homodimers (C87) ...... 118

Figure B.3: N-Linked Glycan Core Structure. All N-linked glycans contain a common Man3GlcNAc2 pentasaccharide core structure attached to an asparagine residue...... 118

Figure B.4: NGAL forms expressed by SEB-1. SEB-1 cells were treated with 1 μM 13-cis RA or varying doses of IL-1β or TNFα for 72 hours. Total cell lysates were analyzed for NGAL using western blotting ...... 120

Figure B.5: Detection of glycosylation in recombinant NGAL. Samples of NGAL-(M), apo- NGAL-(B), and holo-NGAL-(B) were run on gels and stained for glycans and then for total protein. Additionally, a sample of NGAL-(M) was denatured and deglycosylated with PNGase F enzyme as described. Horseradish peroxidase (HP; a glycosylated protein) and soy trypsin inhibitor (TI; an unglycosylated protein) were run as controls for the glycan stain...... 121

Figure B.6: PNGase F enzymatic reaction. PNGase F removes N-linked glycans by hydrolyzing the bond between the anchoring asparagine and the base sugar residue N-acetylglucosamine (GlcNAc) to produce the free glycan and an aspartic acid residue ...... 121

Figure B.7: Treatment of native NGAL with PNGase F. Recombinant NGAL-(M) was treated with PNGase F enzyme without prior denaturation. Whole PNGase F/NGAL reactions were analyzed by SDS-PAGE and stained for glycans (top) and total protein (bottom). RNase B was similarly treated with PNGase F as a control for enzyme activity ...... 122

xii for the deglycosylation reactions. Untreated NGAL-(M) and apo-NGAL-(B) was included on the blot as controls for NGAL detection with western blotting ...... 123

Figure B.9: Purification of NGAL with MagneHis beads. MagneHis beads were used to purify NGAL from PNGase F reactions. Both whole reactions (left side) and MagneHis purified samples (right side) were analyzed by SDS-PAGE and staining for total protein...... 124

Figure B.10: Purification of NGAL by immunoprecipitation with Dynabeads. Mouse anti- NGAL antibody was cross-linked to Dynabeads, and complexes were used to purify NGAL from PNGase F reactions. IP eluents and supernatants were analyzed by SDS-PAGE and stained for total protein. A sample of anti-NGAL antibody was included as a control ...... 125

Figure B.11: MS analysis of NGAL glycosylation. a) Recombinant NGAL-(M) was treated with PNGase F as described. Reactions were analyzed by SDS-PAGE, and the indicated bands were cut out and sent for analysis by mass spectrometry. b) NGAL was immunoprecipitated from treated SEB-1 lysates and media samples, and the eluted product was analyzed by SDS-PAGE. The indicated bands in the 13-cis RA-treated media IP and the anti-NGAL antibody control lanes were excised and sent for analysis by mass spectrometry ...... 126

Figure B.12: Glycan stain of SEB-1 lysates and media supernatants. SEB-1 cells were treated with 1 μM 13-cis RA, 10 ng/mL IL-1β, or vehicle. Cell lysates and media supernatants were analyzed by SDS-PAGE and stained for glycans and then for total protein ...... 128

Figure B.13: Tunicamycin toxicity in SEB-1. SEB-1 were treated with varying doses of tunicamycin (10 pg/mL to 100 μg/mL) and assayed for cell viability. The percent of cell death compared to vehicle is plotted below. A 100 μg/mL concentration was not done for 48 hour time point ...... 128

Figure B.14: Effect of tunicamycin on glycan staining of total cell protein. SEB-1 were treated with 10 pg/mL, 1 ng/ml, 100 ng/mL, or 10 μg/mL tunicamycin or vehicle for 24 hours. Total cell lysates (left side) and media (right side) were run on gels and stained for glycans (top) and then total protein (bottom). A sample of plain untreated media was included as a control in order to determine which proteins are already present in media and not made by SEB-1. Horseradish peroxidase (HP) and soy trypsin inhibitor (TI) were included as glycan stain controls ...... 130

Figure B.15: SEB-1 secretion of NGAL. SEB-1 were treated for 24 hours with 1 μM 13-cis RA alone, 1 μM 13-cis RA plus 10 ng/mL tunicamycin, 1 μM 13-cis RA plus 10 μg/mL tunicamycin, or 10 μg/mL tunicamycin alone. Media supernatants (top row) and cell lysates (bottom two rows) were analyzed by western blot for NGAL expression. NGAL-(M) (labeled „rhNGAL”) was included on each gel as a positive control, and β-actin expression was used as a loading control for the cell lysate samples ...... 131

Figure B.16: TUNEL assays. SEB-1 were treated with 1 μM normal mammalian-expressed NGAL („normal NGAL‟), deglycosylated NGAL („deglycosylated NGAL rxn‟), or vehicle („vehicle H2O‟) for 24 hours. Treatment controls for the deglycosylation reactions included NGAL that was incubated in the same buffers as the deglycosylation reactions but without PNGase F enzyme („NGAL control rxn‟) and PNGase F and its reaction buffers without NGAL („PNGase F control rxn‟). Assay controls included wells treated for 10 minutes with DNase as a positive control and wells treated with DNase that were not incubated with TdT enzyme as a negative control. Representative fluorescence and brightfield images for each treatment group are shown ...... 132

xiii Figure B.17: Annexin V/PI assays. SEB-1 were treated with 1 μM normal NGAL-(M), the whole NGAL-(M) deglycosylation reaction, or vehicle („H2O vehicle‟) for 24 hours. Cell were then trypsinized, stained with PI and antibodies for annexin V, and analyzed by flow cytometry. Mean (± SEM) percentages of apoptotic cells are displayed for each treatment group. Controls included cells treated with 0.1 μM staurosporine as a positive control for apoptosis, as well as controls for the deglycosylation reactions: „NGAL-(M) control rxn‟ is NGAL that was incubated in the same buffers as the deglycosylation reactions but without PNGase F enzyme, and „Deglyc control rxn‟ is PNGase F and its reaction buffers without NGAL added. Data is representative of four separate experiments...... 133

Appendix C:

Figure C.1: The LCN2 gene promoter. The LCN2 gene promoter contains many different transcription factor binding elements, including a κB binding site and retinoic acid response elements ...... 143

Figure C.2: NF- B translocation in SEB-1 sebocytes. Immunofluorescence staining was performed on cultures treated with IL-1β for one hour using primary antibodies to p65 and DAPI ...... 144

Figure C.3: Timeline of NF-κB translocation induced by IL-1β and 13-cis RA in SEB-1. Immunofluorescence staining for p65 was performed on SEB-1 cultures treated with 10 ng/mL human IL-1β, 1 μM 13-cis RA, or vehicle controls for various time-points. Mean (± SEM) percentages of cells with p65 translocation are shown for each time-point; charts are representative of three separate experiments. * P < 0.05 ...... 145

Figure C.4: p65 phosphorylation in response to IL-1β and 13-cis RA in SEB-1. SEB-1 were treated for various time-points with either 1 μM 13-cis RA (top blots), 10 ng/mL IL-1β (bottom blots), or corresponding vehicle controls (C). Cell lysates were analyzed for phospho-p65 protein by western blot ...... 146

Figure C.5: NGAL induction by IL-1β and TNFα. SEB-1 sebocytes were treated with increasing concentrations of IL-1β or TNFα for 72 hours. Total cell lysates were analyzed for NGAL using western blot ...... 147

Figure C.6: NGAL induction by TLR ligands. SEB-1 sebocytes were treated with 20 μg/mL poly(I:C), 10 μg/mL zymosan, 25 μg/mL LTA, 1 μg/mL LPS, or vehicle controls for 24 hours. Total cell protein was subjected to SDS-PAGE. Blots were incubated with primary antibodies to NGAL as well as β-actin as a loading control ...... 148

xiv

LIST OF TABLES

Chapter 1:

Table 1.1: Human Toll-Like Receptors and Their Characteristics...... 6

Table 1.2: Helper T Cell Subsets and Their Characteristics ...... 14

Table 1.3: Plasma Concentrations of Retinoids in Acne Patients and Normal Volunteers ...... 39

Chapter 2:

Table 2.1: Study Patient/Volunteer Demographics ...... 44

Chapter 3:

Table 3.1: Top Genes Changed with Y-27632 Treatment in pSEB ...... 86

Table 3.2: Rho Pathway-Associated Genes Changed with Y-27632 Treatment in pSEB ...... 87

Table 3.3: Wnt Pathway-Associated Genes Changed with Y-27632 Treatment in pSEB ...... 88

xv LIST OF ABBREVIATIONS

9-cis RA 9-cis retinoic acid 13-cis RA 13-cis retinoic acid ACTH Adrenocorticotropic hormone AMP Antimicrobial peptide APC Allophycocyanin dye protein APC Antigen presenting cell APL Acute promyelocytic leukemia ATRA All-trans retinoic acid BrdU Bromodeoxyuridine BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate CAR Constitutive androstane receptors CCR Chemokine receptor CD- Cluster of differentiation CerLN Cervical lymph node CFU Colony forming unit Ci Curies CLA Cutaneous lymphocyte antigen CPM Counts per minute CRABP Cellular retinoic acid binding protein CTLA-4 Cytotoxic T lymphocyte antigen 4 CXCR Chemokine X receptor CYP Cytochrome P450 DC Dendritic cell DEFB Defensin-beta DHEA Dehydroepiandrosterone DHT Dihydrotestosterone DMEM Dulbecco‟s modified Eagle‟s medium EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth receptor FBS Fetal bovine serum FFA Free fatty acid FITC Fluorescein isothiocyanate FMO Fluorescence minus one Foxp3 Forkhead box P3 FXR Farnesoid X receptors GM-CSF Granulocyte macrophage colony stimulating factor HPLC High pressure liquid chromatography HSD Hydroxysteroid dehydrogenase IBD Inflammatory bowel disease ICS Intracellular staining IFN Interferon Ig Immunoglobulin IGF Insulin-like growth factor IL- Interleukin IP Immunoprecipitation IRF Interferon regulatory factor

xvi IU International units kD Kilodaltons LN Lymph node LPS Lipopolysaccharide LTA Lipoteichoic acid LXR Liver X receptors MFI Mean fluorescence intensity MMP Matrix metalloproteinase MS Mass spectrometry MSH Melanocortin-stimulating hormone NF-κB Nuclear factor kappa-B NGAL Neutrophil gelatinase-associated lipocalin NHEK Normal human epidermal keratinocytes PAGE Polyacrylamide gel electrophoresis PBMC Peripheral blood mononuclear cells PBS Phosphate-buffered saline PE Phycoerythrin PerCP Peridinin-chlorophyll protein complex PI Propidium iodide PKC Protein kinase C PNGase F Peptide: N-glycosidase F PPAR Peroxisome proliferator-activated receptors PXR Pregnane X receptors QRT-PCR Quantitative reverse transcriptase polymerase chain reaction Raldh Retinaldehyde dehydrogenase RAR Retinoic acid receptor RARE Retinoic acid response element RBP Retinol binding protein RDH Retinol dehydrogenase ROR Retinoic acid orphan receptor RXR Retinoid X receptor RXRE Retinoid X response element SDS Sodium dodecyl sulfate SEM Standard error of the mean SREBP Sterol regulatory element binding protein TAG Triacylglyceride Tcm Central memory T cell Teff Effector T cell Tem Effector memory T cell TGFβ Tissue growth factor beta TIR Toll/interleukin-1 receptor domain TLR Toll-like receptor TNFα Tissue necrosis factor alpha TR Thyroid hormone receptors TRAIL Tumor necrosis factor-related apoptosis-inducing ligand Treg Regulatory T cell VDR Vitamin D receptor

xvii ACKNOWLEDGEMENTS

The creation of this thesis was only possible with the guidance and friendship of many people. I am most grateful to my advisor, Dr. Diane Thiboutot, who is a wonderful mentor and an example of what I wish for my own future career. Diane not only allowed, but encouraged me to pursue my interests and follow my instincts. I am lucky and grateful to have had the opportunity to work with her. Thanks also to my committee members Dr. Charles Lang, Dr. Gary Clawson, Dr. Stephen Rannels, and Dr. Robert Bonneau for their advice and guidance. I owe a special thanks to Rob, who not only went above and beyond in advising me, but who also tolerated my constant use of his lab. I would like to express my sincere gratitude to my lab-mates, Zhaoyuan Cong, Kathryn Gilliland, Dr. Kimberly Lumsden (former), Dr. Amanda Nelson (former), and Ellen Wolpert. They have given me valuable assistance and friendship over the years. They were beside me day-in and day-out as my research unfolded, and I appreciated their words of encouragement. Because of them, I truly enjoyed coming to work each day. I would also like to thank Dr. David Stanford and Nate Sheaffer in the Penn State Hershey Flow Cytometry Core for their technical assistance. There were many months when I utilized the flow core nearly every day, and their doors were always open (because they gave me the pass-code to the locks). Thank you for your patience and companionship during my tediously long FACS sessions. I would like to thank my friends and family for all their love and encouragement. There are too many to name, and too much love to acknowledge. Finally, to my husband Kyle: you give me perspective and help me laugh when I need it most. You are my best friend and perfect match, and I am extremely lucky to have you in my life. Thank you for your unfailing love and support during this time.

Chapter 1

Literature Review

1.1 Introduction

Acne is a common human ailment that can cause significant physical and psychological morbidity. Its pathogenesis lies in the interplay between four main factors: follicular hyperkeratinization, excess sebum production, Propionibacterium acnes colonization, and inflammation. However, little is known about the environmental or genetic etiology of each of these factors, or why some people get acne while others do not. Retinoids are vitamin A derivatives that are used in a variety of dermatological diseases. In particular, isotretinoin is the only known agent that can permanently cure acne. It does so by modifying all four factors of acne pathogenesis during treatment, but after the cessation of therapy sebum production and P. acnes colonization can return to baseline levels even in the context of continued clinical remission. The mechanisms of isotretinoin‟s long-term efficacy are not yet understood. The skin is a remarkable organ that must protect the body from environmental stresses and harmful pathogens while maintaining a healthy flora, thus an imbalance in skin immune homeostasis can result in susceptibility to infection or hyper-responsiveness that manifests as allergies. Given the many roles that retinoids play in maintaining immunity, isotretinoin may partially exert its long-term effects through modulation of the host immune response to P. acnes. The first section of this chapter will review human skin physiology. The second section will review the epidemiology and pathophysiology of acne, as well as current acne treatments and model systems for acne research. The final section discusses retinoid biology, metabolism, and functions in immune homeostasis and acne therapy.

2 1.2 Skin

1.2.1 Structure of the Skin

The skin is the largest organ in the human body and is responsible for physical protection from the elements, thermoregulation, and recognition of and protection against pathogens.1 The skin has three main layers, the epidermis, dermis, and hypodermis (Figure 1.1).

1.2.1.1 Epidermis

The epidermis has four main layers: the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum is the outermost layer of the skin and consists of squamous cells that form a protective physical barrier against pathogens and the elements. Keratinocytes that have differentiated and lost their nuclei form the outermost layer of the stratum corneum. These keratinocytes continuously shed and become replaced by new cells from the basal layer. Epidermal keratinocytes also form a cutaneous water barrier with epidermal free lipids. The stratum granulosum consists mainly of keratohyalin granules, and the stratum spinosum contains intercellular bridges called desmosomes that connect to the basal germinal cells in the stratum basale. The epidermis consists primarily of keratinocytes, but it also contains other cell types. Melanocytes reside throughout the basal layer of the epidermis and function to make melanin, a pigment which protects the skin from ultraviolet radiation. Less plentiful in the epidermis are Merkel cells, which are mechanoreceptors that identify stresses in the skin (via mechanical stress or cytokines secreted by keratinocytes) and then send signals to neurons. Langerhans cells are immune cells found in the epidermis; Langerhans cells and other immune functions of the skin are discussed further in Section 1.2.2. The dermal-epidermal junction is where the basal cells of the epidermis are connected to the connective tissue of the dermis by hemidesmosomes. The dermal-epidermal junction has three layers: the basal lamina, lamina lucida, and lamina densa. The basal lamina consists of keratinocytes and fibroblasts, while the lamina lucida contains anchoring proteins that interlock with cross-linking fibers from the lamina densa.

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(http://www.web-books.com/eLibrary/Medicine/Physiology/Skin) ______

1.2.1.2 Dermis and Hypodermis

The dermis is a thicker layer of connective tissue composed mainly of collagen, elastin, and fibrillin. The dermis contains a variety of structures, including nerve endings, sweat glands, sebaceous glands, blood vessels, and lymphatic vessels. Nerve endings detect pain, pressure, and temperature. Temperature is regulated by the dilation and contraction of dermal blood vessels and the secretion of sweat by sweat glands in response to heat or stress. Immune cells such as macrophages, lymphocytes, and mast cells also reside in the dermis. The dermis also contains the major portion of the pilosebaceous unit, which is discussed further below. The hypodermis contains mainly adiopose tissue, which contributes to temperature insulation and energy storage.

4 1.2.1.3 Sebaceous Glands

The pilosebaceous unit consists of a hair follicle and an adjoining sebaceous gland. Sebaceous glands are found in all areas of body except the palms and soles and are typically the largest on the face. The majority of sebaceous glands in the skin are part of a pilosebaceous unit, but free sebaceous glands, those not associated with hair follicles, may be located in the areolae, vermillion border of the lips, and in the mouth. Sebaceous gland development begins in the 13th to 16th week of gestation as a bulge in the follicular primordium, with signaling proteins Wnt and TRAF6 playing a major role. Basal sebocytes on the periphery of the acinus have a flattened or cuboidal in shape and contain very few lipids. Starting post-partum, sebocytes begin to differentiate and travel to the center of the gland and up through the follicle. As they differentiate, they accumulate lipid until they finally undergo holocrine rupture and are secreted onto the surface of the skin. The life span of a sebaceous cell is roughly two to three weeks, with the most active lipid synthesis occurring one week before secretion. Humans have a unique sebum composition compared to other mammals. The major components of human sebum are wax esters (26%), triacylglycerides (TAG; 41%), squalene (16%), cholesterol (2%) and cholesterol esters (1%). Only four other known species secrete squalene, and wax esters have been found in the sebum of about 25% of mammalian species. The main resident bacterium in the human sebaceous gland, P. acnes, also contributes to this unique composition by secreting lipolytic enzymes that break down the TAG into free fatty acids (FFA), which then constitute about 14% of the sebum that reaches the surface of the skin. FFA are the principle source of nutrients for P. acnes. Because humans are the only species which secretes TAG in sebum, they are also the only species whose pilosebaceous units support P. acnes colonization. Many of the lipids found in human sebum are unique to the sebaceous gland, containing unusual double bond positions, methyl branching, and long carbon chains that are not found in lipids made elsewhere in the body.2,3 Sebocytes synthesize TAG, wax esters, squalene, cholesterol, and phospholipids de novo using acetate as a substrate, but other lipids such as linoleic acid and Δ9-FFA are not synthesized by sebaceous glands and are instead taken up from the circulation.4-6 The function of sebum is not entirely understood, though several possibilities have been proposed. Generally, sebum provides a vehicle for delivering pheromones, antimicrobial peptides, and antioxidants to the surface of the skin, and may also contribute to the cutaneous water barrier in addition to epidermal lipids.7-9 Sebum may also play a part in innate immune defenses; it contains significant levels of immunoglobulin (Ig)-A, and certain sebaceous lipids have antimicrobial activity against specific bacteria.10 Alternatively, some researchers suggest that sebaceous glands are merely vestigial and serve

5 little or no function in humans. Whatever their physiological role, sebaceous glands can cause various pathologies. Acne is a disease that is partially caused by excess sebum production, whereas seborrheic dermatitis occurs when sebum irritates the skin, though sebum production is normal. Xerosis is a condition characterized by very low sebum production, which causes symptoms such as dryness, scaling, and itching of the skin. Sebum production is high at birth and then low during childhood until puberty begins. Maximum sebum secretion occurs in the late teens to early twenties and declines slowly during adulthood at 20-30% decrease per decade.11-13 This pattern of production is partially caused by systemic levels of androgens and IGF-1, both of which stimulate sebocytes to produce lipids. Human skin and sebocytes can also make androgens locally from cholesterol by expressing enzymes involved in androgen synthesis such as 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and 5α-reductase.14 Androgens regulate lipogenesis by increasing expression of sterol response element binding proteins (SREBP)-1a and -1c as well as lipogenic enzymes.15 Acting on the same pathway, insulin-like growth factor-1 (IGF-1) and insulin have been shown to increase lipogenesis through binding to the IGF-1 receptor and activating SREBP-1 downstream.16,17 Fasting or caloric deprivation reduces plasma IGF-1 levels and thus decreases sebum production with specific decreases in wax esters and TAG.18 Conversely, high-glycemic diets may stimulate sebum production through raising insulin and IGF-1 levels, though the evidence that this in turn exacerbates acne is controversial.19-21 Other hormones can also regulate sebum production. Melanocortins including melanocortin- stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) increase sebum production in rats.22 Treatment of human primary sebocytes with MSH increases lipid droplet accumulation; accordingly, the melanocortin-5 receptor is expressed in human sebocytes and is now considered a differentiation marker.23,24 Peroxisome proliferator-activating receptors (PPARs) are important regulators of sebocyte growth and differentiation. PPARs are nuclear receptors that dimerize with retinoid receptors to control gene expression. All three PPAR isoforms (α, β, and γ) play roles at various stages of lipogenesis.25-28 Hormonal contraceptives and oral isotretinoin are the only treatments available that decrease sebum production. Isotretinoin is further discussed in Section 1.3.3.3.

1.2.2 Immune Function of the Skin

The skin is the body‟s first protective barrier against harmful environmental pathogens and must therefore be adept at recognizing pathogens, mounting appropriate local immune responses, recruiting

6 white blood cells, and healing infected or damaged areas. Normal non-inflamed skin contains a variety of resident leukocytes including macrophages, dendritic cells (DC), memory T cells, and mast cells, all of which aid in the maintenance of tolerance to normal flora while providing surveillance for pathogenic antigens. Non-immune cells also participate in innate immunity by recruiting and activating leukocytes in response to pathogen exposure.

1.2.2.1 Innate Immunity in the Skin

The innate immune system has the ability to recognize pathogens and mount immune responses quickly while creating signals that will recruit and activate components of the adaptive immune system. Pattern recognition receptors on resident skin cells, antimicrobial peptides, and sebum lipids all contribute to the innate immune functions of the skin.

______

Table 1.1: Human Toll-Like Receptors and Their Characteristics

Activated Expression in Localization Ligands Signal Pathway Receptors Skin Bacterial Keratinocytes TLR-1 cell surface MyD88/MAL NF-κB and AP-1 lipoproteins Leukocytes Bacterial Keratinocytes lipoproteins TLR-2 cell surface MyD88/MAL NF-κB and AP-1 Sebocytes (MALP-2 and LTA) Leukocytes Peptidoglycan Keratinocytes NF-κB, AP-1, TLR-3 endosomes dsRNA TRIF Sebocytes and IRF-3 Leukocytes LPS (Gram- Keratinocytes negative bacteria) MyD88/MAL/ NF-κB, AP-1, Sebocytes TLR-4 cell surface LTA (Gram- TRIF/TRAM and IRF-3 Melanocytes positive bacteria) Leukocytes NF-κB, AP-1, Keratinocytes TLR-5 cell surface Flagellin MyD88 and IRF-3 Leukocytes Bacterial Keratinocytes TLR-6 cell surface lipoproteins MyD88/MAL NF-κB and AP-1 Sebocytes MALP-2 Leukocytes ssTNA TLR-7 endosomes R-848 MyD88 NF-κB and AP-1 Leukocytes imidazoquinolones

ssRNA (G rich) NF-κB, AP-1, TLR-8 endosomes MyD88 Leukocytes imidazoquinolones and IRF-3

Keratinocytes TLR-9 endosomes CpG DNA MyD88 NF-κB and IRF-3 Leukocytes TLR-10 unknown unknown unknown unknown unknown

______

7 Pattern recognition receptors are an important component of the innate immune system and are expressed on nearly every type of cell found in the body. Toll-like receptors (TLR) are the main class of pattern recognition receptors in the skin and immune cells that recognize and bind to specific pathogenic antigens. All mammalian TLRs are type I transmembrane proteins consisting of multiple leucine-rich repeats in the extracellular domain and a conserved Toll/interleukin-1 receptor (TIR) homology domain in the cytoplasmic tail. There are ten known TLRs in humans (Table 1.1): TLR-1, -2, -4, -5, and -6 reside on the cell surface and recognize mainly bacterial cell wall components, and TLR-3, -7, -8, and -9 reside in endosomes and recognize viral nucleic acids. Some TLRs (such as TLR-2) are internalized after ligation. TLR activation results in a signal cascade that initiates the transcription of antimicrobial peptides and inflammatory cytokines, which activate neighboring cells and recruit leukocytes to the area of infection. Most TLRs signal through MyD88 to activate nuclear factor-kappa B (NF-κB) and interferon regulatory factors (IRF), which are nuclear receptors that initiate the transcription of inflammatory mediators (Figure 1.2). Keratinocytes express TLR-1, -2, -3, -4, -5, -6, -9, and -10, and the levels of TLR-2 and TLR-4 expression increase with differentiation or stimulation.29-32 Melanocytes express TLR-4, and sebocytes express significant levels of TLR-1, 2, -3, -4 and -6 and low levels of the other TLRs.33 TLR-2 is thought to be the main receptor stimulated by P. acnes, the bacterium involved in acne. TLR-2 recognizes Gram-positive bacterial cell wall components such as lipotechoic acid and peptidoglycan as well as viral envelope proteins. The human TLR-2 gene is located on chromosome 4q32 and has three exons. Nearly all cells in the body express TLR-2 in its full-length form, but monocytes express three different mRNA splice variants, the proportions of which vary between people and vary according to monocyte differentiation stage. The human TLR-2 promoter has no sequence homology with the murine promoter and contains SP1, SP2, C/EBP, NF-κB, and PU.1 transcription elements. The lack of homology in promoter binding elements may explain the differences in TLR-2 tissue expression between mice and humans. For example, murine T cells express TLR-2, but human T cells do not.34 Additionally, murine TLR-2 is up-regulated by any pro-inflammatory stimuli (including LPS, a TLR-4 ligand) through activity of NF-κB and STAT5 binding sites. In human cells, however, NF- κB initiation of TLR-2 transcription requires additional recruitment of CREB-binding protein and p300 to initiate histone acetylation and chromatin remodeling, explaining the variations in TLR-2 regulation between mice and humans.35-37

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www.cellsignal.com

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9 TLR-2 polymorphisms have been implicated in many different types of infections and diseases. The most widely studied TLR-2 polymorphism is TLR-2 Arg753Gln, in which a glutamic acid is substituted for an arginine in the intracellular TIR domain. This polymorphism results in decreased stimulation by bacterial lipopeptides and has been associated with increased risk of infection by Gram- positive bacteria, M. leprae, M. tuberculosis, and cytomegalovirus, as well as atopic dermatitis.38-43 One study showed that up to 9.4% of Caucasians are heterozygous for this particular polymorphism.44 The TLR-2 Arg677Trp polymorphism has also been shown to increase risk of infection with Gram-positive bacteria and tuberculosis.45,46 TLR-2 over-expression has been implicated in the pathogenesis of multiple diseases characterized by chronic inflammation. In some of these diseases, the associated inflammation is sterile, such as in psoriasis, sarcoidosis, Behcet‟s disease, rheumatoid arthritis, and spondylarthropathy.47-50 In others, inflammation is thought to have been originally triggered by an infectious agent that then stimulates a chronic hyper-inflammatory state, such as acne vulgaris and acne inversa (hidradenitis suppurativa).51,52 The involvement of TLR-2 in acne pathogenesis is further discussed in Section 1.3.2.3. Antimicrobial peptides (AMP), including defensins, cathelicidins, and lipocalins, are innate immune modulators that are released in response to inflammatory signals. In addition to direct bacteriostatic or bacteriocidal effects, they also have immunomodulatory properties.53 Neutrophil gelatinase-associated lipocalin (NGAL) is an antimicrobial peptide that can directly kill Gram-negative bacteria by binding to bacterial siderophores and sequestering iron.54-56 NGAL has also been shown to induce apoptosis in various types of mammalian cells including sebocytes.57 The exact mechanism by which NGAL induces apoptosis in mammalian cells is unknown, though a mammalian siderophore has been recently discovered that may play a role in NGAL‟s apoptotic actions.58 NGAL is further discussed in Appendices B and C. Defensins are AMPs that can be induced by infection with bacteria or inflammatory cytokines such as IL-1β, IFN , and TNFα. Human β-defensins (hBD)-2, -3, and -4 are expressed in human skin. Aside from antimicrobial properties, hBDs also increase vascular permeability and mast cell degranulation and are chemotactic for macrophages and neutrophils.59,60 hBDs stimulate keratinocyte migration, proliferation and release of IL-8, IL-18, and IL-20.61,62 Defensins have been implicated in the pathogenesis of several dermatological diseases: psoriasis patients have higher number of copies of hBD genes and have higher expression of hBD in psoriasis lesions than non-affected skin, atopic skin contains low levels of hBD expression, and hBD-4 mRNA has been found to be up-regulated in acne lesions by studies in our lab.63-65 The role of defensins in dermatological diseases is not yet understood. Cathelicidin is an AMP that depends on intracellular proteolytic processing to cathelin and LL-37 in order to obtain full activity.66 Many different cell types secrete cathelicidin to directly kill bacteria:

10 keratinocytes can produce it to directly kill S. aureus and group A Streptococcus, and sebocytes can secrete it to kill P. acnes.67 Additionally, cathelicidin is synergistic with hBD and lysozyme in killing S. aureus and E. coli.53 Cathelicidin can also directly affect immune cells; it has been shown to promote the differentiation of human macrophages into an inflammatory phenotype and increased the release of inflammatory mediators by neutrophils.68,69 Cathelicidin has been shown to be involved in the pathogenesis of some skin diseases such as rosacea and psoriasis. Rosacea patients have higher levels of cathelicidin in their skin, and also express a different post-translationally processed form than is found in normal skin, causing an exacerbated innate immune response.70 Sebum lipids have also been shown to have antimicrobial properties and can thus regulate skin flora. This idea was first proposed in the 1940‟s, but did not gain mechanistic evidence until recently.71 Sebum FFAs enhance innate immunity in human keratinocytes by up-regulating hBD-2.72 Middle to long-chain FFAs (C8-C18) polar lipids and glycosphingolipids have all been shown to have activity against various organisms including S. aureus and Salmonella, and lauric acid has antimicrobial effects on P. acnes.73-78 These studies suggest that sebaceous and epidermal lipids may help to maintain normal skin flora by selectively inhibiting the growth of pathogenic organisms. While the cutaneous innate immune system is essential for protection against pathogens, it can also cause pathology when not sufficiently regulated. Disorders characterized by sterile inflammation such as acne, psoriasis, psoriatic arthritis, Behcet‟s disease, and rosacea have been linked to dysregulation of innate immune signaling.47,48,79 In the case of acne, psoriasis, and rosacea, these conditions are thought to represent disorders of dysbiosis characterized by an imbalance of microorganisms within the cutaneous microbiome that leads to altered innate immune responses and noninfectious skin inflammation.80 Although most studies have focused on inflammatory events within the skin, patients with acne, psoriatic arthritis and Behcet‟s disease have increased expression of TLR-2 on circulating monocytes implying that the dysregulation of innate immune signaling extends beyond the skin.47,48,51

1.2.2.2 Antigen-Presenting and Phagocytic Cells

Innate immune cells serve two important functions in skin immunity: they participate in innate immunity by recognizing and engulfing pathogens, and they help activate adaptive immune cells by presenting antigen and co-stimulatory signals. Phagocytic cells are often the first line of defense against pathogens. They express TLRs and other pattern-recognition receptors on their surface which allow them to recognize and respond to certain pathogens. Stimulation of TLRs instructs phagocytes to engulf foreign pathogens or dead tissue material

11 by receptor-mediated phagocytosis. Enzymes and reactive oxygen species in phagolysosomes then degrade the material into fragments which can then serve as antigens for presentation to other immune cells. Pathogen recognition and phagocytosis also stimulate phagocytes to secrete inflammatory cytokines, chemokines, and antimicrobial peptides that activate neighboring cells and recruit other leukocytes. There are three types of phagocytic cells in the skin: macrophages, DCs, and neutrophils. Neutrophils are the most abundant white blood cell in the body. They rapidly migrate to sites of tissue damage and infection where they phagocytose and kill pathogens and then die at infection sites to form pus. Macrophages and DC are further discussed below. Antigen presenting cells (APC) bridge the innate and the adaptive immune system branches by activating lymphocytes. Once APCs engulf pathogens in infected tissues, they migrate to draining lymph nodes (LN) where they present peptides from the degraded pathogens on their surface. APCs give T cells three distinct signals required for activation: 1) they present peptide antigen bound to major histocompatibility complex (MHC) class-II molecules, which the T cell recognizes with its T cell receptor (TCR); 2) they express co-stimulatory molecules such as CD80 and CD86 which bind to receptors on T cells (such as B7 or CTLA-4); and 3) they secrete cytokines that direct the T cell‟s activation and differentiation. T cell activation is discussed further below in Section 1.2.2.3. The most important APCs in skin, macrophages and DCs, both differentiate from peripheral blood monocytes. Monocytes are bone-marrow derived immune cells that make up three to eight percent of total blood leukocytes. They have both phagocytic and antigen-presenting abilities even in their immature form. They express all human TLRs as well as co-stimulatory molecules CD80 and CD86, but can be distinguished from other immune cells by the expression of surface markers such as CD33, CD11a, and the LPS-receptor CD14.81 Monocytes typically circulate in the blood for roughly one to three days until they are recruited to sites of inflammation, pathogen infection, and tissue damage by following inflammatory cytokine and chemokine gradients released by other resident cells. Once in peripheral tissues, monocytes mature into macrophages or DCs, depending on the inflammatory stimuli and environment. Macrophages are relatively long-lived innate immune cells that are often the first cells to respond to pathogens in the skin. They specialize in presenting processed antigens from ingested pathogens. Unlike neutrophils, macrophages are also resident in healthy tissues such as skin, the linings of the gastrointestinal and respiratory tracts, connective tissue, and the liver. Macrophages express CD14, CD68, and CD11b integrin. DCs are key APCs in the skin and mucosal sites. Multiple different subpopulations of DCs exist in the skin. Langerhans cells (LC) are the only DCs found in the epidermis during the non-inflamed steady state. They extend their dendritic processes up into the stratum corneum, where they can acquire

12 antigens at or near the skin surface. Like macrophages, LCs uptake antigens by receptor-mediated endocytosis, but they also constantly sample the environment by uptaking surrounding fluid by pinocytosis. Inflammatory stimuli such as infection, ultraviolet light, and wounding activate LCs to migrate from the epidermis to the cortex area of skin-draining LNs where they present antigens to circulating naïve T cells. LCs express most TLRs, including high levels of TLR-2, -3, -4, -8, and -10. They also express the C-type lectin Langerin (CD207), CD11b, and epithelial cell adhesion molecule (EpCam). Another subtype of DC in the skin, dermal dendritic cells (dDC), reside in the dermis. Like LCs, dDCs are present in the dermis in the steady-state and migrate to LN upon activation. Unlike LC though, dDCs can cross-present antigens to activate cytotoxic T cell responses. Most dDCs do not express Langerin, but a subset (about 20%) of dDCs are Langerin-positive and CD11b-negative.

1.2.2.3 T cells

T lymphocytes, or T cells, are the key players in cell-mediated immunity. T cells develop in the thymus, where they acquire either CD4 or CD8 expression and a specific TCR on their cell surface. Naïve undifferentiated CD4+ or CD8+ T cells leave the thymus and circulate in the peripheral blood and LNs where they interact with APCs. APCs in the skin engulf pathogens and migrate to skin-draining LNs to present antigen epitopes on MHC molecules to naïve T cells. Naïve T cells whose TCR is an exact match for a particular presented antigen then differentiate and undergo clonal expansion to become effector cells, which migrate into the infected tissues. As the immune response to a pathogen resolves, many of these newly expanded effector cells undergo deletion by apoptosis, but some survive to become effector memory cells (Tem) or central memory T cells (Tcm). Upon re-encountering their specific pathogen, memory T cells do not need co-stimulation from APC to activate and initiate immune responses. This process forms the basis for long-term immunological memory. Tem created from vaccinations can exist for up to 75 years post immunization, with a half-life of seven to fifteen years. T cells express adhesion and homing molecules on their surface that direct their migration patterns. Naïve T cells express LN-homing molecules such as CCR7 and L-selectin which allow them to circulate in the blood and lymph, spending significant amounts of time in LN so they will interact with APC. During activation, APCs direct T cells to express homing receptor patterns that are specific to the microenvironment of activation. If activated in gut lymphoid tissues such as GALT or Peyer‟s patches, T cells will acquire the gut-homing receptors α4β7-integrin and CCR9. If activated in skin-draining LNs or in the periphery, T cells acquire the skin-homing receptors α4β1, CCR4, CCR10, and cutaneous lymphocyte antigen (CLA). E-selectin, which is expressed on all post-capillary venules in skin, binds to

13 CLA on skin-homing T cells to initiate lymphocyte rolling and then extravasation into the surrounding dermis.

About 90% of Tem expressing skin-homing receptors reside in the skin, but 10% circulate in the 82 blood. Tem originating from a cutaneous immune response migrate through the bloodstream to distribute in all parts of the skin, but a significant portion will permanently remain at the original site of pathogen exposure. This is a possible mechanism for how certain skin diseases repeatedly erupt in the same areas but spare others. In psoriasis, for example, it is thought that skin-resident auto-reactive Tem remain quiescent until a neighboring APC stimulates them by producing IFNα, thus causing plaques to form in the same places.83 CD4+ T cells, also called „helper‟ T cells, aid in the activation and function of other immune cells. Helper T cells can be one of several different phenotypes: TH1, TH2, TH17, and Treg (Table 1.2). The surrounding microenvironment influences which phenotype a naïve T cell becomes upon activation. Cytokines released by surrounding cells and APCs determine the differentiation of the T cell. Activated effector T cells remain plastic, meaning that they can be converted from one subset phenotype to another, or from skin to gut-homing and vice versa if re-stimulated by specific antigen-presenting cells.84

TH1 cells aid in initiating „cell-mediated‟ immunity, the principle branch of the immune response that combats viral infections and tumors. TH1 produce IFN , tumor-necrosis factor-α (TNFα), and IL-12 when activated, and subsequently activate macrophages and CD8+ cytotoxic cells.

TH2 cells aid in initiating „humoral‟ immunity by activating B cells to produce neutralizing antibodies against pathogens such as bacteria and parasites. TH2 cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13, which have varying effects on the immune system. IL-4 causes B cells to produce IgE antibodies, and IL-5 activates eosinophils, which can be pathogenic in the case of allergies and asthma that are mediated by a TH2-induced IgE antibody production. IL-10 is generally immunosuppressive and inhibits multiple types of immune cells.

TH17 cells produce IL-17, IL-21, and IL-23 and are important for defense against parasites, but are more often pathogenic in western cultures where parasite infections are rare. TH17 cells are implicated in the pathogenesis of various autoimmune and chronic inflammatory diseases such as psoriasis and inflammatory bowel disease (IBD).

Regulatory T cells (Treg) are antigen-specific CD4+ T cells that suppress other immune cells. They are important for the maintenance of tolerance to self antigens and normal flora, and also function to dampen active immune responses. Treg are further discussed in Section 1.2.2.4. A summary of helper T cell subtypes and their attributes is shown in Table 1.2.

14 ______

Table 1.2: Helper T Cell Subsets and Their Characteristics

TH1 TH2 TH17 Treg IL-2, IL-12, IL-4, IL-5, IL-1α, IL-1β, Induced by IL-10, TGFβ IFN , IL-18 IL-9, IL-10 IL-6, TGFβ IL-6, IL-1β, IL- Inhibited by IL-10 IL-2, IFN IL-2, IL-4, IL-10 21, IL-23 Characteristic Transcription Tbet GATA3 ROR t Foxp3 Factors IL-4Rα, IL- IL-23R, IL-1R1, CD25, CTLA4, Characteristic IL-12Rβ2, 33Rα, CCR3, IL-18Rα, CCR6, CCR5, (CD127 Surface Markers CXCR3, CCR5 CCR4, CCR8 CCR4 negative) Cytokines IFN , TNFα, IL-4, IL-5, IL-6, IL-17, IL-21, IL-10, TGFβ Produced IL-12 IL-10, and IL-13 IL-23, IL-27

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In the past, methods of isolating T cells from human skin such as enzymatic digestion and mechanical dissociation yielded small numbers of T cells, leading researchers to believe that normal skin contains very few T cells. Newer methods have proven different. Recently-developed migration methods use chemokines produced by dermal fibroblasts to draw T cells out of skin biopsies in culture, yielding much higher numbers of recovered cells than seen before. We now know that normal non-inflamed human skin contains about 1 million T cells per square centimeter.85 Skin as a whole contains about 20 billion T cells, more than twice the total number found in the blood. Immunohistochemical analysis of normal human skin has shown that more than 95% of T cells in non-inflamed human skin are memory T cells, and less than 5% are naïve. Most Tem in normal skin express CLA, about 50% express CCR8, and a 82 subset express CCR7 and CCR10. More than 95% of skin-resident CD4+ Tem cells are TH1 polarized.

1.2.2.4 Regulatory T cells

A significant proportion of skin-resident T cells are regulatory T cells. Treg suppress inflammatory responses, create tolerance to normal flora, and prevent autoimmunity. For this reason, the manipulation of Treg in humans is being researched as a potential therapy for immune disorders such as allergies, type 1 diabetes, multiple sclerosis and graft versus host disease. However, the mechanisms of

15 Treg differentiation and function are not yet fully characterized. Classic Treg can be identified by their expression of the transcription factor Foxp3 and the cell surface marker CD25. In humans, Treg are thought to be created via two separate pathways. The first is in the thymus, where CD4+CD25+Foxp3+ naïve T cells are resistant to clonal deletion and are thus positively selected and released to the periphery.

When activated by their specific antigen, these cells are already of the Treg phenotype and will thus suppress the activation and proliferation of other T cell subtypes by producing inhibitory cytokines such as IL-10 and TGFβ.

The second pathway of Treg differentiation occurs in the periphery, where naïve undifferentiated CD4+ T cells leave the thymus and circulate in the blood. Upon first activation, signals from APCs and the surrounding microenvironment direct them to differentiate into Treg. This second pathway, called „peripheral conversion,‟ is thought to be dependent on all-trans retinoic acid (ATRA) with or without 86-88 TGFβ. Additionally, IL-10 and IL-2 have been implicated as players in promoting Treg differentiation.

Treg conversion of naïve T cells can happen in 2-4 days in vitro or 8-14 days in vivo.

Like other CD4+ T cells, Treg have antigen-specific αβ TCRs and need TCR-specific stimulation to become activated, but once activated they suppress other immune cells in an antigen non-specific fashion. It is known that Treg can suppress CD8+ T cells and TH1 or TH2 helper T cells, but they have not yet been shown to directly suppress TH17. Suppression of other lymphocytes is dose-dependent, though it does not necessarily need equal ratios of Treg to Tem. Studies in vitro have shown that suppression can be both cell-cell contact dependent (via CTLA-4) and contact independent mechanisms (via IL-10 and TGFβ production). CTLA-4 is expressed on most Treg and acts as an inhibitor by directly binding to effector T cells and competing for binding to B7 molecules on APCs. Treg also produce TGFβ which acts on neighboring cells, though some studies have shown that Treg production of TGFβ is so low that it is more likely that they exert suppression by inducing TGFβ production in other cells.

Despite a diverse TCR repertoire, Treg specificity is predominantly directed to self-antigen and are thus the main players in inhibiting autoimmunity. It is not known what factors cause a self-recognizing T cell to either undergo clonal deletion, anergy, or differentiation into Treg, though it is thought that the avidity of the TCR for self-antigens plays a role: high avidity for a self-antigen would cause a T cell to undergo deletion, and moderate avidity for a self-antigen would cause it to differentiate into a Treg.

Once activated and differentiated, Treg undergo clonal expansion in the same way as other T cells.

Treg that have been activated in skin-draining lymph nodes acquire skin-homing receptors. In normal human skin and peripheral blood, CD4+Foxp3+ Treg represent about 5-10% of resident T cells. Upon re- exposure to antigen, memory Treg proliferate just as other memory CD4+ T cells. It has been shown that in human skin, secondary cutaneous immune responses (using intradermal injection of tuberculin purified

16 protein derivative) induce Treg proliferation at the same rate as effector T cells, suggesting that skin- 89 resident Treg likely maintain immune homeostasis and dampen inflammatory responses in skin. Several diseases have been shown to be caused by impaired function or decreased numbers of

Treg. Children with allergic asthma have significantly reduced numbers of peripheral Treg than normal patients, and children with persistent or severe allergies had fewer Treg than children with intermittent allergies; however, no functional difference has been found in these conditions.90 In contrast, patients with active rheumatoid arthritis have normal numbers of Treg, but their Treg are dysfunctional due to reduced surface expression of CTLA-4. Infliximab, an anti-TNFα drug used to treat rheumatoid arthritis, 91-94 works in part by inducing a new subset of Treg that suppress via TGFβ-dependent mechanisms.

Research on psoriasis suggests that psoriasis patients‟ Treg have decreased suppressive function compared to Treg from normal volunteers. Additionally, much research is being done on the protective role of Treg in 95 inflammatory bowel disease (IBD). In mouse models, adoptive transfer of Treg can prevent the onset of 96-98 IBD, and human IBD patients have a lower proportion of Treg to TH17 T cells in their peripheral blood.

However, no studies have yet investigated the role of Treg in acne pathogenesis or resolution.

1.3 Acne

1.3.1 Epidemiology

Acne vulgaris is one of the most common human ailments, affecting nearly everyone at some point in their lives (Figure 1.3). Though „physiological‟ acne is considered normal during puberty, „clinical‟ acne may affect up to 54 percent of adults beyond the adolescent period.99 At any given time 40 to 50 million Americans are afflicted by acne, and one-third of these require specialist treatment by a dermatologist.100 In the United States, acne is the second most costly dermatological disease, representing $2.5 billion per year in direct medical costs and $1.2 billion in systemic prescription drug costs.101 Acne can not only cause significant physiological morbidity such as permanent scarring, but it can also cause significant psychological morbidity including low self-esteem, depression, and anxiety.102,103 Acne patients have significantly higher suicide ideation levels (5.6%) compared to the normal population (2.4 to 3.3%).104,105 Additionally, acne is associated with discrimination and decreased rates of employment in adulthood.103 Acne typically begins at puberty and wanes during late teens or early twenties. The age of onset for females is typically lower (12 to 13 years of age) than for males (14 or 15 years of age) due to the age

17 at onset of puberty. Males generally have more severe acne as a result of higher androgen levels. Ethnic differences also exist in acne prevalence; acne is more common in westernized cultures, with Caucasians being affected more than African Americans. Individuals of Asian descent tend to have very little acne. Acne has a partially genetic etiology; twin studies showed that 81% of the variance of the disease was attributable to genetic factors.106

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Figure 1.3: Acne vulgaris. Acne usually affects areas of high sebum production such as the face, chest, and back.

NIH Medline Encyclopedia ______

1.3.2 Pathophysiology

The pathophysiology of acne is thought to have root in the interplay between four factors: 1) follicular hyperkeratinization, 2) excess sebum production, 3) colonization with P. acnes, and 4) inflammation.

1.3.2.1 Follicular Hyperkeratinization

The initiating event in the development of an acne lesion is the formation of a microcomedone. Consequently, the number of microcomedones correlates with acne severity. Keratinocytes at the opening of the follicle hyperproliferate and physically block the opening to the surface of the skin, allowing lipids and other material to accumulate in the follicle. Microcomedones are only visible by

18 histology and are not clinically evident until they transform into an open or closed comedone (Figure 1.4). Closed comedones, typically called „whiteheads,‟ result from the distention of the follicle whose orifice is closed, creating a small palpable lesion. Closed comedones are typically the most common non- inflammatory lesions in acne patients, and also have the most potential to become inflammatory. Open comedones, commonly referred to as “blackheads,” usually develop from closed comedones. They can be up to three millimeters in diameter and get their nickname from melanin found in the plug that gives the lesion a black color. Most acne patients have blackheads, but some patients may have very few or none at all. Acne vulgaris cannot be separated into exclusively inflammatory or non- inflammatory, as most patients will have at least some of both types.

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Figure 1.4: Types of acne lesions. Acne lesions are formed from blockages of the hair follicle called microcomedones. Microcomedones can develop into (a) inflammatory pustules, (b) non-inflammatory open comedones, or (c) closed comedones.

NIH Medline Encyclopedia ______

1.3.2.2 Excess Sebum Production

There is a correlation between sebum production and acne severity; on average, acne patients produce 59% more sebum than individuals who have never had acne.12,107,108 This pattern is partially explained by systemic androgen and IGF-1 levels which increase sebum production. Males have higher androgen levels and are afflicted by acne more than females, and women with acne have higher serum IGF-1, DHEAS, androstenedione, and testosterone than women without acne.109,110 Acne is also a symptom of exogenous administration of androgens and diseases characterized by increased androgen levels, such as polycystic ovary syndrome and adrenal carcinomas. However, acne usually wanes in early

19 adulthood, while serum androgen levels remain stable until around age 40 when they begin to gradually decline, indicating that systemic androgen levels only have a partial effect on acne pathogenesis. Local androgen production by the skin likely contributes to acne pathogenesis. Levels of 17β- HSD were found to be higher in skin of acne subjects than in non-acne prone subjects, though not statistically significant.111 5α-reducatase activity is significantly greater in acne-prone skin (facial skin and scalp) than in non-acne-prone skin (arm, abdomen, or leg) in normal patients.112 However, 5α- reducatase activity does not differ between acne patients and non-acne patients, and 5α-reductase has not proven to be an effective direct target for acne therapy.109,111 A systemic type-1 5α-reductase inhibitor (Compound A) did not clinically improve acne in a randomized, controlled, double-blinded trial, nor did it augment the beneficial effects of minocycline.113 Additionally, 5α-reductase inhibitors do not reduce overall sebocyte activity or proliferation in SZ95 sebocytes in vitro.114 One way that sebum contributes to acne pathophysiology is by supporting the growth of P. acnes, which releases enzymes that convert TAG into FFA, its principle nutrient. Sebum composition may also play a role in acne. Certain lipids found in sebum may be directly comedogenic, such as squalene and FFA, and acne patients have higher proportions of these in their sebum than normal people.107 Peroxidated squalene can also activate NF-κB signaling, contributing to the inflammatory milieu in acne lesions.115 Antimicrobial agents that inhibit P. acnes growth also change sebum composition by reducing the amount of FFA present in the follicle, thereby reducing the inflammatory and comedogenic effects of these lipids.

1.3.2.3 P. acnes and Inflammation

Inflammatory lesions are usually the most troubling to acne patients because they are the most visually evident and can cause significant physical discomfort and scarring. They develop from microcomedones or closed comedones and become papules, pustules, or deep nodules. P. acnes stimulates keratinocytes and sebocytes in the follicular duct, causing expression of inflammatory cytokines such as IL-1α, IL-1β, IL-8, IFNγ, TNFα, and TGFα.116 These cytokines subsequently promote infundibular hyperkeratinization and recruitment of immune cells. T cells are among the first cells recruited to the site within the first six hours of inflammatory lesion formation in a CD4+ to CD8+ ratio of nearly three to one.117 Neutrophils follow within 24 hours, followed by macrophages, which migrate into the surrounding dermis. As a result of this inflammatory recruitment, multiple inflammatory genes and cytokines including TNFα, IL-1β, IL-8, IL-10, and matrix metalloproteases are up-regulated in active acne lesions compared to normal skin.65,118

20 Because inflammation represents only one part of acne pathogenesis, relieving inflammation does not abolish acne completely. For example, agents such as corticosteroids relieve cutaneous inflammation in the short term, but overall they make acne worse with repeated topical or systemic use.119 Successful acne therapies often target P. acnes instead. Propionibacterium acnes (previously called Corynebacterium parvum) is a Gram-positive anaerophilic rod that is part of the normal flora of human skin, oral cavity, large intestine, external ear, and conjunctiva. Though it is not directly pathogenic, it can be an opportunistic pathogen for various infections such as synovitis, osteitis, sarcoidosis and other isolated cases.120 P. acnes resides in the anaerobic environment of the sebaceous glands where it feeds off of lipids in sebum. Members of the Propionibacterium genus get their name from the propionic acid that they produce as an end product of fermentation. P. acnes produces a unique peptidoglycan that contains cross-linkages between L,L- diaminopimelic acid residues, creating a unique structure which helps P. acnes resist phagocytosis by macrophages. P. acnes also makes phospatidyl inositol, which is a feature of eukaryotes and is uncommon in bacteria.121 P. acnes has specific effects on immune responses. P. acnes stimulates both classical and alternative complement pathways and induces certain classes of immunoglobulins over others. P. acnes can also augment cytotoxic T cell responses to viruses and tumors as well as skew an otherwise TH2- 122-126 inducing antigen response towards a TH1-type. As such, a substantial amount of immunology research uses P. acnes as an adjuvant for vaccines to viruses such as rabies.122 In mice, P. acnes co- infection can enhance resistance to various pathogens including malaria, Listeria monocytogenes, Mycobacterium leprae, trypanosoma, and leishmaniasis, but it can also be damaging by increasing the severity of LPS-induced endotoxic shock through hyper-production of TNFα.127-133 Finally, there is evidence that P. acnes can induce maturation of DC by up-regulating CD80, CD86, and CD83 in peripheral blood monocyte-derived DC.134 The exact component(s) of P. acnes that stimulates immune responses has not been elucidated, but several studies have implicated a surface polysaccharide as the primary immune antigen. Squaiella et al. showed that a soluble polysaccharide of P. acnes is one of the major antigens that modulates Type I hypersensitivity reactions to OVA in mice, and Nakatsuji et al. reported surface sialidase to be the responsible antigen for mediating P. acnes vaccine responses in mice.135-138 Nonetheless, P. acnes likely contains additional antigens that impact immune responses.139,140 For example, in a study investigating P. acnes sonicate versus its purified polysaccharide in stimulating anti-tumor responses, it was found that the whole P. acnes sonicate stimulated both macrophages and NKT cells, whereas the purified polysaccharide stimulated only macrophages.126 These observations suggest the possibility that P. acnes contains multiple antigens that play a role in acne.

21 In acne pathogenesis, P. acnes stimulates both immune cells and keratinocytes. P. acnes treatment of keratinocytes in vitro increases Ki67 expression, implying that P. acnes could directly cause hyperkeratinization in the sebaceous follicle.141 However, the major role of P. acnes in acne is thought to be activation of the innate immune system through TLRs. It stimulates monocytes, keratinocytes, and sebocytes through activation of TLR-2/TLR-1 dimers, resulting in the release of inflammatory cytokines such as IL-6, IL-8, IL-1β, IL-1α, IL-12p40, and TNFα.142 In keratinocytes, P. acnes induces expression of GM-CSF and BD- 2, whereas our lab has shown that it causes increased expression of NGAL in sebocytes.143-146 P. acnes stimulation of TLR-2 also induces increased TLR-2 expression.141 Along with neutrophil-released mediators, P. acnes components are the main chemoattractants for monocytes in acne lesions, and evidence shows that P. acnes has direct T-cell mitogenic activity.147 By these mechanisms, P. acnes contributes to the inflammation associated with acne. The involvement of P. acnes in acne is seemingly paradoxical. Its role in acne pathogenesis is supported by the fact that reducing the number of P. acnes colonizing the skin with antibiotics can be effective at treating acne, and that antibiotic resistance of P. acnes correlates with treatment failure.148 However, the strain and number of P. acnes does not always correlate with disease severity, and only some people with P. acnes colonization get acne.145,149-151 P. acnes by itself induces little or no inflammation in immunologically naïve mice, but in combination with other facultative bacteria it can be synergistic in causing pathogenic immune responses, suggesting that P. acnes only becomes pathogenic in the context of a precipitating immune event.152 Thus, it is debatable as to whether acne is considered to be an infection by P. acnes or an exaggerated immune response to this commensal organism. Indeed, recent studies suggest that a patient‟s specific immune response to P. acnes may play a larger role in acne than the pathogenicity of P. acnes itself. When challenged with an intradermal bolus of live P. acnes, acne patients produce am exaggerated local inflammatory reaction compared to people without acne.153 Additionally, peripheral blood mononuclear cells (PBMC) from acne patients release more IL-8, IL- 12p40, and IFN in response to in vitro stimulation with viable P. acnes than PBMC from normal volunteers, regardless of whether the P. acnes strain is isolated from the skin of an acne patient or a normal patient.154 Finally, acne patients have higher serum levels of antibodies to P. acnes than normal patients or patients with skin diseases other than acne, but they do not differ from normal subjects in their serum levels of antibodies to other flora such as S. epidermis.155,156 Acne patients‟ exaggerated immune response to P. acnes may be explained by recent studies which report that acne patients‟ peripheral blood monocytes and skin keratinocytes express higher levels of TLR-2 compared to normal controls.51,141 Though one study has found that acne vulgaris is associated with the TLR-2 Arg753Gln polymorphism in a Chinese Han ethnic group, most studies have found that TLR-2 and TLR-4 polymorphisms are not associated with acne, suggesting that in western populations

22 TLR-2 expression levels rather than functional differences are responsible for the hyper-responsiveness of acne patients‟ monocytes to P. acnes.157,158 These findings suggest that altering acne patients‟ immune responses to P. acnes could be a potential target for future therapies.

1.3.3 Current Treatments for Acne

A number of different therapies are used to treat acne, including keratolytic agents, hormonal therapy, and antibiotics. Most treatments for acne focus on only one or two of the four different pathophysiological causes, and almost all of them provide only transient benefits; once the medication is stopped, acne returns. Keratolytic agents reduce the hyperkeratinization associated with acne, thus reducing the number of comedones that would have the potential to become inflammatory. Other agents aim at reducing inflammation or P. acnes colonization.

1.3.3.1 Topical Treatments

Topical therapy may be useful for mild acne cases or in conjunction with systemic treatments. There are many topical treatment options available for acne in the form of creams, lotions, toners, and cleansers, many of which are available over-the-counter. Benzoyl peroxide, an antimicrobial agent, is widely used in over-the-counter acne medications. While effective at reducing P. acnes colonization and alleviating inflammation, benzoyl peroxide can cause skin irritation and bleaching of hair and clothing. Prescription topical treatments include antibiotics such as erythromycin or clindamycin that reduce P. acnes counts. At least one study has shown that killed P. acnes is still capable of inducing inflammatory responses in human skin, which may explain how bactericidal agents are not completely curative for acne without concurrent use of other therapies.144 Topical keratolytic agents such as salicylic acid, alpha-hydroxy acids, and azelaic acid are sold in over-the-counter products. Prescription retinoids are more effective in preventing hyperkeratinization. Tretinoin (ATRA; brand name Retin-A) was the first retinoid to be used in this way, though newer generation retinoids such as adapalene and tazerotene have shown to be equally effective with fewer side effects like redness and irritation. Newer formulations of tretinoin have allowed the drug to be delivered in a low dose over time to reduce side effects.

23 1.3.3.2 Systemic Treatments

Systemic treatments for acne may be indicated in severe cases or in patients who have been unresponsive to topical therapies. Antibiotics are prescribed as systemic therapy for acne and act in the same way as topical antibiotics do, by reducing the numbers of P. acnes colonizing the sebaceous gland. Commonly prescribed oral antibiotics include erythromycin and tetracyclines. However, erythromycin also inhibits P450 enzymes in the liver, in particular 3A4, which results in many potentially dangerous interactions with other drugs. For this reason the tetracyclines have become a more popular choice for acne treatment. The tetracycline family of antibiotics (including tetracycline, doxycycline, and minocycline) inhibit bacterial protein synthesis by binding with the 30S ribosomal subunit. They also have direct effects on human cells, including reducing leukocyte migration by inhibiting proteases like matrix metalloproteinases (MMP). Some antibiotics such as roxithromycin and nadifloxacin may also have anti-androgen effects by suppressing androgen receptor transactivation.159 Minocycline has best log- reduction in P. acnes growth, but side-effects such as skin discoloration limit its use. Systemic antibiotics are more effective than topical antibiotics for treating acne, but have more side-effects and are also more likely to contribute to the creation of antibiotic-resistant bacterial strains, which have become a significant health concern in recent years. Indeed, studies have shown that the overall incidence of P. acnes antibiotic resistance has increased from 20% in 1978 to 62% in 1996 due to the use of antibiotics as acne therapy.160 For this reason, it is recommended that systemic antibiotics be used for less than six months if possible, and never as a monotherapy. The concurrent use of topical benzoyl peroxide can help to reduce the chance of resistance formation in P. acnes, but does not prevent resistance formation in bacteria elsewhere in the body. Oral hormonal therapy is an effective systemic acne treatment for females. Hormonal contraceptives contain estrogens and progestins that antagonize androgens, thus decreasing sebum production.161,162 Certain progestins even have specific anti-androgen activity. Spironolactone is a progestin component of certain oral contraceptives that inhibits 17β-HSD/5a-reducatase activities and antagonizes aldosterone. Cyproterone acetate is a progestin that acts as an androgen receptor blocker and is also used with estrogen in oral contraceptives. However, anti-androgens induce gynecomastia in men and thus can only be used in females, and acne frequently returns at the cessation of therapy. The only other therapy that affects sebum production is oral isotretinoin.

24 1.3.3.3 Isotretinoin

Isotretinoin (13-cis retinoic acid; 13-cis RA) was developed by Roche Pharmaceuticals and first used as a treatment for ichthyosis in the 1970s, when researchers noted a remission in patients‟ acne during treatment.163,164 In 1982, the FDA granted approval for the use of isotretinoin to treat severe or recalcitrant acne. Marketed under the brand names Accutane, Amnesteem, Claravis, and Sotret, isotretinoin is also prescribed „off-label‟ for a variety of other dermatological diseases including psoriasis, Gram-negative folliculitis, pyoderma faciale, and severe rosacea as well as some cancers, including acute promyelocytic leukemia (APL), cutaneous T-cell lymphoma, and some solid tumors.165 Isotretinoin is the only known agent that affects all four components of acne pathogenesis, reducing sebum production up to 90% and hyperkeratinization by 50% as well as reducing P. acnes colonization and inflammation. Isotretinoin is also the only known agent capable of inducing a permanent cure; up to 80% of patients stay in remission long-term after the cessation of therapy, though about 41% will eventually experience an acne relapse that will need second course of isotretinoin.166,167 Though relatively expensive, isotretinoin is taken for only six months and is the only therapy that alters the course of acne long-term, making it one of the most cost-effective therapies. Conventional dosing for acne is typically 0.5 to 1.0 mg/kg/day for 16 to 32 weeks of therapy, reaching a cumulative dose of 120 to 150 mg/kg. There is evidence that lower dosing for longer periods can have equal efficacy with potentially less-severe side-effects, but this then increases the amount of time that females are exposed to teratogenic levels of the drug.168 Though isotretinoin is very effective at treating acne, it has many serious side-effects, including birth defects. Common mild side-effects include chelitis, pruritis, and photosensitivity of the skin. Isotretinoin can also cause many serious side-effects such as insulin resistance, hypertriglyceridemia, hepatotoxicity, and pseudotumor cerebri. Though most side-effects such as moderate to severe elevations in serum lipid and transaminase levels are transient in most subjects (more than 79%), up to 45% of patients may experience a side-effect serious enough to warrant cessation of therapy.169 Isotretinoin has also been linked to depression and suicidal behaviors, though a causal link has not yet been established mainly due to the compounding underlying association between severe acne and suicide ideation.170 Because of its teratogenic potential and serious side-effect prolife, the distribution and use of isotretinoin are regulated by the US Food and Drug Administration via an online registration program called iPLEDGE, making isotretinoin one of two drugs in the United States (along with thalidomide) to be so strictly controlled. Its indications for use in acne are thus restricted to the most severe cases: nodulocystic acne that is unresponsive to other prescription treatments or that causes scarring and/or undue psychological stress to the patient.

25 Isotretinoin belongs to a class of compounds called retinoids. Retinoids and their functions are discussed further in Section 1.4.

1.3.4 Model Systems for Acne Research

1.3.4.1 Cell Lines

There are various cell lines available for each type of cell found in the skin. Primary sebocytes are nearly impossible to culture in vitro because they differentiate and rupture before they can proliferate to any substantial confluency. Our lab created an immortalized sebocyte cell line (SEB-1) by transfecting primary sebocytes from the ear of a 55 year-old male patient with SV40 large T antigen under the control of a CMV promoter.14 Large T antigen protein binds to and hyperphosphorylates Rb, p53, and hsc70, which collectively override signals for senescence and apoptosis, allowing cells to be passaged continuously in culture in numbers great enough for use in any assay.171 Another immortalized sebocyte cell line called SZ95 has been created by Zouboulis et al., also using large T antigen.172 Recently, the reversible immortalization of primary sebocytes has been accomplished in our lab using the drug Y-27632, which is an inhibitor of Rho kinase. Primary sebocytes dissected from human skin biopsies are treated with Y-27632 in culture along with a feeder layer of 3T3 fibroblasts. In these conditions, sebocytes lose contact inhibition and grow to confluency. The development of this model is further discussed in Chapter 3. There are numerous keratinocyte cell lines commercially available for research. The most common primary cells are normal human epidermal keratinocytes (NHEK). The term NHEK does not refer to a single cell line, but is a general term for primary keratinocytes isolated from adult human skin or neonatal foreskin that are then normalized and cryopreserved. NHEK can undergo only a limited number of population doublings, making them a relatively expensive and time-sensitive cell line to use for research use. In contrast, immortalized keratinocyte cell lines can be passaged continuously. HaCaT keratinocytes are immortalized human keratinocytes that were taken from the edge of a tumor, whereas KH-SV are keratinocytes that were created by transformation with SV-40 large T antigen.

26 1.3.4.2 Animal Models

There is no comparable animal model for inflammatory acne because humans are the only animals that are colonized by P. acnes. Humans sebum has a high level of triglycerides which serve as the main food source for P. acnes. Most animals do not produce any triglycerides in their sebum, whereas other animals such as rabbits, hamsters, and mice have only very low concentrations.173 Various animal models have been used for acne research, but each model can mimic only one or two aspects of acne pathogenesis. For researching the physiology of the pilosebaceous unit, mouse and rat preputial glands are sometimes used as a model. Preputial glands are pheromone-secreting eccrine glands that are found near the genitals in some mammals, and those found in mice and rats are similar in structure to human sebaceous glands. The M2-SMO mouse has increased sonic-hedgehog activity in the skin, causing increased sebaceous gland development.174 The Hairless mouse is homozygous for the „hairless‟ gene (hr/hr) which regulates the transition between growth phases in the hair cycle, making the Hairless mouse a useful model for research involving the hair follicle. One model for acne research has been bioengineered to represent the pilosebaceous microenvironment using mouse dermal skin explants seeded with human sebocytes in a tissue chamber.135 For researching acne comedogenesis, rabbit ears and hairless mice have been used as models. The Rhino mouse has a characteristic hyperkeratinization of the epidermis and so is often utilized in research involving keratolytic agents.175 However, these models do not have the inflammation that is associated with acne. To simulate the inflammation and P. acnes colonization associated with acne, researchers often inject P. acnes intradermally or subcutaneously into rabbit or hamster ears to cause inflammation. De Young et al. has shown that the injection of formalin-killed P. acnes intradermally into the ears of rats caused an inflammatory response similar to an acne lesion.176,177 This same group also demonstrated that intradermal immunization with formalin-killed P. acnes can create a hypersensitivity response when rats are later challenged intradermally with live P. acnes.177 Mouse models have been particularly useful for studying the physiology and function of retinoids. Because systemic retinoid treatment has potentially dangerous side effects and is teratogenic, the administration of systemic retinoids to healthy human subjects is unethical. Animal studies on retinoids frequently employ a special diet to induce vitamin A deficiency, which is then treated with exogenous ATRA. Thus, animal models have been used to study the physiology of retinoids as well as the pharmacokinetics of different formulations or derivatives.

27 There are several drawbacks to animal models, the primary one being that P. acnes cannot grow in the pilosebaceous unit of animals other than humans. Injecting P. acnes intradermally or subcutaneously into animals is merely an approximation of the inflammation that occurs in acne, and does not exactly replicate the microenvironment of a comedone. Additionally, mouse skin is much thinner and thus more permeable to drugs than human skin, which somewhat limits conclusions drawn from topical drug testing in mice.178

1.3.4.3 Human Subjects

Human subjects are the ideal model for acne research because there is no comparable animal model. Certain procedures in humans are limited for the obvious reasons of safety and plausibility, but topical drug testing is readily accomplished in humans because it is generally low-risk and non-invasive. Human skin samples can be obtained by punch biopsy (three or four millimeters in diameter) for assays such as tissue culture, gene-chip analysis, or immunohistochemistry. Less invasive procedures can be employed to examine surface proteins or sebum production. Tape stripping removes the surface layer of keratinocytes using sticky tape; protein can be extracted from these tape strips and studied using various techniques. Sebum can be easily measured using neutral solvents to extract sebaceous lipids from the surface of the skin, which are then analyzed by gravimetric or chromatographic techniques. Alternatively, absorptive surfaces such as cigarette paper or frosted glass change transparency when they come in contact with lipids, and the corresponding change in light transmission can be quantified as a function of sebum amount.179,180

1.4 Retinoids

1.4.1 Retinoid Biology, Functions, and Metabolism

Retinoids are vitamin A metabolites that have many important functions in development, vision, immunity, and cell growth. Retinoids are characterized chemically as compounds containing a retinyl group, consisting of a beta-ionone group attached to an isoprenoid chain. Vitamin A (retinol) is absolutely essential for vision and is converted into 11-cis retinal for incorporation into retina pigments in the eye. Retinol and retinaldehyde (retinal) can be irreversibly converted to retinoic acid, which is the biologically active endogenous retinoid.

28 Vitamin A deficiency is a global health problem. The World Health Organization estimates that 250,000 to 500,000 children in developing countries become blind each year due to vitamin A deficiency.181 Reduced plasma retinoid levels have also been associated with increased risk of mortality in children and cancer in adults.182-184 In developed countries, however, vitamin A toxicity is becoming more prevalent due to over-supplementation. The Recommended Dietary Allowance of vitamin A is 3,000 IU per day for male adults and 2,310 IU for female adults, whereas the Tolerable Upper Intake Level for vitamin A is 10,000 IU. Many over-the-counter vitamin supplements sold in the United States contain as much as 25,000 IU. Because retinoids are fat soluble, they are stored in the liver and adipose tissue in the body and can easily reach toxic levels with retinol overdose. Acute vitamin A toxicity may result in nausea, vomiting, blurred vision, dizziness, and impaired muscle coordination. Chronic vitamin A toxicity, also called Hypervitaminosis A, is characterized by liver abnormalities, reduced bone mineralization, birth defects, hair loss, skin desquamation, and central nervous system disorders. Retinoids are obtained from the diet in the form of retinol or retinyl esters from animal sources or as carotenoids (such as beta-carotene) from plant sources and are absorbed in the small intestine with the aid of bile salts (Figure 1.5). Retinyl esters undergo hydrolysis to produce more retinol, and retinol is esterified (usually with palmitic acid) and transported to the liver in a lipoprotein complex. Retinol dehydrogenase (RDH) in liver hepatocytes and other tissues converts retinol to retinal and releases it into circulation, where it binds to retinol binding proteins (RBP). Cells take up plasma retinal and irreversibly convert it to ATRA using retinaldehyde dehydrogenases (Raldh1, Raldh2, and Raldh3). Cells that do not contain these enzymes cannot use retinal and must therefore depend on other cells in their microenvironment for ATRA. Cellular retinoic acid-binding proteins (CRABP) is a carrier protein that transports ATRA from the cytosol to the nucleus of cells, where it has access to its nuclear receptors, though there is also evidence that CRABP can sequester excess ATRA away from nuclear receptors as well as regulate ATRA‟s metabolism.185-187 ATRA can be metabolized by many different enzymes into ketones, epoxides, and hydroxylated derivatives, but the major route of ATRA elimination from the body is thought to be via metabolism by cytochrome P450 (CYP) enzymes and then excretion in urine and bile.188 The CYP26 subfamily is the main group of enzymes that hydroxylates the retinoic acid isomers. CYP26A1 and CYP26B1 primarily hydroxylate ATRA, but may also metabolize 13-cis RA and 9-cis RA.189 ATRA is the most thermodynamically stable retinoic acid isomer, but it can readily isomerize to 13-cis RA or 9-cis RA, or vice versa in biological systems (Figure 1.6).190 Thus all three isomers can be present in cells at the same time. 9-cis RA can also be formed through oxidation by Raldh, but this has not been shown to occur in vivo.191

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ATRA exerts its effects through binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR), which are nuclear transcription factors. Both ATRA and 9-cis RA act as ligands for RAR, but 9- cis RA is currently the only known endogenous ligand for RXR. RAR and RXR receptors form dimers with each other to bind to retinoic acid response elements (RARE) in gene promoter regions and induce transcription of target genes. RXR receptors can also form dimers with other nuclear transcription factors including the vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPAR), liver X receptors (LXR), thyroid hormone receptors (TR), farnesoid X receptors (FXR), pregnane X receptors (PXR), and constitutive androstane receptors (CAR) (Figure 1.7). Almost every human tissue expresses at least one RXR subtype, indicating the importance of these receptors in cell signaling.192 Interaction

31 with RXR increases the ligand-binding affinity for the partner receptor and is essential for its function. Some of these receptors (PPAR, LXR, FXR, and PXR) can be activated by RXR ligands in the absence of their own ligands, whereas others (RAR, TR, and VDR) require binding of their own ligands for activation. By this mechanism, retinoids can influence gene expression in many different pathways. Interestingly, the intracellular ratio of CRABP-II to fatty acid binding protein has been shown to determine whether ATRA acts through RAR or PPARβ nuclear receptors, which results in distinct functional outcomes.193 A third type of retinoid receptor, the retinoic acid-related orphan receptor (ROR), has high homology to RAR and has many functions in immune development and responses. ROR receptors do not form dimers with other receptors and appear to function as monomers when binding to response elements. ATRA binds to RORβ and ROR with high affinity, and melatonin has been proposed as a ligand for RORα.194,195 X-ray crystallography has implicated cholesterol as an additional ligand for RORα, suggesting that this receptor may play a role in cholesterol homeostasis.196

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32 During embryonic development, endogenous ATRA modulates the expression of HOX gene clusters by acetylating local histones and opening the chromatin structure to transcription factors.197 Spatial and temporal expression of individual HOX genes control anteroposterior and lateral patterning of tissues. As a result, exogenous administration of retinoids is extremely teratogenic because it disrupts the endogenous ATRA gradient in the developing embryo. Even high supplementation with vitamin A raises serum ATRA levels, increasing risk of teratogenesis.198,199 Retinoic acid embryopathy includes birth defects such as craniofacial, cardiac, thymic, and central nervous system malformations. Retinoids are frequently used clinically for treating cancers and dermatological disorders. Human skin expresses all forms of RARs and RXRs, though RARγ and RXRα are the most common.200-203 While supplementation with high levels of vitamin A can improve acne slightly, the risk of Hypervitaminosis A is high.204-206 Certain vitamin A derivatives and synthetic retinoids can be used clinically with fewer or less severe side-effects. Pharmacologically-used retinoids are divided into three generations or classes. The first generation of retinoids includes retinol, ATRA (tretinoin), 13-cis RA (isotretinoin), and 9-cis RA (alitretinoin). ATRA is an effective topical, but not oral, therapy for acne. In contrast, isotretinoin is an effective oral therapy for acne but loses its sebostatic effect when applied topically.207 The second generation of retinoids includes the synthetic compounds etretinate and acitretin, both of which are used systemically for the treatment of psoriasis. The third generation retinoids bexarotene, tazarotene, and adapalene were developed to have similar efficacy in treating cancers and cutaneous disorders with reduced side-effects compared to the first generation retinoids. Tazarotene and adapalene are used topically for acne, showing nearly equal efficacy with less skin redness, peeling, and irritation.208,209 Much of the clinical pharmacological data for ATRA has come from studies using ATRA as a systemic chemotherapy agent for cancers such as acute promyelocytic leukemia (APL). There is great variability in retinoid metabolism between patients; as such, ATRA has a highly variable peak plasma concentration, which peaks about one to two hours after oral dosing.210 ATRA has a relatively short half- life in serum, about 45 minutes.211 The early observation that some APL patients showed reduced responsiveness with repeated ATRA administrations suggested that ATRA can induce its own catabolism.212 The mechanism for the reduced responsiveness to therapy was found to be decreased plasma ATRA levels caused by a combination of reduced intestinal absorption, increased enzymatic catabolism, plasma lipid peroxide levels, and increased plasma CRABP levels.211,213 As a result of these findings, much of the recent clinical trials with retinoids have focused on isotretinoin as an alternative to ATRA because isotretinoin has more favorable pharmacokinetic properties. Isotretinoin has a higher peak plasma concentration and significantly longer half-life (about 22 hours) compared to ATRA, possibly because it is less readily metabolized by CYP enzymes.214 Isotretinoin is thought to be a pro- drug for ATRA by acting as a stable reservoir for biologically active isomers or metabolites.215

33 Differences in metabolism and bioavailability may help explain why these isomers differ in their efficacy in treating acne. CRABP may also play a role in acne therapy efficacy by targeting retinoids to skin. CRABP-II is the most abundant retinoid carrier protein expressed in normal human skin.216 Topical treatment with ATRA, isotretinoin, or adapalene increases CRABP-II expression in the skin, and systemic treatment with acitretin and the synthetic retinoid Ro137410 also increases CRABP-II in the skin.216-218 In contrast, systemic isotretinoin does not induce overall skin expression of CRABP-II to the levels found with systemic acitretin or Ro137410, but it does induce CRABP-II specifically in the sebaceous glands compared to the rest of the epidermis.219-222 If CRABP-II acts as a delivery protein for retinoic acid isomers, then this could represent one way by which systemic isotretinoin therapy specifically targets sebaceous glands.

1.4.2 Retinoid Effects on the Immune System

Retinoids play many roles in modulating the immune system, which is clinically evident by the fact that vitamin A deficiency is paradoxically linked to both autoimmune disease and immune deficiencies. Vitamin A‟s actions in immunity are tightly regulated within microenvironments. Local ATRA concentrations are controlled by DC, monocytes, and intestinal epithelial cells, which are the only immune cells known to express Raldh enzymes. By controlling the conversion of retinal to ATRA, these cells control T cell subtype differentiation in local microenvironments such as the gut and skin. DC can also accumulate and store ATRA for subsequent release during antigen presentation.223 Retinoids have many effects on the adaptive immune system. ATRA has pronounced effects on T cell subset differentiation by acting both directly on T cells as well as through APC priming. ATRA has significant influence on TH1/TH2 balance, though there are conflicting reports as to whether ATRA promotes TH1 or TH2. The general consensus is that ATRA promotes TH2 responses and suppresses TH1, which has been shown in vitro using normal mouse and human cells as well as in vivo in mice.224,225 However, cancer trials using ATRA as a chemotherapy drug in humans suggest that ATRA enhances endogenous defenses against tumors in part by inducing cytotoxic T cell responses through production of IFNγ.226 This discrepancy might be explained by the presence or absence of IL-2 in the differentiating microenvironment: treatment of normal human PBMC with either ATRA or 13-cis RA plus IL-2 induces IFNγ and IL-12p40, but these retinoids do not induce IFNγ or IL-12p40 on their own.227

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ATRA also mediates immune homeostasis by suppressing autoimmune TH17 responses and promoting Treg. Peripheral conversion of naïve T cells into Treg is thought to be dependent on ATRA with or without TGFβ (Figure 1.8a).86,87,228 It has been shown that in vitro ATRA treatment converts purified adult human peripheral naïve T cells into CD4+CD25+Foxp3+ Treg cells, both directly through RAR 86,87,229 activation and indirectly through the inhibition of cytokines that prevent Treg conversion. 230,231 Additionally, retinoids suppress the differentiation of naïve T cells into the TH17 phenotype. ROR t is necessary for the differentiation of TH17 T cells and is activated by downstream actions of IL-6 and TGFβ, but ATRA blocks the RORγ induction by these cytokines.228,229,232 Retinoids also have distinct effects on APC. Monocyte-derived DC express high levels of RARα and RXRα receptors, and also express high levels of RXR partner receptors such as VDR, LXRα, PPARγ,

35 and PPARδ.233 In the absence of inflammatory signals, retinoids induce apoptosis in DC via a RARα/RXR-dependent mechanism. In the presence of inflammatory cytokines, however, retinoids promote the maturation of DC and enhance antigen presentation to T cells, which is mediated by an RXR- dependent but RAR-independent pathway.234 Human monocytes and macrophages express RARα, RXRα, LXRα, and PPARγ receptors. Previous studies have demonstrated the ability of ATRA treatment in vitro to decrease the production of inflammatory cytokines by antigen-challenged monocytes. ATRA treatment of human umbilical cord PBMC increases IL-10 and decreases IL-12 and TNFα levels upon LPS challenge, but had no effect on IL-8 or TGFβ secretion.235,236 Additionally, ATRA treatment of monocytes in vitro down-regulates TLR-2, but not TLR-4 expression in human PBMC stimulated with TLR-2 ligands.237 These effects on APC have downstream consequences for T cell activation and differentiation. When monocytes and naïve T cells are cultured together, ATRA treatment can affect the subset differentiation of T cells by modulating the cytokines produced by the stimulated monocytes.238,239 Retinoids are especially important for maintaining mucosal immunity and the induction of oral tolerance to antigens. ATRA promotes B cell differentiation and class switching to IgA through RAR- dependent signaling (Figure 1.8b); accordingly, vitamin A deficiency impairs mucosal antibody responses.240-244 ATRA also controls lymphocyte homing to mucosal sites. In vitro, ATRA treatment of purified naïve T cells promotes expression of gut-homing receptors and suppresses the expression of skin- homing receptors upon stimulation (Figure 1.9).245-247 Concentrations as low as the picomolar range are 248 sufficient for the induction of α4β7 and CCR9 expression on newly differentiated Treg in vitro. Additionally, incubation of naïve T cells with ATRA-pretreated DC induces T cell expression of α4β7.223 Thus, it had been assumed until recently that ATRA worked primarily to induce gut-homing in activated lymphocytes. However, it has since been shown that in vivo, specialized populations of DC expressing Raldh exist in both the gut lymphoid tissues and skin-draining lymph nodes. Both of these specialized types of DC (CD103+ in the gut and CD103- in the skin) can generate ATRA to promote local differentiation of Treg, and both DC types direct newly-differentiated Treg to express homing molecules specific to the environment of activation.249-252 This finding suggests that ATRA may promote the expression of gut-homing receptors as a default, but that additional microenvironment signals in the skin can override default pathways to promote skin-homing receptors instead. For example, co-culture of T cells with dermal fibroblasts overrides gut-imprinting to promote expression of skin-homing receptors.253 It was recently demonstrated that Raldh enzyme expression in gut mucosal DC decreases with vitamin A deficiency and increases with vitamin A supplementation.254 It is currently unknown if a similar regulatory pathway exists for DC in cutaneous environments.

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Figure 1.9: Retinoid effects on lymphocyte homing. a) ATRA treatment of naïve T cells in vitro promotes gut- homing receptor expression and suppresses skin-homing receptor expression. However, new evidence suggests that DC in skin-draining lymph nodes can also produce ATRA to induce a skin-homing phenotype in T cells. b) ATRA treatment also promotes a gut-homing phenotype in B cells.

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37 Through the promotion of regulatory T cell responses, suppression of autoimmune TH17 responses, and control of lymphocyte gut-homing, retinoids have been shown to have a possible therapeutic role for inflammatory bowel disease. Systemic ATRA treatment increases Treg and decreases

TH17 T cells in colon tissue of mice with TNBS-induced colitis, alleviating the colonic inflammation and systemic weight loss associated with colitis.255 On the same note, a diet high in retinyl acetate decreases inflammation and increased the number of Treg in the intestines of a mouse model of Crohn‟s Disease, but 247 does not affect Treg function compared to a normal diet. ATRA treatment of colon biopsies from 255 human colitis patients increases Treg and decreases TH17 T cell differentiation. These data are all the more intriguing because isotretinoin has recently been implicated as a causative factor for Crohn‟s disease in humans.256-261 This link is highly controversial and has not yet been proven; nevertheless, isotretinoin manufacturers have been successfully litigated. This association may be explained by new research into the role of gut microflora in maintaining gut immune homeostasis. Disturbances in gut flora have been shown to promote intestinal TH17 inflammation through the activation of TLR-9, and probiotics are able to prevent TNBS-induced colitis in mice.262,263 Accordingly, very recent evidence has emerged that links antibiotic use, particularly doxycycline, to a higher risk of Crohn‟s disease.264 Because isotretinoin is not usually prescribed until all other treatment options have been proven ineffectual, acne patients are likely to have been through multiple courses of systemic antibiotics before being prescribed isotretinoin. This could explain the apparent relationship between isotretinoin and Crohn‟s disease, though more studies are needed to fully elucidate the association. Finally, retinoids affect innate immune responses and antimicrobial peptide production in many cell types without the involvement of leukocytes. ATRA has been shown to induce cathelicidin protein in bone-marrow progenitor cells as well as its proteolytic processing enzymes in keratinocytes.265,266 Our lab has shown that 13-cis RA induces expression of NGAL in sebocytes, and other labs have shown NGAL induction by 4-HPR and 9-cis RA in cancer lines.267

1.4.3 Mechanisms of Retinoids in Acne Therapy

Both systemic and topical retinoids have proven to be efficacious in the treatment of a variety of dermatological disorders, including acne, rosacea, psoriasis, actinic keratosis, hidradenitis suppurativa, pyoderma faciale, and non-melanoma skin cancers. ATRA is used as a topical medication for acne and is effective at reducing hyperkeratinization, thereby reducing the number of comedones that have the potential to become inflammatory. ATRA limits hyper-proliferation of keratinocytes through the regulation of cAMP, EGF, TGFα, PKC, and TGF-β2.268 However, ATRA does not affect sebum

38 production through topical application, possibly because it cannot penetrate deep enough into skin to affect cells below the epidermis. In contrast, systemic isotretinoin treatment modulates all four factors of acne pathogenesis and can successfully treat severe recalcitrant acne. Isotretinoin‟s short-term mechanisms of action are well understood. Isotretinoin reduces follicular hyperkeratinization and comedone formation in vivo, most likely through mechanisms similar to topical ATRA.269 Additionally, patients on isotretinoin experience up to 90% reduction in sebum production by 12 weeks of therapy, whereas ATRA and 9-cis RA do not affect sebaceous glands.270,271 During isotretinoin therapy, structural changes in sebaceous glands in vivo show smaller size, fewer numbers of immature cells, and an absence of mitotic figures.272 Our lab has previously shown that NGAL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) are the primary mediators of 13-cis RA-induced apoptosis in sebocytes.57,273 Isotretinoin reduces P. acnes colonization as well, presumably by reducing sebum, which is its main food source. However, sebaceous gland size and P. acnes colonization increase again after the cessation of isotretinoin therapy, even in the context of continued clinical remission, and the long-term degree of sebum suppression is not related to relapse after isotretinoin therapy.274,275 These observations demonstrate that other factors play a role in isotretinoin‟s long-term mechanism of action. While sebaceous gland size and P. acnes colonization can return to baseline levels after the cessation of isotretinoin therapy, inflammation generally does not return. This suggests that modulation of inflammatory responses may play a role in isotretinoin‟s ability to induce long-term remissions of acne. Many in vitro studies using ATRA have shed light on the mechanisms by which isotretinoin reduces acne-associated inflammation. As discussed above, studies have shown that ATRA decreases expression of TLR-2 in human monocytes, keratinocytes, and sebocytes. Treatment of monocytes with ATRA in vitro reduces P. acnes-induced release of inflammatory cytokines such as TNFα and IL-12p40.237 Adapalene treatment decreases TLR-2 expression in skin explants from normal individuals and acne patients.276 However, these effects have not been demonstrated with isotretinoin therapy in vivo. The efficacy of retinoid therapy for acne has provoked the question of whether or not people with acne have different levels of circulating retinoids or retinoid metabolites than people without acne. Acne patients have been shown to have lower concentrations of retinol in their plasma and skin compared to normal volunteers (Table 1.3).277,278 Both ATRA and 13-cis RA are found in concentrations ranging from 1 to 3 ng/mL in normal human plasma, but the levels of these isomers in normal volunteers versus acne patients has not yet been determined.279 One study has shown that systemic isotretinoin therapy temporarily increases 13-cis RA and 4-oxo-13-cis RA in acne patients‟ serum and skin, but these increases completely reverse two to four weeks after the cessation of therapy, indicating that an enduring accumulation of retinoids in serum or skin is not responsible for isotretinoin‟s long-term efficacy.221

39 Additionally, acne patients may have fundamental differences in retinoid metabolism or transport compared to those without acne. Levels of CYP26A activity in skin do not differ between acne patients and healthy subjects without acne, though CYP26A is inducible with isotretinoin therapy.280 Two studies have reported decreased levels of serum RBP in acne patients compared to healthy subjects or those with only mild acne. However, the levels of CRABP proteins in the skin of untreated acne patients versus normal volunteers have not been studied.277,281

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Table 1.3: Plasma Concentrations of Retinoids in Acne Patients and Normal Volunteers. The chart below summarizes reports in the literature on levels of retinoids in untreated normal volunteers, untreated acne patients, and acne patients who had been on isotretinoin therapy (0.5 mg/kg/day) for 1 month. A dash indicates that the parameter has not yet been reported.

Retinoid (ng/mL) Patients on or Protein (μg/mL) Normal Volunteers Acne Patients isotretinoin therapy 1.94 ± 0.54a, 1.4 ± all-trans RA - - 0.2f, 1.32 ± 0.46g 1.51 ± 0.35a, 1.4 ± 13-cis RA - 171 ± 23.2d 0.3f, 1.63 ± 0.85g 0.67 ± 0.16a, 4-oxo-all-trans RA - - not detectableg 2.86 ± 1.32a, 3.68 ± 4-oxo-13-cis RA - 594 ± 60.4d 0.99g 645 ± 220a, 518 ± retinol 387 ± 56c, ~420h 62.6 ± 6.16d 142c, 541h 1130 ± 42b, 1082 ± carotenoids 770 ± 308c - 437c retinol binding protein 45.7 ± 9.8c, 38.9 ± 37.5 ± 6.0c, 40.9 ± 39.2 ± 1.98d (RBP) 4.7e 1.6d, 30.4 ± 4.5e

References: a) Muindi et al.211; b) Phillips et al.282; c) Rollman et al.277; d) Rollman et al.221; e) Michaëlsson et al.281; f) Tang et al.283; g) Eckhoff et al.198; h) Mier et al.278

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40 1.5 Significance of Research Project

Retinoids including isotretinoin have shown promise for the treatment of dermatological diseases and cancers. However, because these drugs have a severe side-effect profile including teratogenicity, it is unethical to administer these drugs to healthy volunteers for the purpose of research. Thus, mechanistic studies on retinoid treatments in humans in vivo are lacking because they are limited to studying only patients who are prescribed retinoids as a necessary therapy. Studies suggest that ATRA can modulate the immune system in multiple ways, but its ability to do so in humans in vivo is largely unproven. Additionally, conflicting reports regarding ATRA‟s actions in vitro have made it difficult to discern how it works in vivo. For example, though it is generally thought to promote TH2 responses, ATRA has been shown induce TH1 responses in cancer patients. The reasons behind these discrepancies are unknown, but they underscore the need for in vivo studies characterizing how retinoids work as therapies for various human diseases.

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Isotretinoin is the only known therapy that induces durable remissions of acne. However, isotretinoin‟s long-term mechanisms of action are poorly understood. In the short-term, it induces apoptosis of sebaceous glands and decreases numbers of colonizing P. acnes, though these factors partially reverse after the cessation of therapy. A substantial amount of research suggests that the ability of isotretinoin to modulate the immune system may represent one mechanism for inducing long-term remission of acne. Treatment of normal human monocytes with ATRA in vitro decreases surface expression of TLR-2, thereby decreasing inflammatory cytokine production in response to stimulation

41 with P. acnes. However, these effects have not been shown with isotretinoin therapy in acne patients in vivo. Additionally, several studies have suggested that acne patients‟ monocytes are hyper-responsive to P. acnes. These data led us to hypothesize that isotretinoin creates a long-lasting immune tolerance to P. acnes as part of its mechanism in permanently curing acne (Figure 1.10). This is the first study to characterize isotretinoin‟s in vivo effects on peripheral blood monocytes and lymphocytes in acne patients.

42 Chapter 2

Systemic Isotretinoin Treatment Modulates Acne Patients’ Immune Response to P. acnes

2.1 Chapter Abstract

Isotretinoin is the only agent that induces a permanent remission of acne, but the mechanism underlying its long-term efficacy is unknown. We hypothesized that modulation of the immune response to the bacterium involved in acne, P. acnes, is key to isotretinoin‟s ability to induce long-term or permanent remissions of acne. This study is the first to investigate changes in peripheral immune cells associated with isotretinoin treatment in acne patients in vivo. Peripheral blood samples were obtained from acne patients before starting isotretinoin therapy (baseline) and at 1, 4, 8, and 20 weeks after starting isotretinoin therapy. Monocytes and lymphocytes were isolated, stimulated in vitro, and analyzed by flow cytometry. Acne patients‟ monocytes expressed higher levels of TLR-2 and secreted more IL-1β, IL-6, IL-8, IL-10, and IL-12p70 in response to P. acnes than monocytes from normal volunteers. Isotretinoin therapy significantly decreased TLR-2 expression on acne patients‟ monocytes compared to their baseline values, and this decrease was maintained for at least six months after the cessation of therapy. Additionally, isotretinoin therapy significantly decreased the secretion of IL-1β, IL-6, IL-8, IL-10, and IL-12p70 by acne patients‟ untreated and P. acnes-treated monocytes compared to baseline. However, isotretinoin therapy did not affect peripheral lymphocyte subset differentiation or cytokine production in response to either P. acnes treatment or global activation. Acne patients‟ lymphocytes proliferated less in response to P. acnes-stimulation than lymphocytes from normal volunteers, though isotretinoin did not affect lymphocyte proliferation during the course of therapy. These results suggest that modulation of the innate immune response to P. acnes may represent one mechanism of isotretinoin‟s long-term efficacy.

2.2 Introduction

Isotretinoin, a pro-drug for all-trans retinoic acid (ATRA), is a retinoid that is used as a systemic therapy for a variety of cancers and dermatological diseases. Isotretinoin is the only agent that induces a permanent remission of acne, but the mechanism underlying its long-term efficacy is unknown. Studies suggest that patients‟ specific immune responses to P. acnes play a larger role in acne than the pathogenicity of P. acnes itself. We hypothesized that modulation of the immune response to P. acnes is key to isotretinoin‟s ability to induce long-term or permanent remissions of acne.

43 ATRA has been shown to have multiple immunomodulatory effects. It is proposed to have immunosuppressive effects on antigen-presenting cells such as monocytes and dendritic cells. These changes have subsequent effects on antigen presenting cells‟ co-stimulation of T cells and can thus alter

TH1/TH2 balance. Additionally, ATRA acts directly on T cells to promote differentiation of naïve T cells into regulatory T cells (Treg) and inhibit their differentiation into pro-inflammatory TH17 cells. While these effects have been demonstrated in vitro by treating cells with ATRA, they have not yet been studied in vivo. Because the exogenous administration of retinoids causes severe side-effects including birth defects, these drugs are strictly regulated and studies characterizing the action of retinoids in humans in vivo are lacking. Acne patients are generally healthy subjects that represent over 6,000 isotretinoin prescriptions per year in the United States.284 Therefore, this group presents a unique opportunity for characterizing isotretinoin‟s actions in vivo. This study is the first to investigate changes in peripheral immune cells associated with isotretinoin treatment in patients in vivo. This study included male and female subjects ages 12 to 40. Acne patients who were scheduled by their dermatologist to receive isotretinoin for their acne were recruited through the Penn State Hershey Dermatology clinic. Normal volunteers who had never taken isotretinoin and who currently did not have acne were also recruited from the community to serve as control subjects. For all subjects, a peripheral blood sample was taken by venipuncture at each visit. PBMC were isolated from blood samples, and monocytes and lymphocytes were treated ex vivo with antigens and then analyzed for surface markers and cytokine production. Serum levels of cytokines and retinoids and their metabolites were also assayed. This project is innovative because it is the first to identify the immunomodulatory effects of isotretinoin in treated acne patients. An understanding of isotretinoin‟s actions in vivo are critical to the development of alternative therapies for severe acne as well as other immune disorders.

2.3 Results

2.3.1 Subject demographics

Table 2.1 shows a summary of characteristics for the acne patients and volunteer subjects enrolled in this study. The study enrolled a total of 27 acne patients, 10 males and 17 females, with a mean (± SEM) age of 17.9 ± 1.4 years. One patient was of Hispanic ethnicity; the rest were Caucasian. The mean (± SEM) number of inflammatory lesions at baseline was 41 ± 6.3, and the mean (± SEM) number of non-inflammatory lesions was 68 ± 17 (Figure 2.1). Of the 27 acne patients treated with isotretinoin, 10 completed the study through 20 weeks of isotretinoin therapy. Of the patients that did not complete the study through 20 weeks, 1 stopped taking isotretinoin at the direction of his dermatologist, 2

44 stopped taking isotretinoin due to medication costs, 1 withdrew consent, and 6 patients were lost to follow-up between 8 and 20 weeks. A total of 19 normal volunteers were recruited for the study, 8 males and 20 females, with a mean (± SEM) age of 22.4 ± 1.5 years. Because different experimental methods were used for some of the early enrolled subjects, their samples were not included in the data analysis. Adjusted numbers for the actual data analysis are also shown in Table 2.1.

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Table 2.1: Study Patient/Volunteer Demographics. This study included male and female subjects ages 12 to 40. Acne patients who were scheduled to start isotretinoin therapy were recruited through Penn State‟s Department of Dermatology. Normal volunteers without acne were recruited from the community to serve as controls. Demographic information for all enrolled subjects (top table) and the subjects included in the data analysis (bottom table) is displayed.

Total enrolled:

Total Age Average Males/ No. who have previously enrolled Range Age Females taken isotretinoin

Acne patients 31 12 - 35 18.7 ± 5.6 Males: 12 8 Females: 19

Healthy 28 12 - 34 24.0 ± 6.0 Males: 8 2 Volunteers Females: 20

Subjects included in data analysis:

Total Age Average Males/ No. who have previously enrolled Range Age Females taken isotretinoin

Acne patients 27 12 - 35 17.9 ± 4.7 Males: 10 6 Females: 17

Healthy 19 12 - 34 22.4 ± 6.3 Males: 6 0 Volunteers Females: 13

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45 2.3.2 All enrolled patients responded clinically to isotretinoin therapy

As a measure of patients‟ clinical response to isotretinoin, acne severity was monitored using lesion counts at each time-point of the study. Inflammatory and non-inflammatory lesion totals were counted for the facial area (excluding the nose) using standard visual counting techniques. Acne patients had significantly fewer total lesions at all time-points compared to their baseline counts (P = 0.0167, 0.0012, 0.0006, and 0.0007 at 1, 4, 8 , and 20 weeks, respectively), demonstrating clinical improvement of their acne with isotretinoin treatment (Figure 2.1). Patients showed a significant reduction in non- inflammatory lesions at just 1 week of isotretinoin therapy compared to baseline, whereas they did not have a significant reduction in inflammatory lesions until 4 weeks of isotretinoin therapy.

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Figure 2.1: Lesion counts of acne patients on isotretinoin therapy. Mean (± SEM) inflammatory and non- inflammatory lesion counts are displayed for acne patients prior to (n = 25) and during isotretinoin therapy (1 week, n = 19; 4 weeks, n = 19; 8 weeks, n = 17; 20 weeks, n = 10). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with patients‟ baseline counts.

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46 2.3.3 Serum concentrations of retinoids do not differ between acne patients and normal volunteers

The efficacy of retinoids in treating acne has evoked the question of whether or not acne patients differ from people without acne in either serum levels of retinoids or the metabolism of retinoids. Though it has previously been shown that acne patients have slightly lower serum levels of vitamin A (retinol) compared to normal volunteers, the levels of retinoic acid isomers and their metabolites in acne patients‟ serum have not yet been reported.277,278 To investigate possible differences in serum retinoid concentrations between acne patients and normal volunteers, serum samples were assayed for levels of the three retinoic acid isomers (ATRA, 13-cis RA, and 9-cis RA) and their respective 4-oxo metabolites using HPLC/mass spectrometry (Figure 2.2). Acne patients at baseline did not differ from normal volunteers in serum levels of ATRA, 13-cis RA, 9-cis RA, or their 4-oxo metabolites (P = 0.9599, 0.5273, and 0.5775 for the isomers; P = 0.3294, 0.8827, and 0.8914 for the 4-oxo metabolites, respectively). As expected, acne patients had elevated levels of all three isomers and metabolites in their serum at all time-points during isotretinoin therapy compared to baseline. However, there was a large degree of intra- and inter-patient variability for all measured retinoids during isotretinoin therapy, which was likely due to lack of standardized timing of dosing in clinical practice.

2.3.4 Acne patients have higher serum concentrations of IL-10, but not inflammatory cytokines, compared to healthy volunteers

Though serum levels of inflammatory cytokines are increased in other skin diseases such as psoriasis, the levels of inflammatory cytokines in the serum of acne patients have not yet been assessed.285 To investigate whether or not acne patients differ from people without acne in circulating levels of inflammatory cytokines, serum samples were assayed for cytokine concentrations using bead arrays and flow cytometry. Results indicate that acne patients at baseline did not differ from normal volunteers in serum levels of IL-1β, IL-6, IL-8, or IL-12p70 (P = 0.4832, 0.9890, 0.9340, 0.5308, respectively), nor did isotretinoin therapy significantly affect serum levels of these cytokines (Figure 2.3). Interestingly, acne patients did have had significantly higher levels of serum IL-10 compared to normal volunteers (P = 0.0016), though isotretinoin did not affect serum IL-10 levels at any time-point during therapy. Serum concentrations of TNFα were either very low or below the assay detectable limit (2 pg/mL) for all samples.

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49 2.3.5 Isotretinoin therapy down-regulates expression of TLR-2 on acne patients’ monocytes in vivo

Volunteers‟ and patients‟ PBMC were isolated from blood samples using centrifugation gradients and separated into monocytes and lymphocytes using standard adherence methods. Monocytes were treated with vehicle (unstimulated) or 1 μg/mL P. acnes sonicate for 20 hours. At the end of the incubation period, cell culture supernatants were decanted and stored for later cytokine analysis. To examine surface expression of TLR-2, cells were harvested and stained using fluorescently-conjugated antibodies and then analyzed by flow cytometry. Monocytes were gated by forward and side scatter and then CD14 expression (Figure 2.4). The mean fluorescence intensity (MFI) for TLR-2 and TLR-4 on CD14+ cells was calculated and normalized to unstained controls for each sample at each time-point (Figure 2.5). At baseline, acne patients‟ unstimulated monocytes expressed higher levels of TLR-2 than normal volunteers (p = 0.03627) (Figure 2.6a). P. acnes sonicate treatment induced TLR-2 expression in both patients‟ and volunteers‟ monocytes, but P. acnes-induced TLR-2 expression was significantly greater in acne patients‟ monocytes (P = 0.04087). Isotretinoin therapy significantly decreased TLR-2 expression in acne patients‟ untreated monocytes within 1 week (P = 0.00040), and continued to decrease TLR-2 expression at each time-point examined compared to baseline (4 weeks, P = 0.0038; 8 weeks, P = 0.0014; 20 weeks, P = 0.0403). Additionally, isotretinoin significantly blunted the induction of TLR-2 expression in monocytes by P. acnes treatment at all time-points during therapy compared to baseline (1 week, P = 0.0037; 4 weeks, P = 0.0170; 8 weeks, P = 0.00010; 20 weeks, P = 0.0059). Similar results were observed in monocytes treated with whole heat-killed P. acnes (data not shown). When possible, acne patients were reanalyzed six months after the cessation of their isotretinoin therapy. At six months post-treatment, levels of TLR-2 expression in unstimulated monocytes from all eight analyzed patients were still significantly lower than their baseline and 1 week levels (P = 0.0004 and 0.0076, respectively), and were not significantly different from their 4 week, 8 week, or 20 week levels (P = 0.1692, 0.4104, and 0.7480). TLR-2 expression in P. acnes-stimulated monocytes at six months post-treatment was significantly lower than baseline (P = 0.0041), but was not significantly different from the other time-points (1 week, P = 0.0899; 4 weeks, P = 0.1660; 8 weeks, P = 0.5804; 20 weeks, P = 0.4902). These data suggest that the long-term suppression of TLR-2 expression could represent a possible mechanism for isotretinoin‟s ability to induce long-term remission of acne.

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To further characterize the decrease in TLR-2, the cumulative percent change in TLR-2 over time was graphed (Figure 2.6c). In unstimulated monocytes, TLR-2 MFI decreased 44% by just one week of isotretinoin therapy and continued to decrease until 20 weeks of isotretinoin therapy, when TLR-2 MFI had decreased by an average of 38 % compared to baseline. The same pattern was observed in P. acnes- stimulated monocytes, with an average decrease in TLR-2 expression of 29% by 1 week of therapy and 60% by 20 weeks. Six months after the cessation of therapy, TLR-2 expression was still decreased by 72% in unstimulated and 67% in P. acnes-stimulated monocytes.

2.3.6 TLR-2 expression on peripheral blood monocytes does not correlate with age

The increased expression of TLR-2 on acne patients‟ monocytes prompted us to speculate whether reduced TLR-2 expression could be a mechanism by which acne naturally regresses with age. To investigate the relationship between TLR-2 expression and age, we performed correlation analyses for TLR-2 MFI against age for both normal volunteers and acne patients at baseline (Figure 2.7). No correlation was found between TLR-2 expression and age in either unstimulated or P. acnes-treated monocytes from normal volunteers (Pearson correlation = 0.429, P = 0.076; Pearson correlation = 0.391, P = 0.135, respectively) or acne patients at baseline (Pearson correlation = 0.255, P = 0.265; Pearson correlation = 0.204, P = 0.375, respectively). The lack of correlation between TLR-2 expression and age suggests that decreases in monocyte TLR-2 expression is unlikely to represent a mechanism for the spontaneous remission of acne with age.

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Figure 2.6: TLR-2 expression in acne patients’ peripheral monocytes during isotretinoin therapy. Blood samples were taken from normal volunteers (Vols) (n = 22) and acne patients at baseline (n = 25), 1 week (n = 19), 4 weeks (n = 19), 8 weeks (n = 17), and 20 weeks (n = 10) of isotretinoin therapy as well as six months after the cessation of therapy (n = 8). Isolated peripheral monocytes were treated with no antigen (unstimulated; blue bars) or 1 μg/mL P. acnes sonicate (red bars) for 20 hours and analyzed by flow cytometry. a) Mean (± SEM) relative TLR- 2 MFI values are displayed for both normal volunteers and acne patients. b) Mean (± SEM) cumulative percent changes in TLR-2 MFI are displayed for acne patients‟ monocytes over the course of isotretinoin therapy. * denotes significance compared to acne patients‟ baseline, P < 0.05; ** P < 0.01; *** P < 0.001. + denotes significance compared to normal volunteers, P < 0.05; ++ P < 0.01; +++ P < 0.001

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Figure 2.7: TLR-2 expression versus age in the study population. TLR-2 MFI values in unstimulated (left) and P. acnes-treated (right) monocytes are plotted against age for both normal volunteers (Vols) and acne patients at baseline.

Unstimulated Monocytes P. acnes-treated Monocytes

25000 25000

20000 20000

Acne Pts

2 - Vols 15000 15000

10000 10000 MFITLRof

5000 5000

0 0 0 10 20 30 40 0 10 20 30 40 Age Age

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2.3.7 Acne patients’ monocytes express lower levels of TLR-4 than normal volunteers’

To determine if isotretinoin could affect other TLRs, or if its effects were specific for TLR-2, monocytes were also analyzed by flow cytometry for surface expression of TLR-4. Isotretinoin therapy did not affect levels of surface TLR-4 expression on acne patients‟ monocytes at any point during therapy in any of the treatment groups (Figure 2.8). Interestingly, normal volunteers‟ unstimulated monocytes tended to have higher levels of surface TLR-4 expression than acne patients‟ at baseline (though not significant; P = 0.1079), and had significantly higher TLR-4 expression than acne patients‟ at 1, 4, and 8 weeks of isotretinoin therapy (P = 0.0325, 0.0245, and 0.0226). Similar results were found for all treatment groups, including both LTA and LPS-treated monocytes (data not shown).

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2.3.8 Isotretinoin therapy decreases inflammatory cytokine production by acne patients’ monocytes in response to P. acnes

Media supernatants from cultured monocytes were assayed for concentrations of inflammatory cytokines using bead arrays (Figure 2.9). At an unstimulated basal level, acne patients‟ monocytes secreted significantly more TNFα than monocytes from normal volunteers, but did not differ in secretion of other inflammatory cytokines. However, in response to stimulation with P. acnes sonicate, acne patients‟ monocytes at baseline secreted significantly more IL-1β, IL-6, IL-10, and IL-12p70 than monocytes from normal volunteers. Just 1 week of isotretinoin therapy significantly decreased acne patients‟ monocytes‟ secretion of IL-1β, IL-6, IL-10, and IL-12p70 in response to P. acnes compared to baseline. IL-1β, IL-6, and IL-10 continued to decrease through 20 weeks of therapy. This suppression was sustained at six months after the cessation of therapy, suggesting that isotretinoin suppresses inflammatory cytokine secretion by monocytes long-term. Similar results were observed in monocytes treated with whole heat-killed P. acnes (data not shown).

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Figure 2.9: Inflammatory cytokine production by monocytes from acne patients treated with isotretinoin. Mean (± SEM) concentrations of inflammatory cytokines are displayed for unstimulated (blue bars) and P. acnes- treated (red bars) monocytes from normal volunteers (Vols) and acne patients during isotretinoin therapy. * denotes significance compared to acne patients‟ baseline, P < 0.05; ** P < 0.01; *** P < 0.001. + denotes significance compared to normal volunteers, P < 0.05; ++ P < 0.01; +++ P < 0.001

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To characterize the relationship between TLR-2 expression and cytokine secretion in acne patients‟ and volunteers‟ monocytes, correlation analysis was performed. In P. acnes-stimulated monocytes from volunteers and acne patients across all time-points of isotretinoin therapy, high correlations were found between TLR-2 expression and secretion of IL-1β (Pearson correlation = 0.673, P < 0.001), IL-6 (Pearson correlation = 0.568, P < 0.001), IL-8 (Pearson correlation = 0.348, P < 0.001), and IL-10 (Pearson correlation = 0.529, P < 0.001), but not for IL-12p70 (Pearson correlation = 0.147, P = 0.189). No significant correlations were found between TLR-2 expression and cytokine secretion in unstimulated monocytes, nor were any significant correlations observed between TLR-4 expression and cytokine secretion. These analyses indicate that the reduction in cytokine secretion in acne patients‟ monocytes in response to P. acnes stimulation may be linked to TLR-2 expression, but not to TLR-4. This further supports the hypothesis that the down-regulation TLR-2 potentially plays a role in the reduction in the P. acnes-induced inflammation associated with acne.

2.3.9 Isotretinoin therapy does not affect TLR-2 expression in the epidermis of acne patients as determined by immunohistochemistry

Previous studies have shown that retinoid treatment of keratinocytes in vitro can down-regulate TLR-2 expression. To correlate changes in skin TLR-2 expression with the changes seen in acne patients‟ peripheral monocytes, immunohistochemistry was performed on formalin-fixed paraffin- embedded skin biopsies obtained in an earlier study from eight acne patients at baseline and 8 weeks of isotretinoin therapy.57 Biopsies were taken from normal (non-lesional) skin at both time-points. Immunohistochemistry for TLR-2 was performed using the avidin-biotin complex method. Representative fields for the epidermis from each patient are shown in Figure 2.10. TLR-2 expression (brown areas) did not appear to be different in either the dermis or epidermis after 8 weeks of isotretinoin treatment compared to baseline using this method.

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2.3.10 Isotretinoin therapy does not affect proportions of Treg in peripheral blood

Regulatory T cells (Treg) suppress inflammatory responses and are thus of great interest as potential therapies for autoimmune and chronic inflammatory diseases. Several localized inflammatory diseases such as asthma have been associated with reduced proportions of Treg in the peripheral circulation. We therefore sought to compare the peripheral blood proportions of Treg in from acne patients to normal volunteers. To investigate T cell subset proportions in acne patients and normal volunteers, lymphocytes were treated for five days with no antigen, anti-CD3/anti-CD28 antibodies, or P. acnes sonicate. At the end of the incubation period, lymphocytes were harvested, stained with fluorescently- conjugated antibodies, and analyzed by flow cytometry. Lymphocytes were first gated on scatter properties and then by CD4 expression before analysis of subtype markers (Figure 2.11a). Acne patients‟ lymphocytes at baseline did not have different proportions of CD25+Foxp3+ T cells compared to normal volunteers in response to any antigen (Figure 2.11b), indicating that acne patients at baseline do not differ from normal volunteers in proportions of circulating Treg.

In vivo, Treg can arise from two distinct pathways: 1) they can differentiate in the thymus and enter peripheral circulation as CD4+CD25+Foxp3+ cells, or 2) circulating naïve T cells can undergo

„peripheral conversion,‟ where they differentiate into Treg upon activation in the peripheral circulation or 86 tissues. Peripheral conversion of Treg is thought to be dependent on ATRA. We therefore next investigated whether in vivo priming with isotretinoin renders peripheral naïve T cells more likely to differentiate into Treg upon activation. Lymphocytes from acne patients before and during isotretinoin therapy were cultured and analyzed as above. Proportions of Treg resulting from stimulation with either P. acnes sonicate or anti-CD3/anti-CD28 antibodies for five days did not differ at any time-point of isotretinoin therapy compared to baseline (Figure 2.11b), suggesting that systemic isotretinoin therapy does not necessarily impart Treg-differentiating signals on naïve T cells in the periphery. In order to exclude the possibility that Treg were de-differentiating in culture, lymphocytes were also analyzed 20 hours after activation, and similar results were obtained (data not shown).

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61 2.3.11 Lymphocyte proliferation in response to P. acnes is blunted in acne patients compared to normal volunteers

Because Treg suppress the proliferation and activity of leukocytes in a non-specific manner, lymphocyte proliferation in response to antigens can be used as a measure of Treg numbers or function.

To determine if isotretinoin affects Treg suppressive function in response to global stimulation or specifically in response to P. acnes antigens, lymphocyte proliferation assays were performed. Isolated peripheral lymphocytes from patients were plated with either no antigen, anti-CD3 and anti-CD28 antibodies, or P. acnes sonicate for five days. For the last four hours of incubation, 3H-thymidine was added to each well. Cells were then harvested and analyzed by scintillation counting. Acne patients‟ lymphocytes proliferated significantly less than normal volunteers‟ lymphocytes in response to P. acnes sonicate at baseline (P = 0.0089), 1 week (P = 0.0196), 4 weeks (P = 0.0409), and 8 weeks (P = 0.0319) of therapy (Figure 2.11). Acne patients‟ lymphocytes tended to proliferate less than normal volunteers‟ in response to global stimulation by anti-CD3 and anti-CD28 antibodies, but this effect did not achieve statistical significance. Isotretinoin therapy did not change acne patients‟ lymphocyte proliferation in any treatment group at any time-point compared to baseline.

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Figure 2.13: Cytokine secretion by acne patients’ lymphocytes after 20 hours stimulation. Displayed below are mean (± SEM) media concentrations of secreted cytokines from acne patients‟ and normal volunteers‟ (Vols) lymphocytes after 20 hours of stimulation with a) anti-CD3 and anti-CD28 antibodies or b) P. acnes sonicate. a) Anti-CD3 and anti-CD28 antibody-treated:

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Figure 2.14: Cytokine secretion by acne patients’ lymphocytes after five days stimulation. Displayed below are mean (± SEM) media concentrations of secreted cytokines from acne patients‟ and normal volunteers‟ (Vols) lymphocytes after five days of stimulation with a) anti-CD3 and anti-CD28 antibodies or b) P. acnes sonicate.

a) Anti-CD3 and anti-CD28 antibody-treated:

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66 2.3.12 Isotretinoin therapy does not affect peripheral blood lymphocyte cytokine secretion

Retinoid treatment of T cells can skew helper T cell differentiation and responses. In the presence of TGFβ, ATRA promotes Treg differentiation and inhibits TH17 differentiation. However, in the absence of TGFβ, ATRA treatment of normal human PBMC and purified T cells promotes TH2 responses and inhibits TH1. To investigate the effects of isotretinoin therapy on the secretion of TH1, TH2, and

TH17-associated cytokines by activated lymphocytes, media supernatants from P. acnes sonicate and anti- CD3/anti-CD28-stimulated lymphocytes were analyzed at 20 hours and five days of treatment. Isotretinoin therapy did not significantly affect IL-2, IL-4, IL-5, IL-10, IL-17A, TNFα, or IFNγ secretion by lymphocytes after 20 hours (Figure 2.13) or 5 days (Figure 2.14) of stimulation with either P. acnes sonicate or anti-CD3 and CD-28 antibodies. Acne patients‟ lymphocytes at baseline did not differ from normal volunteers‟ in the secretion of inflammatory cytokines in either incubation time.

2.4 Discussion

Many of isotretinoin‟s short-term mechanisms of action in suppressing acne have been previously elucidated. Within the first few weeks of therapy, isotretinoin induces apoptosis of sebocytes to shrink sebaceous glands and reduce P. acnes colonization.57,286 However, these factors partially reverse within weeks after the cessation of therapy, and in some cases return to baseline levels even in the context of continued clinical remission. Isotretinoin can induce permanent remissions of acne, but the mechanisms of its long-term efficacy are unknown. This is the first study to show isotretinoin‟s ability to modulate immune responses in vivo. We have shown here that acne patients‟ peripheral blood monocytes at baseline express higher levels of TLR- 2 than monocytes from normal volunteers, both at a basal (unstimulated) level as well as in response to treatment with P. acnes sonicate. Systemic isotretinoin significantly decreased both basal and P. acnes- induced expression of TLR-2 in acne patients‟ monocytes within just 1 week of beginning therapy and continued to suppress TLR-2 levels through 20 weeks of therapy. This suppression was still evident up to six months after the cessation of the course of isotretinoin, with cumulative decreases in TLR-2 expression of 72% in unstimulated monocytes and 67% in P. acnes-stimulated monocytes compared to baseline. This data suggests that the down-regulation of TLR-2 expression and stimulation by P. acnes may represent one mechanism by which isotretinoin induces a durable remission. It must be noted that the range of TLR-2 expression for acne patients‟ monocytes at baseline overlapped with the range of TLR-2 expression by normal volunteers. Some acne patients‟ monocytes had baseline TLR-2 expression levels within the „normal‟ range, yet isotretinoin therapy still decreased TLR-2 expression in these

67 patients‟ monocytes. The fact that some patients do not necessarily have „abnormally‟ elevated TLR-2 expression on their monocytes illustrates the involvement of other factors involved in acne pathogenesis such as local TLR-2 expression in the skin, sebum production, and androgen excess. We attempted to correlate our findings in patients‟ monocytes with changes in TLR-2 expression in patient skin biopsies using immunohistochemistry, but did not detect differences in TLR-2 expression in either the dermis or epidermis of acne patients‟ skin after 8 weeks of isotretinoin therapy. However, given that immunohistochemistry is a relatively insensitive method, more quantitative assays may be needed to determine if isotretinoin therapy affects TLR expression levels in the skin. The mechanism by which retinoids affect TLR-2 expression is unknown. Because published data has demonstrated that treatment of normal human monocytes with ATRA in vitro down-regulates TLR-2 mRNA, we hypothesized that isotretinoin operates by a similar mechanism in acne patients‟ monocytes in vivo.237 Whether the reduction in TLR-2 mRNA arises from decreased TLR-2 gene transcription or decreased TLR-2 mRNA stability is unknown, for retinoids have been shown to affect both.287 The gene for TLR-2 (also called CD282) contains multiple transcription factor binding elements in the promoter region, including SP1, SP2, NF-κB, C/EBP, and PU.1. Notably absent from the promoter regions are retinoic acid receptor-binding elements (RAREs), indicating that direct activation of retinoic acid receptors (RARs) is likely not the working mechanism of isotretinoin therapy. We have shown that isotretinoin therapy effectively raises serum concentrations of all three retinoic acid isomers. Therefore, it is possible that isotretinoin may affect TLR-2 expression through 9-cis RA activation of retinoid-X- receptors (RXRs). RXRs dimerize with many different nuclear transcription factors, many of which can influence immune responses, such as the vitamin D receptor. A non-canonical physical association of RXRs with NF-κB receptors has been proposed as a way that retinoids can block the transcription of NF- κB target genes induced by TLR-4 ligands.288 Additionally, ATRA is classically known for controlling the expression of the HOX genes during embryogenesis through local histone acetylation, thereby directing gene transcription by rendering gene promoters physically available for transcription factor binding.197 Any one or a combination of these mechanisms could play a role in isotretinoin‟s down- regulation of TLR-2 in monocytes. We subsequently found that peripheral blood monocytes in acne patients at baseline secrete increased amounts of inflammatory cytokines IL-1β, IL-6, IL-10, and IL-12p70 in response to P. acnes stimulation compared to monocytes from normal volunteers. Our IL-8 data contrasts with previously published studies that showed that acne patients‟ P. acnes-stimulated monocytes secrete more IL-8, IL- 12p70, and IFNγ than monocytes from normal volunteers, but these methods differed from ours in that one study analyzed monocytes from very small numbers of human subjects (two normal volunteers and

68 three acne patients) and the other study cultured patients‟ total PBMC together with P. acnes without first separating monocytes and lymphocytes.154,289 This is the first study to show that systemic administration of isotretinoin affects the secretion of inflammatory cytokines by peripheral monocytes. Isotretinoin therapy decreased secretion of IL-1β, IL-6, IL-10, and IL-12p70 in acne patients‟ monocytes at both an unstimulated level and in response to P. acnes stimulation in as little as 1 week of therapy. This suppression continued through 20 weeks of therapy, at which point acne patients‟ monocytes secreted „normal‟ levels of cytokines. As with TLR-2 expression, these changes were maintained at least six months after the cessation of therapy. Though we did not detect differences in IL-8 secretion between volunteers‟ and acne patients‟ monocytes at baseline or during isotretinoin therapy, lower secretion of IL-8 was observed at the six month post-treatment time- point compared to baseline. These results are in accordance with data that has shown that treatment of normal human PBMC in vitro with ATRA decreases secretion of IL-6, IL-12p70, and TNFα in response to P. acnes.237 A plausible mechanism for the reduced cytokine response would be that there are fewer molecules of TLR-2 present on the surface of monocytes to bind to P. acnes antigens and initiate the inflammatory signal cascade, which is supported by the strong correlation between TLR-2 expression and cytokine production in patients‟ monocytes. With this theory, the fact that isotretinoin therapy did not affect cytokine secretion in monocytes treated with LPS, a TLR-4-binding antigen (data not shown), can be explained by the observation that TLR-4 levels did not change over the course of isotretinoin therapy. It is interesting to note that in our study IL-10, an immunosuppressive cytokine, followed the same pattern as inflammatory cytokines, in that acne patients‟ baseline monocytes produced more IL-10 than healthy volunteers‟ in response to P. acnes treatment, and isotretinoin therapy decreased IL-10 production in acne patients‟ monocytes over time. Acne lesions express 46-fold higher IL-10 mRNA compared to normal skin, and increased lesional expression of IL-10 has been recently reported in acne inversa (hidradenitis suppuritiva).118,290,291 This increase in IL-10 is thought to represent a compensatory response to the intense inflammation.118 Our findings, however, differ from reports that acne patients‟ PBMC secrete less IL-10 in response to P. acnes than PBMC from healthy volunteers.235,289 These differences may be due to the increased severity of acne in our patient population. As a whole, the data presented here suggest that isotretinoin induces long-term remission of acne in part by „normalizing‟ acne patients‟ immune response to P. acnes. Local reductions in P. acnes- induced inflammatory cytokine production may have direct benefits on the cells within the pilosebaceous unit. For example, it has been shown that inflammatory cytokines promote infundibular hyperkeratinization, contributing to comedogenesis.141 It is also possible that the down-regulation of TLR-2 on monocytes has additional down-stream effects on other immune cells in the skin microenvironment. It has been shown in other disease contexts that TLR expression levels on monocytes

69 or dendritic cells can modulate the adaptive immune system by directing the differentiation of naïve T cells during antigen presentation. For example, in chronic hepatitis B patients, the HepB virus alters TLR-2 and TLR-4 expression on peripheral monocytes, thus imparting enhanced differentiation and suppressive function on CD4+CD25+ Treg cells. It would be interesting to investigate whether the down- regulation of TLR-2 on monocytes skews P. acnes-specific T cell responses.

Along these lines, it is possible that excessive T cell activity or impaired Treg suppressive function contributes the chronic inflammation associated with acne. Several cutaneous diseases such as psoriasis and atopic dermatitis have been associated with abnormal numbers or function of Treg in the skin, but the involvement of Treg in acne pathogenesis or resolution has not yet been studied. We addressed this by investigating proportions of peripheral Treg in normal volunteers and acne patients. We did not detect any differences in peripheral Treg proportions between acne patients as baseline and normal volunteers.

Additionally, isotretinoin therapy did not alter peripheral proportions of Treg in acne patients, nor did it render naïve T cells more likely to differentiate into Treg upon activation ex vivo. These data are especially intriguing given the fact that ATRA has been shown to be potent and sufficient for inducing 86,292 Foxp3+ Treg differentiation from naïve T cells. Although ATRA concentrations of 10 nM are sufficient for promoting naïve T cell differentiation into Treg in vitro, we found no effect on Treg as a result of systemic administration of isotretinoin that raised serum levels of ATRA in the range of 200 nM for several months. We present here the first evidence that oral retinoid therapy for up to 20 weeks does not affect peripheral proportions of Treg. This is particularly relevant to the use of isotretinoin or ATRA in chemotherapy or chemoprevention. Because Treg play a pivotal role in suppressing immune responses, the induction of Treg activity may be detrimental in cancer where CD8+ cytotoxic T cell activity is needed to kill tumor cells. Indeed, in multiple cancers the numbers or activity of Treg are increased both in the periphery and the tumor microenvironment, and higher numbers of Treg correlate with a worse prognosis.293-298 However, our study did not investigate T cell changes in the skin microenvironment. It is possible that acne patients have fewer Treg in their skin than normal patients or that their Treg have functional deficits that aid in acne pathogenesis. We attempted to use immunohistochemistry to analyze numbers of Foxp3+ Treg in skin biopsies from acne patients at baseline and after 8 weeks of isotretinoin therapy, but this method was not sufficiently quantitative to draw conclusions from the assay (data not shown). Future experiments could isolate T cells and monocytes from fresh skin biopsies in order to investigate changes in TLR expression or subtype differentiation associated with isotretinoin therapy in vivo.

While we did not find abnormal proportions of Treg in acne patients, we did find elevated serum concentrations of IL-10 in acne patients compared to normal volunteers. IL-10 is an anti-inflammatory cytokine that is produced by multiple cell types including Treg, TH2 helper T cells, and monocytes. The

70 clinical significance of marginal increases in serum IL-10 is unknown, but it may represent evidence of a compensatory response to sustained inflammation. Increased serum levels of IL-10 have been noted in patients with laryngeal cancer where 20 percent of patients had levels in excess of 1 pg/ml whereas control subjects had no detectable IL-10.299 In order to mimic the in vivo environment as closely as possible, we stimulated acne patients‟ monocytes and lymphocytes in the presence of their own serum instead of FBS. The elevated IL-10 levels in the serum could be partially responsible for the decreased lymphocyte proliferative response to stimulation seen in acne patients at baseline and through 20 weeks of isotretinoin therapy (proliferation assays were not done at six months after the cessation of therapy). The efficacy of retinoid therapy in acne provokes the question of whether or not acne patients differ from normal subjects in retinoid absorption, transport, or metabolism. Previous studies in the literature have shown that acne patients have lower plasma levels of diet-derived retinoids (retinol and carotenoids) and circulating retinol-binding protein compared to normal volunteers. However, we did not detect differences in plasma levels of the three retinoic acid isomers or their 4-oxo metabolites between acne patients at baseline and normal volunteers, suggesting that in the periphery, at least, acne patients do not have abnormal conversion or metabolism of retinol to its active forms. However, this does not preclude the possibility that acne patients have differences in retinoid metabolism in their skin. Analysis of the serum levels of retinoids and metabolites in isotretinoin-treated patients showed that treatment levels of retinoids vary greatly over the course of therapy. This variation may arise from differences in the timing of taking the medication. Because isotretinoin is highly lipophilic, its absorption vastly increases when taken with a high-fat meal; ATRA concentrations in serum can be up to 36% higher after a meal than in fasting subjects.283 Additionally, strict regulation by the iPLEDGE program frequently causes patients to miss several days of medication between monthly prescriptions. As a result of factors such as these, serum retinoid levels during the course of therapy were highly variable and could not be correlated with other parameters such as clinical response or TLR-2 expression. The relationship between TLR-2 expression and acne susceptibility requires further investigation. Our study found that acne patients‟ monocytes express higher surface levels of TLR-2 compared to normal volunteers. This raises the question of whether or not endogenous TLR-2 regulation plays a part in the spontaneous remission of acne seen in most people in early adulthood. We did not observe a significant correlation between age and monocyte TLR-2 expression in either normal volunteers or acne patients at baseline, suggesting that TLR-2 does not naturally decrease after adolescence. Additionally, it is not known if acne patients‟ monocytes have innately higher levels of TLR-2 throughout their lives, or if TLR-2 expression is up-regulated sometime before or during puberty. Analysis of pre-pubescent subjects would help to determine the age at which acne patients start to express abnormally high TLR-2 levels, and if this in turn correlates with the onset of acne. The time-course of increased TLR-2 expression may also

71 give insight into why acne patients have such high TLR-2 expression on their PBMC. If TLR-2 levels rise sometime during childhood or puberty, then there is a possibility that a precipitating event or illness would play a role. TLR-2 is known to be able to recognize and initiate immune responses to viral envelope glycoproteins from VZV and HSV.300-302 It would therefore be interesting to correlate TLR-2 over-expression with the onset of typical childhood illnesses such as chicken pox (Varicella zoster), strep throat, or mononucleosis. However, if acne patients‟ monocyte TLR-2 expression is high from birth, then investigating epigenetic mechanisms of TLR-2 gene expression would be a logical next step. Evidence shows that the maternal environment can impact the development of the fetal immune system. For example, one study has shown that maternal allergies correlate with greater cord blood PBMC responses to TLR-2, -4, and -5 ligands, and the inflammatory responsiveness of these TLRs to their ligands is higher in newborns who subsequently develop allergies.303 If maternal allergies can be linked to TLR-2 expression and/or function in children, then this could partially explain the high prevalence of acne in western cultures that also have a high prevalence of allergies. In conclusion, we have shown that systemic isotretinoin therapy decreases TLR-2 expression and cytokine production in peripheral monocytes in humans in vivo. This suppression continues through 20 weeks of isotretinoin therapy and persists for at least six months after the cessation of therapy. It will be important for future studies to investigate the duration of TLR-2 suppression. By studying patients several years after their isotretinoin course, we can understand if TLR-2 down-regulation represents part of isotretinoin‟s short-term mechanism of action, its long-term mechanism for inducing permanent remission, or both. It is possible that TLR-2 down-regulation is not permanent, but is nevertheless maintained long enough to either modulate the adaptive immune response or to merely keep acne in remission until other age-related factors level out.

2.5 Methods

2.5.1 Human Subjects

Human studies were done in accordance with Penn State‟s Institutional Review Board (Penn State IRB protocol #23784EP) and the basic principles of the Declaration of Helsinki. The study included male and female patients ages 12 to 40 who had been prescribed isotretinoin for their acne by their dermatologist. Patients were recruited through the Penn State Department of Dermatology located at the

72 Hershey Medical Center, Hershey, PA and were consented for the study by approved and trained study coordinators. A 30 mL peripheral blood sample was obtained from patients before starting isotretinoin therapy (baseline) and at 1, 4, 8, and 20 weeks after starting their isotretinoin therapy (0.5 to 1 mg/kg/day). When possible, an additional blood sample was obtained from these patients six months after the cessation of their isotretinoin therapy. Healthy volunteers ages 12 to 40 without acne and without previous exposure to isotretinoin served as controls for comparison to acne patients. A one-time 30 mL peripheral blood sample was obtained from the volunteers. All blood samples were drawn into sodium-heparin Vacutainer tubes (BD Biosciences) and left horizontal at room temperature in the dark until processing. At each visit, patients and volunteers filled out a health questionnaire regarding factors that may influence their immune systems at the time of blood sampling (such as having a head cold or receiving a vaccination within the previous month). Data on chronic medical conditions and current medications was also collected. When possible, changes in patients‟ isotretinoin dosage (as ordered by their dermatologist) were recorded. Additionally, lesion counts were performed (inflammatory, non-inflammatory, and total) at each visit as an indicator of clinical response to isotretinoin therapy.

2.5.2 P. acnes Cultures

P. acnes strain ATCC 6919 (American Type Culture Collection) was grown on Brucella broth agar supplemented with 5% (v/v) sheep blood, 50 μg/mL hemin, and 5 μg/mL vitamin K1 (BD Diagnostics) under anaerobic conditions at 37° C. A single colony from this plate was used to inoculate

Difco Reinforced Clostridal Broth (BD Biosciences). The culture was overlaid with N2 gas and grown overnight at 37° C in a shaker until bacteria reached their log growth phase. Bacteria were then washed twice and resuspended in sterile PBS. Sonicates were made by sonicating suspensions (at setting #9) three times for 30 seconds each. All sonicate preparations were stored at -20° C.

2.5.3 Cell Culture

All sample processing and cell culture took place in a sterile tissue culture hood under yellow light. Blood samples were processed using a density gradient to separate red blood cells, PBMC, and serum: blood was mixed with an equal volume of 1x RPMI 1640 (made in the Penn State Department of Microbiology and Immunology central core facility), overlaid onto a 12 mL layer of Ficoll-Paque (GE

73 Healthcare) in a 50 mL conical tube, and spun at 1200 x g for 20 minutes according to manufacturer‟s guidelines. Each blood sample was divided equally into two Ficoll-Paque gradients. After centrifugation, serum was carefully decanted from the top of the buffy coat and set aside. An aliquot of serum was stored at -80° C for later analysis. The buffy coat containing the PBMC was carefully removed and transferred to a new 50 mL conical tube, washed three times with RPMI, and resuspended in RPMI/10% (v/v) patient serum/antibiotics. PBMC were separated into lymphocytes and monocytes using the standard adherence method.304 In summary, total PBMC were plated in 12-well plates at 5x106 cells/well in RPMI/10% (v/v) patient serum/antibiotics for 1.5 hours, when the monocytes were gently washed off with two RPMI rinses. For QPCR samples, PBMC were plated in 24-well plates at 2.5x106 cells/well and then separated as described above. Monocyte samples consistently achieved at least 95% purity with minimal contamination by lymphocytes (data not shown). Monocytes and lymphocytes were then separately treated in RPMI/10% (v/v) patient serum/antibiotics. Leftover monocytes were treated as described below, and lymphocytes were spun and resuspended for re-plating in 12-well plates at 25x105 cells/well. Monocytes were treated with vehicle (no antigen), 1 μg/mL P. acnes sonicate, 1 μg/mL heat-killed P. acnes, 1 μg/mL Escherichia coli LPS (Sigma), or 10 ng/mL Bacillus subtillus LTA (Sigma) for 20 hours. Lymphocytes were then re-plated and treated in duplicate with vehicle (no antigen), 1 μg/mL P. acnes sonicate, or 1 μg/mL anti-CD3 antibody plus 1 μg/mL anti-CD28 antibody for 20 hours or five days. For the last four hours of incubation, cells were treated with 10 μg/mL brefeldin-A.

2.5.4 Antibodies and Flow Cytometry

Treated monocytes were trypsinized, washed once with PBS, and resuspended at 1x106 cells in 50

μL of PBS/2% (v/v) FBS/0.1% (w/v) NaN3 for staining. Monocytes were incubated with mouse anti- human TLR-4-biotin (clone HTA125), mouse anti-human TLR-2-AlexaFluor 647 (clone 11G7), mouse anti-human CD33 - FITC (clone Sp2/0), and mouse anti-human CD14-APC-H7 (clone MφP9) or isotype controls (all BD Biosciences) for 30 minutes at 4° C. At the end of the incubation cells were washed once with PBS/2% (v/v) FBS/0.1% (w/v) NaN3, resuspended in 50 μL, and incubated with Streptavidin-PE (BD Biosciences) for 30 minutes at 4° C. Cells were then washed and resuspended in PBS for flow cytometry. To detect intracellular expression of TLR-2 and TLR-4, one set of monocytes from each sample was permeabilized before staining using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer‟s directions.

74 Lymphocytes were collected and washed once with PBS before fixing. Lymphocytes were first surface stained using panels containing the following antibodies: mouse anti-human CD4 - PE-Cy7 (clone L200), mouse anti-human CD25 - PerCP-Cy5.5 (clone M-A251), mouse anti-human CD127 - FITC (clone HIL-7R-M21), mouse anti-human CD152 - APC (clone BNI3), mouse anti-human CD124 - PE (clone hIL4R-M57), and rat anti-human CD212/IL-12 receptor - PE (clone 2B6/12β2) or isotype controls (all BD Biosciences) for 30 minutes at room temperature. Cells were then fixed and permeabilized using the Foxp3 Staining Buffer Set (eBiosciences) according to the manufacturer‟s directions. Intracellular staining was performed in the Foxp3 Staining Buffer Set Permeabilization Buffer using panels containing the following antibodies: mouse anti-human Foxp3 - PE (clone 259D/C7), mouse anti-human IL-4 - APC (clone 8D4-8), mouse anti-human IFNγ - APC (clone 4S.B3), mouse anti-human IL-17A - AlexaFluor 488 (clone N49-653), mouse anti-human IL-21 - PE (clone 3A3-N2.1), mouse anti-human IL-5 - PE (clone JES1-39D10), and mouse anti-human IL-2 - PE (clone MQ1-17H12) or isotype controls (all BD Biosciences) for 30 minutes at room temperature. Cells were then washed and resuspended in PBS for flow cytometry. All samples were analyzed using an LSR II flow cytometer using FACSDiva Software (BD Biosciences). Anti-mouse IgG-κ CompBeads-Plus (BD Biosciences) were used for compensation controls. Data was analyzed using FlowJo Flow Cytometry Analysis Software (Tree Star).

2.5.5 Cytokine Detection Using Bead Arrays

Supernatants from the treated monocytes and lymphocytes were assayed for cytokines using

Human Inflammatory Cytokines and Human TH1/TH2/TH17 Cytokines Cytometric Bead Array kits (BD Biosciences) according to manufacturer‟s directions. Samples were run on a LSR II flow cytometer using FACSDiva Software and analyzed using FCAP Analysis Software (both BD Biosciences). The theoretical minimum limit of detection for each cytokine in samples was approximately 2 pg/mL.

2.5.6 Lymphocyte Proliferation Assays

Lymphocytes were stimulated with anti-CD3 and anti-CD28 antibodies or P. acnes sonicate in RPMI/10% (v/v) patient serum/antibiotics as described above. Cells were plated at 1x105 cells/well in 96-well round-bottom plate in triplicate wells for five days. For the last four hours of incubation, cells were pulsed with 1 μCi/well of tritiated thymidine (Perkin-Elmer). At the end of the incubation, cells

75 were collected onto filter paper discs using a PhD Cell Harvester machine (Brandel, model #290) and placed in scintillation vials with 2mL of CytoScint biodegradable scintillation fluid (Fisher). The degree of 3H-thymidine incorporation (as a measure of DNA proliferation) was determined by liquid scintillation analysis.

2.5.7 High-Performance Liquid Chromatography and Mass Spectrometry

Concentrations of retinoids and metabolites in patients‟ serum samples were assayed by high performance liquid chromatography (HPLC) and mass spectrometry. Revisions were made to an established method of sample extraction, and liquid chromatography conditions were modified from the literature.305,306 Acitretin was used as the internal standard for the retinoic acid isomers. Because there is no commercially available standard for the 4-oxo metabolites, these compounds were measured as „relative peak areas‟ relative to their parent isomer. Samples were mixed and loaded onto Waters Oasis HLB Extraction Columns (3 cc, 60 mg) (Waters) preconditioned with 2 x2 mL of methanol, 2 mL of water, and 2 mL of 25% (v/v) methanol. The sample tube was rinsed with 2 mL of water and the rinsing solution was transferred to the column. Each column was washed with 3 mL of 25% (v/v) methanol and 3 mL of 50% (v/v) methanol. Samples were eluted from the column with 3mL of 90% (v/v) acetonitrile, dried down under nitrogen gas, and reconstituted with 50 μL of acetonitrile followed by the addition of 50 μL of water. Samples were then transferred to 250 μL glass inserts, centrifuged at 2000 x g for 20 minutes, and analyzed. Retinoic acid isomers and their metabolites were separated on a Supelco ABZ +PLUS column (100 mm x 2.1 mm, 3 μm) using an Agilent 1100 series HPLC. Mobile phase A consisted of 40% acetonitrile/30% methanol/0.1% formic acid (all v/v). Mobile phase B consisted of 55% acetonitrile/30% methanol/0.1% formic acid. A linear gradient was generated at 250 μL/min: 5 minutes, 100% A to 100% B; 5 to 13 minutes, 100% B; 13 to 13.1 minutes, 100% B to 100% A; 13.1 to 20 minutes, and re- equilibrated with 100% A with an injection volume of 20 μL. An Applied Biosystems/MDS Sciex 4000 QTrap (triple quadrupole) mass spectrometer with electrospray ionization was operated in selected reaction monitoring mode. The dwell time for ATRA and its metabolites was 50 milliseconds, and optimum positive ESI conditions included: curtain gas, 40; collision gas, 10; ion spray voltage, 5500; temperature, 500º C; ion source gas #1, 40; ion source gas #2, 35. Compound-dependent parameters were as follows: declustering potential, 50; entrance potential, 10; collision energy, 25; and collision cell exit potential, 8.

76 Each run of patient samples included a standard curve using the above mentioned pooled pretreatment serum:RPMI matrix, spiked with 50 ng/mL acitretin internal standard and 0, 0.3, 1, 2, 5, 10, 50, 100, 250 ng/mL each of 13-cis RA, 9-cis RA, and ATRA. Spiked quality control samples were run at 0.3, 1, 40, 125, and 200 ng/mL. The average R-squared value for the standard curves was 0.998. Eight-point calibration curves were constructed using AB Sciex analyst software (version 1.5.1) by plotting the peak area ratios of each of the three retinoids (13-cis RA, 9-cis RA, and ATRA) and acitretin versus the corresponding concentrations and fitting the data using linear regression with 1/x2 weighting factor. The methods were found to be highly accurate with less than 6.6% deviation from the nominal values and highly precise with inter-assay precision less than 9.86%, and intra-day precision was less than 9.42% for all three retinoids. A paired t-test was used to analyze patient sample baseline data with other time points in their treatment, and volunteer and patient baseline data were analyzed using an unpaired t-test, with P < 0.05 being considered significant.

2.5.8 Human Skin Biopsies and Immunohistochemistry

Human skin biopsies from acne patients were obtained from a previous study.57 Punch biopsies were taken the back skin of acne patients before therapy (baseline) and after 8 weeks of isotretinoin therapy. Biopsies were taken from normal non-lesional skin at both time points. For immunohistochemistry, sections were deparaffinized in xylene and rehydrated with washes in pure ethanol, 95% (v/v) ethanol, and nanopure water. For Foxp3 staining, sections were incubated in Tris/EDTA buffer (10mM Tris/1mM EDTA/0.05% (v/v) Tween20, pH 9.0) at 90° C for 30 minutes and then cooled at room temperature for 20 minutes. For TLR-2 staining, sections were incubated in Trilogy Buffer (CellMarque) at 90° C for 20 minutes and then cooled at room temperature for 20 minutes.

Endogenous peroxidase activity was blocked by incubating sections in 3% (v/v) H2O2 for 10 minutes at room temperature. Sections were then blocked for one hour in 1% (w/v) BSA/TBS at room temperature and incubated in monoclonal rabbit anti-human TLR-2 antibody (R&D Systems) diluted in 1% (w/v) BSA/TBS overnight at 4° C. The next day sections were stained with secondary biotinylated antibody using Vectastain ABC and developed using Vectastain AEC kits (Vector Laboratories), counterstained with hematoxylin, and mounted to glass cover slips with aqueous mounting medium.

77 2.5.9 Statistical Analysis of the Data

Monocyte sample data was first gated on forward and side-scatter properties, and then gated on CD14 expression to identify monocytes. TLR-2 and TLR-4 expression was calculated using the mean fluorescence intensity of each marker for CD14+ cells. MFI values were normalized to negative controls for each patient at each time-point. Lymphocyte sample data was first gated on forward and side-scatter measurements, and then gated on CD4 expression to identify the CD4+ helper T cells. The percent of CD4+ T cells expressing markers of each subset in each isotretinoin sample was compared to baseline for each patient. For all assays, each patient served as his/her own control, with data from each time-point during isotretinoin treatment being compared back to his/her own baseline value. All patient data was analyzed using paired Student‟s t-tests for comparison of patient data across isotretinoin therapy, and unpaired t- tests for comparison between patients and healthy volunteers, with a P value < 0.05 being considered significant. Two-factor ANOVA tests could not be performed due to various missing time-points (many patients missed one or more study visits).

78 Chapter 3

Development and Characterization of a Primary Sebocyte Cell Line (pSEB)

3.1 Chapter Abstract

The use of primary human cells for scientific research is often hampered by the inability to grow substantial amounts of cells for use in experimental assays. Conversely, the use of cell lines immortalized by SV40 Large T or other oncogenes raises concerns over the validity of such models in terms of enhanced proliferation at the expense of diminished differentiation. We describe here a novel method of reversibly immortalizing primary sebocytes using the small molecule Rho-kinase inhibitor Y-27632. Primary human sebocytes grown in the presence of Y-27632 and 3T3 fibroblast feeder layers can be expanded for over 30 passages and exhibit characteristics of normal human sebocytes, whereas removal of the drug allows growth arrest and differentiation. This method represents a valuable new model system for research on human sebocyte biology.

3.2 Introduction

Acne is a common skin disease that is partially caused by excess sebum production, which supports the growth of P. acnes and may also directly contribute to inflammation and comedogenesis. Research on sebocyte biology has been hampered in the past by difficulties in creating appropriate model systems. There is no comparable animal model for inflammatory acne because humans are the only animals that are colonized by P. acnes. Primary sebocytes are nearly impossible to culture in vitro because they differentiate and rupture before they can proliferate to any substantial confluency. Thus, the use of cell lines for sebaceous gland research was difficult until SV40-immortalized sebocyte cell lines were created. We previously created an immortalized sebocyte cell line (SEB-1) by transfecting primary sebocytes from the ear of a 55 year-old male patient with SV40 large T antigen under the control of a CMV promoter.14 Large T antigen protein binds to and hyperphosphorylates Rb, p53, and hsc70, which collectively override signals for senescence and apoptosis, allowing cells to be passaged continuously in culture in numbers great enough for use in any assay.171 Another immortalized sebocyte cell line called SZ95 has been created by Zouboulis et al., also using large T antigen.172 While these cell lines have been useful systems for basic research, the comparison of primary cells to Large T transformed cells presents several problems. Large T imparts tumor-like qualities on cells, which may render SV40-transformed

79 cells more relevant for use in cancer research than for research on basic physiology. Large T also integrates randomly into the host cell‟s genome, and can affect any number of different genes in the process. Finally, SV40 transformation is irreversible, which somewhat limits the scope of use for SV40- transformed cells. Many cells in the human body are senescent or not actively proliferating unless an external signal is given, such as wounding. Thus, the ability to induce proliferation and subsequently cease it would allow for the creation of more biologically-relevant model systems using human cells. We describe here a new model system for sebocyte research which reversibly immortalizes primary sebocytes using the Rho-kinase inhibitor Y-27632. Y-27632 is a small molecule that selectively inhibits both isoforms of Rho-associated protein kinase (ROCK) (Figure 3.1). ROCK belongs to a family of serine/threonine kinases that interact with Rho GTPases. The Rho pathway mediates signaling from cell adhesion molecules such as integrins on the cell surface and converges on genes involved in cytoskeleton reorganization to control cellular contraction, adhesion, migration, and proliferation.307 Y- 27632 binds to ROCK‟s catalytic site to inhibit its kinase activity, thus blocking the Rho pathway and overriding integrin-mediated cell contact inhibition.308,309 Y-27632 has been previously shown to efficiently immortalize primary human keratinocytes.310 It has also been used to extend the life of embryonic stem cells and de-differentiate them from multipotent stem cells into pluripotent.311,312 In this Chapter we describe methods for using Y-27632 treatment and 3T3 fibroblast feeder layers to efficiently expand primary sebocytes (pSEB) in culture. ______

a) b)

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80 3.3 Results

3.3.1 Methods for growing pSEB from human sebaceous glands

Primary sebocytes were dissected from the sebaceous gland from the scalp of a 65 year-old male and placed in tissue culture plates with murine 3T3 fibroblasts that had been pretreated with mitomycin-c to inhibit their proliferation. Sebaceous glands and 3T3 were grown together for three days before adding fresh medium containing 5 μM of Y-27632. Media was replaced every other day with fresh media containing Y-27632, and cultures were passaged when they reached confluency. For splitting, 3T3 were first washed off using gentle pipetting with a dilute trypsin/EDTA solution. pSEB were then trypsinized and washed before replating with fresh 3T3 that had been pretreated with mitomycin-c. In culture, pSEB grown in the presence of Y-27632 tend to gather together into small round colonies with 3T3 fibroblasts surrounding them (Figure 3.2a,b). These colonies are three-dimensional and multi-layered, a growth pattern that is characteristic of some types of primary cells when grown in culture. At 40x magnification, yellow lipid droplets are visible in the cytoplasm (Figure 3.2c).

3.3.2 pSEB can be successfully expanded with Y-27632 for over 30 passages

To observe the effects of Y-27632 on proliferation, growth curves were created using pSEB population doublings. pSEB were continuously passaged in the presence of Y-27632 for over 200 days in the presence of Y-27632 and 3T3 fibroblast feeder layers. At each passage, pSEB were counted and re- seeded at an exact density. pSEB exhibited steady growth and proliferation with an average of one population doubling every 1.7 days (Figure 3.3). Periodically, some cultures were deprived of Y-27632, 3T3, or both. Most of these cultures exhibited growth arrest or death after only a few passages without Y- 27632 and/or 3T3 cells. The growth arrest observed in cultures deprived of Y-27632 confirmed that pSEB had not spontaneously immortalized in the presence of the drug. To further examine the proliferation in pSEB grown with Y-27632, bromodeoxyuridine (BrdU) incorporation assays were performed. pSEB were grown with Y-27632 plus 3T3 as above or were deprived of Y-27632 for 11 days before BrdU assays. Results show increased BrdU incorporation in cultures continuously treated with Y-27632 compared to cultures that had been without drug for 11 days (Figure 3.4), indicating an increased rate of proliferation induced by Y-27632. As controls, some cultures included the 3T3 in the incorporation assay (they were not washed off before BrdU addition). Interestingly, the 3T3 also incorporated BrdU, indicating that they were also actively proliferating in culture even though they had been pretreated with mitomycin-c.

81 ______

Figure 3.2: pSEB in culture. pSEB grown in the presence of Y-27632 are shown at 20x magnification with (a) and without (b) 3T3 fibroblasts present. At 40x magnification (c), lipid droplets are visible (yellow dots) in pSEB colonies.

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82 ______

160 p39 140 p33 120 +Y27632 +3T3 100 p23 +3T3 only 80 p18 60 +Y27632 p14 only 40 p9 20 p5

Population Population doubling p3 0 0 100 200 300 Days ______

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83 3.3.3 pSEB grown with Y-27632 show characteristics of primary sebocytes

Sebocytes produce more lipid as they differentiate and migrate up the pilosebaceous follicle, eventually undergoing holocrine rupture onto the skin surface. Lipogenesis can therefore be assayed as a measure of maturity and responsiveness to lipogenic hormones. We performed Oil Red O staining of cultures to visualize lipid droplets (Figure 3.5) as well as lipogenesis assays using 14C-acetate incorporation. Total lipogenesis assays showed that pSEB grown in the presence of Y-27632 produce a similar amount of lipids as SEB-1, but produced significantly more lipids when grown without the presence of the drug for five days (Figure 3.6). This data suggests that when Y-27632 is removed from culture, pSEB differentiate and produce more sebum as they cease proliferation. We tested pSEB response to androgen by treating cultures with R1881 (methyltrienolone), a synthetic androgen that binds strongly to the AR. Total lipogenesis assays showed that R1881 does not induce lipogenesis in pSEB cultured with or without Y-27632 (Figure 3.7). Accordingly, western blotting showed that pSEB do not express AR, even when treated with R1881 for 72 hours (data not shown).

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Figure 3.5: Oil Red O staining for lipids in pSEB. Oil Red O staining shows lipid droplets (red) in SEB-1 cells and pSEB that had grown without Y-26732 for two days.

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84 ______

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3.3.4 Gene array analysis of Y-27632’s effects in pSEB

To explore the major pathways by which Y-27632 acts to promote proliferation, we performed gene expression arrays on pSEB cultures. Four treatment groups were tested in triplicate samples: 1) pSEB grown continuously with Y-27632 and 3T3; 2) pSEB grown with Y-27632 but deprived of 3T3 for 10 days; 3) pSEB grown with 3T3 but deprived of Y-27632 for 10 days (all three groups were passage 6); and 4) pSEB at passage 23 that had spontaneously immortalized and grown successfully without Y-27632 or 3T3 for over 30 days. It should be noted that because pSEB could not grow in the absence of both Y- 27632 and 3T3 at passage 6, this control group could not be obtained for array analysis. Cut-off values for significant gene changes were set at 1.2 fold-change or greater. When comparing pSEB grown with Y-27632 and 3T3 to pSEB grown with only 3T3 for 10 days, we found levels of over 2500 gene transcripts changed. The 10 most up-regulated and down-regulated genes are displayed in Table 3.1. As expected, many genes known to be directly involved in the integrin- Rho pathway are changed on the gene chip with Y-27632 treatment. Notably, mRNA of the major Rho- pathway kinases such as RhoA and ROCK are not changed with Y-27632 treatment. A summary of genes changed in the Rho pathway is displayed in Table 3.2. Pathway analysis of the gene expression data has highlighted a few major pathways that are affected by Y-27632. In particular, numerous components of the Wnt pathway have significant changes in mRNA levels with Y-27632 treatment. Several Wnt isoforms are changed with Y-27632, as well as β- catenin and a few other mediators of the Wnt pathway (Table 3.3). Future studies will elucidate the involvement of this pathway and others in the immortalization effects of Y-27632 in pSEB.

86 ______

Table 3.1: Top Genes Changed with Y-27632 Treatment in pSEB

Top ten up-regulated genes

Gene Symbol Gene Name Fold-Change with Y-27632 WNT4 Wingless-type MMTV integration site family, member 4 +92.4 HAL Histidine ammonia-lyase +36.5 LY6H Lymphocyte antigen 6 complex, locus H +31.8 TEX101 Testis expressed transcript 101 +31.0 PLA2G4D Phospholipase A2, group IVD (cytosolic) +26.8 IGFBP5 Insulin-like growth factor binding protein 5 +25.3 KRT2 Keratin 2 +22.2 Glycosylphosphatidylinositol specific phospholipase D1, GPLD1 +19.4 transcript variant 1 SLC47A1 Solute carrier family 47, member 1 +16.9 WNT3A Wingless-type MMTV integration site family, member 3A +16.4

Top ten down-regulated genes

Gene Symbol Gene Name Fold-Change with Y-27632 LOC728285 Similar to keratin associated protein 2-4 -84.1 ROPN1 Ropporin, rhophilin associated protein 1 -62.8 ALOX5AP Arachidonate 5-lipoxygenase-activating protein -33.3 Tissue factor pathway inhibitor (lipoprotein-associated TFPI -31.6 coagulation inhibitor), transcript variant 1 RGS4 Regulator of G-protein signalling 4 -31.5 CYGB Cytoglobin -28.8 NLRP3 NLR family, pyrin domain containing, transcript variant 3 -27.0 DUSP8 Dual specificity phosphatase 8 -20.6 Retinoic acid receptor responder (tazarotene induced), RARRES1 -17.2 transcript variant 2 NLRP3 NLR family, pyrin domain containing 3 -15.4 RBMS3 RNA binding motif, single stranded interacting protein -15.2

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87 ______

Table 3.2: Rho Pathway-Associated Genes Changed with Y-27632 Treatment in pSEB

Gene Symbol Gene Name Fold-change with Y-27632 Up-stream of IGTA2 Integrin α2 -2.1 ROCK: IGTA5 Integrin α5 -4.4 IGTB6 Integrin β6 -4.7 IGTB8 Integrin β8 -2.5 ICAM2 Intercellular adhesion molecule 2 -2.4 CERCAM Cerebral endothelial cell adhesion -3.8 molecule CEACAM6 Carcinoembryonic antigen-related cell -2.2 adhesion molecule 6 CEACAM1 Carcinoembryonic antigen-related cell -3.6 adhesion molecule 1 L1CAM L1 cell adhesion molecule -2.9 JAM Junctional adhesion molecule 2 -8.7 HEPACAM Hepatocyte cell adhesion molecule +4.7 CADM4 Cell adhesion molecule 4 +1.7 Down-stream RND3 Rho-family GTPase 3 -1.6 of ROCK: ARHGAP25 Rho-GTPase activating protein 25 -2.0 ROPN1 Ropporin, rhophilin associated protein 1 -62.8 ROPN1B Ropporin, rhophilin associated protein -23.2 1B CDC42EP2 Cdc42 effector protein 2 -1.8 CDC42EP3 Cdc42 effector protein 3 -2.0 CDC42EP4 Cdc42 effector protein 4 +1.8 MPRIP Myosin phosphatase Rho interacting -2.0 protein SYDE1 Synapse defective 1, Rho GTPase, -2.5 homolog 1 CLDN23 Claudin 23 -2.0 CLDN8 Claudin 8 -6.6 CLDN7 Claudin 7 -10.3

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88 ______

Table 3.3: Wnt Pathway-Associated Genes Changed with Y-27632 Treatment in pSEB

Gene Gene Name Fold-change Symbol with Y-27632 Wnt isoforms and WNT4 Wingless-type MMTV integration +94.2 pathway components site family, member 4 WNT3A Wingless-type MMTV integration +16.4 site family, member 3A WNT5B Wingless-type MMTV integration -7.0 site family, member 5B WNT3 Wingless-type MMTV integration +2.1 site family, member 3 WNT7B Wingless-type MMTV integration +2.3 site family, member 7B WNT10A Wingless-type MMTV integration +3.4 site family, member 10A CTNNB1 β-catenin -1.85 CTNNBIP1 β-catenin interacting protein 1 +1.7

WLS Wntless homolog APCDD1 adenomatosis polyposis coli down- +6.5 regulated 1 Cdc42EP2 Cdc42 effector protein 2 -1.8 Cdc42EP3 Cdc42 effector protein 3 -2.0 Cdc42EP4 Cdc42 effector protein 4 +1.8 FZD2 Frizzled homolog 2 +1.7 FZD8 Frizzled homolog 8 -1.9 DACT2 dapper, antagonist of beta-catenin, -9.5 homolog 2 Wnt target genes HAL Histidine ammonia lyase +36.5 PPARA Peroxisome proliferator-activated +4.6 receptor (PPAR) alpha PPARD Peroxisome proliferator-activated -1.8 receptor delta ID2 Inhibitor of DNA binding 2 +10.1 TGFB2 Transforming growth factor β2 END1 Endothelin 1 -3.3 VEGFB Vascular endothelial growth factor B +1.5 VEGFC Vascular endothelial growth factor C -2.4 MMP7 Matrix metallopeptidase 7 -2.1 SOX9 Sex determining region Y-box 9 -2.8

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89 3.4 Discussion

We have shown that the transient immortalization of primary sebocytes is possible by treatment with the ROCK inhibitor Y-26732. The addition of this compound to primary sebaceous glands expanded sebocytes continuously to enough confluency that they could be used for experimental assays. Furthermore, the removal of the drug ceased sebocyte proliferation and allowed them to differentiate and produce more lipid. This is a novel model system for sebocyte research that overcomes the need for transfection by SV40 to achieve immortalization. pSEB cultures were highly dependent on Y-27632 and the 3T3 for growth and survival. As shown in Figure 3.3, removal of the drug ceased proliferation within just one passage. Most of the cultures subsequently died after several passages without Y-27632. A few cultures, though, survived for many passages without Y-27632 treatment, though they ceased to proliferate. It is possible that these cells could have entered an extended stage of senescence before finally dying. Future studies will further characterize patterns of proliferation and cell cycle in the presence of Y-27632 as well as determine the amount of time needed for Y-27632 treatment to induce proliferation when added to cells or subsequently cease proliferation when removed. One drawback to this system is the uncertainty of exactly which primary sebocytes in the parent sebaceous gland responded to Y-27632 to populate the expanded cultures. Y-27632 could preferentially act on cells in one differentiation stage more than others. Consequently, cells in the pSEB cultures could represent a mixture of sebocytes that were synchronized in their cell cycles by Y-27632 treatment, or they could have all come from just one (or a few) cells that were at one particular stage of growth. It is unknown whether or not these factors would create variability in experimental results between cultures. For example, because only basal and immature sebocytes express the AR, it is unclear whether the lack of AR expression in pSEB cultures is an artifact of random cell selection by Y-27632.313 Additionally, both inter- and intra-individuality could affect experimental results. We dissected sebaceous glands from three different individuals. Two of these glands (one from the temple of an 83 year-old male, and one from the cheek of a 65 year-old male) ceased proliferating after about 10 passages with Y-27632, and earlier passages were difficult to recover from frozen stocks. We have therefore exclusively used pSEB from the third gland (from the scalp of a 65 year-old male) for most experimental assays (all data presented in this Thesis is from this third gland). It is unknown if the first two glands failed to continue growing because of individual differences between people, differences between sebaceous gland location on the body (cheek versus scalp, for example), or because of differences in the cells that grew out of the sebaceous gland to populate the culture. These possibilities are difficult to

90 confirm experimentally, but may be overcome by pooling cells from multiple sebaceous glands and/or multiple individuals, much like normal human epidermal keratinocyte stocks that are sold commercially. Many questions remain to be explored with future projects. Listed below are some of the current directions of investigation.

What major pathways does Y-27632 affect? Future work will elucidate the mechanisms by which Y-27632 induces proliferation in sebocytes. According to the gene array data, the Wnt pathway appears to be one of the most promising areas of investigation. As shown in Table 3-3, multiple genes involved in Wnt signaling are changed with Y- 27632 treatment. This data is especially interesting because the Wnt pathway is heavily involved in sebocyte differentiation during embryogenesis. The canonical Wnt pathway involves Wnt signaling through a surface receptor complex that converges on β-catenin, which translocates to the nucleus where it displaces Groucho-related gene or CREB-binding co-repressors from the LEF/TCR transcription complex. In non-canonical Wnt signaling, Frizzled can activate RhoA, which then acts on target genes involved in microtubule organization.314 This cross-talk appears to be mediated primarily by the Wnt3A isoform, which is also one of the top genes changed with Y-27632 treatment. Future studies will investigate the potential role of Wnt in the immortalization of pSEB by ROCK inhibition.

Is Y-23762 inducing a stem cell-like phenotype in primary sebocytes? Sebaceous gland development begins in the 13th to 16th week of gestation as a bulge in the follicular primordium. This bulge region contains epidermal stem cells that divide and populate the epidermal keratinocytes, follicular keratinocytes, and sebaceous glands. Wnt and TRAF6 playing a major role in this fate determination. Increased Wnt signaling promotes differentiation into follicular keratinocytes, whereas decreased Wnt signaling promotes sebocyte differentiation. Recently, B lymphocyte-induced maturation protein 1 (Blimp-1) has been proposed as a marker for a population of progenitor cells that reside in the sebaceous gland.315 Other markers for sebocyte progenitors include the PPAR receptors, which play roles in sebocyte differentiation and maturation, and β4 integrin, which marks the basal row of developing bulge keratinocytes, as well as c-myc and CD34. Several of these markers show changes in mRNA levels with Y-27632, which raises the question of whether or not Y- 27632 can de-differentiate primary sebocytes into sebocyte/bulge cell progenitors.

Is Y-27632 inducing an epithelial to mesenchymal transition in pSEB? Epithelial to mesenchymal transitions (EMT) have garnered interest in cancer research. This term describes the phenotypic changes a cell undergoes when it loses its epithelial cell-cell adhesion properties

91 and gains features of motile, invasive fibroblastoid mesenchyme. EMT is characterized by decreased epithelial markers such as E-cadherin, occludin, and claudins, as well as increased mesenchymal markers such as vimentin, N-cadherin, fibronectin, vitronectin, matrix-metalloproteases (MMPs), and c-myc.316,317 Notably, both Wnt and TGFβ signaling can contribute to EMT, which is also characterized by increased Rho-GTPase activation.318 Many genes that are associated with either epithelial or mesenchymal phenotypes are changed with Y-27632 treatment in pSEB. However, the gene changes do not all fit into one direction of transition or the other. For example, Y-27632 down-regulates mRNA for claudins and mucin and up-regulates Notch, which are characteristic changes seen in EMT. However, Y-27632 also down-regulates vimentin, fibronectin, and β-catenin, which are features of the reverse transition, or mesenchymal to epithelial transition (MET). Future experiments will confirm changes in gene transcripts by Western blotting for proteins to begin to elucidating this discrepancy.

What growth signals do the 3T3 contribute to the cultures? It is interesting to note that removal of 3T3 from the pSEB cultures also ceases proliferation, even in the presence of Y-27632. pSEB require both the 3T3 and Y-27632 to survive and proliferate. 3T3 have been used for years as feeder layers to support the growth of epithelial cells in culture, but their exact contributions to cell survival are unknown. Research has shown that 3T3 feeder layers can delay senescence in primary keratinocytes by maintaining levels of specific transcription factors such as Sp1 and Sp3.319 Additionally, 3T3 enable epithelial cells to retain stem cell markers.320 Even more intriguing is the fact that 3T3 that have been lethally irradiated have similar efficacy in supporting epithelial culture growth as live 3T3 that have been growth-arrested by mitomycin-c. The mechanisms by which 3T3 feeder layers support cell growth are unknown. We have gene expression data on pSEBs that have been grown as described with Y-26732 and 3T3 as well as pSEBs that had been grown with Y-27632 but deprived of 3T3 for 10 days before the array was performed. The analysis of this data could yield insight into how 3T3 contribute to pSEB survival.

In conclusion, we demonstrate herein a novel system for temporarily immortalizing primary sebocytes to achieve adequate expansion for use in experimental assays. This immortalization appears to be reversible, as the removal of the drug ceases proliferation and allows the pSEB to differentiate and produce more lipids. Future studies will give insight into the mechanisms by which Y-27632 and 3T3 feeder layers synergize to sustain pSEB proliferation. This model could be a valuable tool in researching sebocyte biology.

92 3.5 Methods

3.5.1 Sebaceous Gland Dissection and Cell Culture

NIH Swiss strain murine 3T3 fibroblasts (ATCC) were used as feeder layers for sebocytes. Separate 3T3 fibroblast cultures were maintained in order to supply fresh fibroblasts for each pSEB passage. Prior to use with pSEBs, 3T3 fibroblasts were treated with mitomycin-c (Sigma) for 30 minutes to prohibit their proliferation. Primary sebocytes were isolated by the dissection of fresh skin samples obtained from the dermatology clinic at Penn State Hershey Medical Center. Dissected sebaceous glands and 3T3 fibroblasts were placed in 35 mm culture plates containing Bajor‟s Sebocyte medium (with 10% FBS (v/v)). After several days in culture, pSEB were treated with Y-27632. Stocks of Y-27632 dihydrochloride monohydrate (Sigma Aldrich) were made by resuspending powder stock in sterile water at 10 mM., and 200 µL aliquots were stored at -20° C. pSEB cultures were treated with 5 µM of Y-27632 in Bajor‟s Sebocyte medium and given fresh media containing Y-27632 every other day. Cultures were passaged when they reached confluency. For treatment in lipogenesis assays, the synthetic androgen methyltrienolone (compound R-1881; Sigma Aldrich) was resuspended in sterile water and stored in aliquots at -20° C until use. For experimental assays, 3T3 fibroblasts were washed off plates before pSEB were harvested for assays. Plates were gently rinsed using 1.2 mL warmed 0.02% (w/v) EDTA/PBS to dislodge the fibroblasts, aspirated, and then washed twice with PBS. SEB-1 cells were cultured using previously published conditions.14

3.5.2 Oil Red O Staining

Cultures were seeded in 35 mm or 60 mm plates for Oil Red O staining. 1 mg/mL stocks of Oil Red O (Sigma Aldrich) were made by dissolving in 99% (v/v) isopropanol. Before use, Oil Red O stocks were mixed with distilled water and filtered. 3T3 fibroblasts were washed off plates as described above and pSEB cultures were washed once with PBS. Cells were fixed with 10% (v/v) formalin for 30 minutes at room temperature and then washed twice with PBS. Oil Red O was added to cultures and incubated at room temperature for 10 to 15 minutes. Plates were rinsed once with 50% (v/v) isopropanol and once with water before drying or mounting with aqueous mounting medium.

93 3.5.3 Lipogenesis Assays

The incorporation of 14C-acetate into cells was used as a measure of lipogenesis. Cultures were seeded in 35 mm plates with Y-27632 and treated with 10 nM R-1881 for 24 and 48 hours. At the end of the treatment period, plates were washed with 1x PBS and cells were treated with trypsin, pelleted, and washed once with plain media. An aliquot of cells from each sample was saved for counting. 1.5 ml of media containing 1 μCi of 14C-acetate (New England Nuclear) was added to each sample, mixed well, and incubated in a shaker for 2 hours at 37° C. Samples were mixed well before adding 3 mL of ethyl ether, vortexing, and pelleting. Samples were frozen at -80° C for 10 minutes or until the bottom layer was frozen. The top layer was poured into 12 x 75 mm glass tubes and dried. 200 μL of ethyl ether was added to each tube to dissolve the residue from extraction, and then each sample was added to a scintillation vial containing 10 mL of scintillation fluid and analyzed using a scintillation counter.

3.5.4 Gene Chips

Gene array analysis was performed on four different pSEB treatment groups. pSEB at passage 6 were seeded into 60 mm plates with a 3T3 fibroblast feeder layer plus Y-27632, a 3T3 feeder layer only (no Y-27632), or Y-27632 only (no 3T3 feeder layer) and grown for 10 days, with fresh media added every other day. Spontaneously immortalized pSEB at passage 23 that had been grown and passaged for over 30 days without either 3T3 fibroblasts or Y-27632 were also included as a treatment group. At the end of the 10 days of treatment, 3T3 fibroblasts were washed off the plates and all plates were rinsed twice with 1x PBS. RNA was isolated using RNAeasy kits (Qiagen) according to manufacturer‟s directions. RNA samples were aliquotted and stored at -80° C until use. Treatments were set up in triplicate, and RNA isolated from each replicate was treated as one sample for use with a separate gene chip. RNA quality and integrity were checked using Eukaryotic Total RNA Nano Series II chips (Agilent) assayed using an Agilent 2100 Bioanalyzer. Gene expression microarrays were performed in the Penn State Hershey Core Research Facilities using Illumina HT-12 gene chips. Expression levels were quantified from gene chips using NimbleScan software (Roche Nimblegen), and then data analysis was performed comparing treatment groups using GeneSpring GX software (Agilent). pSEB treatment groups were compared using Student‟s t-tests, with P < 0.05 being considered significant. Gene changes were considered significant if absolute fold-change values equaled 1.2 or greater. Fold change data was imported into IPA Software (Ingenuity Systems) for pathway analysis.

94 3.5.5 BrdU Incorporation Assays

pSEB were grown as described in the presence of Y-27632 and grown 24-well plates for four days before BrdU proliferation assay were performed. Some cultures were deprived of Y-27632 for seven days before seeding into 24-well plates (for a total of 11 days without Y-27632). Before adding BrdU, 3T3 cells were washed off some wells using 0.02% (w/v) trypsin. Fresh media containing BrdU was added to each well and incubated for 2.5 hours at 37° C. Cultures were then fixed and permeabilized, and incubated with anti-BrdU primary antibodies at room temperature for one hour. Wells were washed and then incubated with secondary biotinylated antibody for one hour at room temperature. After washing, color reactions were developed using peroxidase substrate, and absorbance was measured using a Synergy HT Multi-Detection Microplate Reader (BIO-TEK).

95 Chapter 4

Discussion

4.1 Introduction

Isotretinoin has been used for nearly 30 years in the treatment of acne as well as other dermatological diseases and some cancers. It is the only known agent that can permanently cure acne, however its long-term mechanisms of action are unknown. In this Thesis, we have shown that isotretinoin has immunomodulatory effects in acne patients in vivo, which may help explain how isotretinoin can induce permanent remissions of acne. However, the relevance of this model extends to other disease contexts for which isotretinoin is used as a therapy.

4.2 Rationale, Hypothesis, and Results of This Work

Because of its severe side-effect profile and teratogenicity, it is unethical to administer isotretinoin to healthy subjects. Thus, in vivo studies on isotretinoin‟s mechanisms of action are lacking. We hypothesized that an induction of immune „tolerance‟ to P. acnes plays a part in isotretinoin‟s mechanism of permanently curing acne.

In this Thesis, we have shown that:

1) Acne patients’ innate immune systems are hyper-responsive to stimulation by P. acnes. Acne patients‟ monocytes express higher levels of TLR-2 and secrete larger amounts of inflammatory cytokines in response to P. acnes stimulation compared to monocytes from normal volunteers. However, no abnormalities in peripheral lymphocytes were detected in acne patients.

2) Isotretinoin therapy normalizes acnes patients’ innate immune responses to P. acnes. This is the first study to show that isotretinoin therapy not only reduces TLR-2 expression in acne patients‟ monocytes at a basal level in vivo, but also inhibits P. acnes-induced up-regulation of TLR-2 expression. Additionally, this is the first study to show that systemic isotretinoin reduces inflammatory cytokine production by acne patients‟ monocytes in response to P. acnes.

96 3) Isotretinoin therapy does not affect peripheral blood proportions of Treg

Though ATRA has been shown to be sufficient for promoting naïve T cell differentiation into Treg in vitro, we present here the first evidence that oral retinoid therapy does not affect peripheral

subsets of Treg, despite raising serum retinoic acid isomer concentrations to over ten-fold the

normal levels for months at a time. Studies investigating Treg in acne patient skin biopsies were inconclusive.

4.3 Explanation of Model

This study has demonstrated that modulation of the innate immune system could be one way in which isotretinoin cures acne. Acne patients‟ monocytes express higher numbers of TLR-2 receptors on their surface and produce larger amounts of inflammatory cytokines in response to TLR-2 stimulation by P. acnes sonicate. The lack of association between acne and TLR-2 polymorphisms as well as our observed correlation between TLR-2 expression levels and cytokine production further support the hypothesis that over-abundance of surface TLR-2 receptors represents the main defect in acne patients‟ monocytes, rather than abnormal function of TLR-2 or any of its down-stream mediators. Systemic isotretinoin therapy normalizes monocytes‟ inflammatory response to P. acnes by reducing the numbers of TLR-2 receptors available on the surface to bind to P. acnes antigens and initiate inflammatory signal cascades. We did not find isotretinoin-associated changes in the adaptive immune cells including peripheral proportions of Treg, but many more aspects of this system need further study.

4.4 Future Directions

This Thesis has shown some of isotretinoin‟s effects on the immune system in vivo. However, many questions remain unanswered. Future research could further elucidate isotretinoin‟s in vivo actions as well as the underlying mechanisms for the factors involved in acne pathogenesis. The following sections review some of the major future directions for this project.

97 4.4.1 What factors are responsible for the higher TLR-2 expression in acne patients’ monocytes?

The reasons behind the abnormal TLR-2 expression on acne patients‟ monocytes are unknown. Acne patients may have innately higher levels of TLR-2 throughout their lives, or TLR-2 expression levels may correlate with age. There are currently no reports in the literature that describe a relationship between age and TLR expression levels. Although we found no correlation between age and monocyte TLR-2 expression in either acne patients or normal volunteers, the age range of our study was limited (12 to 35 years). Our data suggests that spontaneous TLR-2 down-regulation does not represent a mechanism by which age naturally regresses in early adulthood; however, this does not preclude the possibility that TLR-2 expression is up-regulated sometime before or during puberty in some people. TLR-2 expression on monocytes temporarily increases in the presence of its ligands, but the time course of this upregulation has not been well characterized. It is possible that a precipitating immune event could cause a more permanent aberrant TLR-2 expression. Because TLR-2 recognizes antigens from bacteria and viral envelope proteins, it would be interesting to correlate TLR-2 over-expression with antibiotic usage or the onset of typical childhood illnesses such as chicken pox (Varicella zoster), strep throat, or mononucleosis.

4.4.2 How do retinoids work to down-regulate TLR-2 expression?

The mechanism by which retinoids affect TLR expression is unknown. Published data has demonstrated that treatment of normal human monocytes with ATRA in vitro down-regulates TLR-2 mRNA, suggesting that isotretinoin operates by a similar mechanism in acne patients‟ monocytes in vivo.237 The gene for TLR-2 contains multiple transcription factor binding elements in the promoter region, but does not contain any RAREs, thus it is unlikely that retinoids regulate TLR-2 directly though ATRA-activated RAR-RXR mediated mechanisms. Because isotretinoin therapy raises serum 9-cis RA concentrations, it is possible that isotretinoin affects TLR-2 expression through 9-cis RA activation of RXRs in dimers with other nuclear receptors such as VDR. Previous work has shown that the treatment of normal human PBMC and PBMC from Behcet‟s patients with vitamin D3 in vitro decreases TLR-2 and TLR-4 expression.321 However, we did not find that isotretinoin decreased TLR-4 expression in vivo, and vitamin D did not affect TLR-2 expression in normal monocytes in our system when incubated with either FBS or human serum (data not shown), indicating the involvement of other factors in this complex signaling network. Additionally, isotretinoin could modulate TLR-2 expression by a more indirect mechanism. ATRA is classically known for controlling the expression of the HOX genes during embryogenesis

98 through local histone acetylation, thereby directing gene transcription by rendering gene promoters physically available for transcription factor binding.197 The TLR-2 gene has not been shown to be controlled epigenetically by histone acetylation, but this remains a possibility. The TLR-2 gene promoter is completely unmethylated in human PBMC and primary tissues, whereas in leukemias and immortalized cell lines that do not express TLR-2, the promoter has almost complete CpG methylation.36 It is plausible that epigenetic regulation of the TLR-2 gene could play a role in either acne patients‟ over-expression of TLR-2 or in isotretinoin‟s ability to decrease its expression.

4.4.3 Does isotretinoin also affect leukocytes in the skin or other microenvironments?

Though we have found that isotretinoin down-regulates TLR-2 expression in peripheral immune cells, it is unknown whether similar mechanisms occur in the skin microenvironment. It has been shown in other labs that retinoid treatment down-regulates TLR-2 expression in skin keratinocytes in vitro. We therefore attempted to use immunohistochemistry to investigate TLR-2 expression in skin from acne patients before and after 8 weeks of isotretinoin therapy, but this method was not quantitative enough to make conclusions, nor did it allow the analysis of individual cell types. To answer these questions more directly and quantitatively, leukocytes could be isolated from fresh skin punch biopsies from acne patients on isotretinoin therapy for analysis by flow cytometry. However, punch biopsies can be difficult to obtain in large enough numbers for valid statistical analysis. Retinoids have other known effects on the immune system apart from TLR regulation. One major function is the promotion of immunosuppressive Treg responses. DC in skin and gut-draining lymph nodes express Raldh enzymes and can directly promote Treg differentiation by controlling the conversion of retinal to ATRA in immune microenvironments. It will be important to determine whether flooding the system with exogenous retinoids affects these microenvironments, or whether it would be possible to manipulate immune microenvironments through local retinoid availability. We found that isotretinoin did not affect peripheral Treg proportions in acne patients. We attempted to investigate changes in Treg numbers in the skin microenvironment, but immunohistochemistry for Foxp3 on acne patient skin biopsies before and during isotretinoin therapy was inconclusive. As mentioned above, more sensitive assays such as flow cytometry of fresh cells should be used in order to further explore these questions. The investigation of isotretinoin‟s effects on lymphocytes in vivo also has relevance to cancer trials where it is used as a chemotherapy agent.

99 4.4.4 What are the downstream consequences of TLR-2 down-regulation on monocytes?

One puzzling aspect of isotretinoin therapy is its time-line of action. It has long been known that patients need to take the full cumulative dose of 120 mg/kg in order to achieve maximum chance of permanent remission, even in patients who demonstrate marked clinical improvement rapidly within a few months of therapy. Our data shows TLR-2 expression levels in most patients have reached their lowest values by 8 weeks of isotretinoin therapy, and then continue at this low expression level through 20 weeks. If TLR-2 expression is a primary mediator in the inflammatory response, then it is puzzling why 8 weeks of therapy would not be sufficient. This observation suggests that TLR-2 down-regulation could have downstream effects that take time to manifest clinically. As mentioned above, it is possible that the reduction in TLR-2 has downstream consequences for skewing P. acnes-specific T cell responses, or perhaps down-regulation of the gene for an extended period of time allows epigenetic changes such as promoter methylation to take place.

4.4.5 What factors determine if an acne patient will stay in remission with isotretinoin therapy?

Just as we know little about the underlying reasons that some people get acne, we also know little about why some acne patients respond differently to various therapies, including isotretinoin. Most patients experience clinical remission of acne with one course of isotretinoin, but as many as 41 percent will experience a relapse that requires a second course, sometimes years after their first.167 The reasons that some patients relapse are unknown. A few studies have pointed to particular clinical factors that increase a patient‟s risk of recurrence after isotretinoin therapy. Studies have shown that adult female patients are less likely to experience a long-lasting remission of acne with isotretinoin treatment than male patients. Our lab has previously shown that isotretinoin-induced NGAL expression in sebaceous glands was increased by only 1% in female acne patients compared to 12 to 36% in male acne patients, which may partially explain the gender discrepancy in remission time.57 Patients who have had acne less than six years and those who are younger than 19 at the time of isotretinoin therapy are also more prone to recurrence than older patients. Additionally, patients with significant acne on the torso all tend to respond less well than other subjects.322 Finally, subjects who experience a sebum rebound to within 10% of the pretreatment levels may also be less likely to stay in remission. The underlying mechanisms for these predictive factors are unknown. Individual variations in retinoid metabolism or tissue transport may be one reason for differences in acne patients‟ response to therapy. We did not find differences in serum levels of retinoic acid isomers

100 or metabolites between acne patients at baseline and normal volunteers, suggesting that acne patients do not have abnormal retinoid absorption, conversion, or metabolism in the periphery. However, it has been shown that acne patients have lower levels of serum RBP than normals, indicating that they may differ in the transport of retinoids to tissues such as the skin.277,281 As expected, we found that serum levels of retinoic acid isomers and their 4-oxo metabolites were greatly increased during isotretinoin therapy compared to baseline, but due to intra- and inter-patient variability, we were unable to correlate serum retinoid levels with clinical response. Whereas our study was unable to account for timing and dosage amounts for isotretinoin taken by the acne patients, some cancer trials have been able to control these factors while investigating the pharmacokinetics of isotretinoin. These trials have found significant inter- individual variation with up to a five-fold difference between peak and trough plasma concentrations in the same patient.323 Additionally, the time of first peak plasma concentration of isotretinoin varied considerably between patients on the same dose. These findings indicate that significant individual differences exist in baseline retinoid absorption, metabolizing enzymes, or carrier proteins. Because systemic retinoids also affect their own metabolism through the regulation of RBP and CRABP proteins, it may also be possible that patients differ in their feedback responses to exogenous retinoid therapy. It would be interesting to investigate if such differences in retinoid metabolism determine the extent of acne patients‟ response to isotretinoin therapy, or if such factors could predict whether or not a patient will need a second course of isotretinoin to reach full remission.

4.4.6 Could isotretinoin effectively treat other disorders characterized by TLR-2 over-expression?

Many disorders characterized by sterile inflammation have been linked to dysregulation of innate immune signaling.47,48,79 Specifically, TLR-2 over-expression in various cell types has been implicated in the pathogenesis of multiple diseases characterized by chronic inflammation such as psoriasis, sarcoidosis, Behcet‟s disease, rheumatoid arthritis, spondylarthropathy, and atopic dermatitis.47-50 In particular, TLR-2 over-expression is observed on peripheral circulating monocytes in patients with acne, psoriatic arthritis, rheumatoid arthritis, and Behcet‟s disease, implying that the dysregulation of innate immune signaling extends beyond the skin.47,48,51,324 Interestingly, acne is a symptom of some „autoinflammatory‟ syndromes, which are group of systemic inflammatory disorders without an apparent autoimmune, infectious, allergic, or immunodeficient origin. Of these syndromes, severe nodulocystic acne is a symptom of SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome, PAPA (pyogenic sterile arthritis, pyoderma gangrenosum and acne) syndrome, and Behcet‟s disease. Intriguingly, in a subset of SAPHO patients (about 42%), P. acnes is suspected as a causative agent for

101 the osteitis.325,326 Like acne, antibiotics can be effective for treatment of SAPHO syndrome, but efficacy is lost after their discontinuation, suggesting that an exaggerated immune response to P. acnes could play a role in SAPHO pathogenesis.327 Isotretinoin is not currently a routine treatment for any of the autoinflammatory syndromes, but a case report has shown to be successful in treating refractory acne associated with SAPHO in one patient.328 Because isotretinoin effectively down-regulates TLR-2 on circulating monocytes in acne patients in vivo, it could be a potential therapy for other disorders characterized by TLR-2 over-expression or hyper-responsiveness to P. acnes.

4.5 Conclusions

In conclusion, the data presented in this Thesis indicates that isotretinoin modulates acne patients‟ immune systems in vivo, thus „normalizing‟ their immune responses to P. acnes. Future work is needed to determine why acne patients have abnormally high expression of TLR-2 on their monocytes, and whether TLR-2 expression could be manipulated with a safer therapy than oral isotretinoin.

102 Appendix A

Mouse Studies

A.1 Chapter Abstract

As an adjunct to positive data showing that isotretinoin therapy in vivo down-regulates TLR-2 on peripheral blood monocytes in humans, a mouse model for P. acnes hypersensitivity was attempted in order to investigate corresponding changes in responses to P. acnes in the skin and skin-draining lymph nodes during isotretinoin therapy. C57BL/6 mice were sensitized with repeated intradermal injections of heat-killed P. acnes in the skin of the back and then challenged with a subcutaneous injection of live P. acnes in the base of the ear. Ears swelling was measured at baseline and at sacrifice as an indication of overall inflammatory response. Ears and draining lymph nodes were harvested and homogenized, and leukocytes were enriched using centrifugation gradient. Leukocytes were briefly stimulated with mitogens in vitro then stained for surface markers and intracellular cytokines using fluorescently- conjugated antibodies and analyzed by flow cytometry. While mice injected once with live P. acnes in the ear successfully mounted an inflammatory response, mice previously sensitized with repeated injections of heat-killed P. acnes did not show an augmented inflammatory response over mice that were mock-sensitized with saline. Additionally, proportions of CD4 and CD8 positive lymphocytes and their subtypes in ear-draining lymph nodes were not different between P. acnes-sensitized mice and saline- sensitized. Total numbers of leukocytes obtained from ears were not enough to be able to draw conclusions about subtype proportions. Due to these obstacles, the mouse model was not further pursued.

A.2 Introduction

Positive findings in this thesis show that isotretinoin therapy in humans in vivo down-regulates acne patients‟ peripheral monocyte TLR-2 expression and cytokine production in response to P. acnes. These studies confirm findings in vivo that had been suggested by in vitro treatment of human PBMC with retinoids. Peripheral blood samples are minimally invasive and are thus easy to obtain from patients; however, studies using only peripheral blood samples limits the scope of the results. It is still unknown if isotretinoin affects TLR-2 expression on monocytes in skin draining lymph nodes or on mature macrophages or DC in the skin. This Thesis attempted to create a mouse model of P. acnes hypersensitivity for the purpose of answering these questions. A mouse model would allow investigation

103 of the cellular infiltrate in skin and draining lymph nodes in response to P. acnes as well as changes in these cells associated with isotretinoin treatment during P. acnes challenge. This Appendix describes the attempts made at creating such a model.

A.3 Results

A.3.1 Live P. acnes subcutaneous injection successfully induces a cutaneous inflammatory response in C57BL/6 mice.

To first test the inflammatory response to live P. acnes in C57BL/6 mice, mice were injected subcutaneously once in the base of one ear with 450 μg of live P. acnes or saline vehicle in the opposite ear. Ear swelling was measured using micrometers at baseline and 2, 3, 4, 6, and 7 days after injection. This one-time injection with P. acnes induced marked swelling in ears of C57BL/6 mice compared to saline injection. Ear thickness in P. acnes-injected ears was roughly two-fold greater at 2 and 3 days and three-fold greater at 4 days following injection compared to saline-injected ears (Figure A.1).

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104 A.3.2 Prior exposure to P. acnes does not alter ear swelling in response to P. acnes challenge in

C57BL/6 mice.

To hyper-sensitize mice to P. acnes, mice were injected intradermally in the back with 450 μg of heat-killed P. acnes or saline vehicle three times on days -27, -24, and -21 (Figure A.2). Three weeks after the last sensitization injection (on day 0), mice were challenged subcutaneously with either 20 μg or 100 μg of live P. acnes in the right ear and saline vehicle in the left ear. Mice were sacrificed 2, 4, and 7 days after challenge. Ear swelling was measured with micrometers at baseline before challenge and at sacrifice. Within both the P. acnes and vehicle sensitization groups, all P. acnes-challenged ears had significantly higher fold-changes in swelling at the site of injection compared to vehicle-challenged ears at 2 and 4 days after the challenge (Figure A.3). At both of these time-points, P. acnes-challenged ears were roughly two-fold thicker than saline-challenged ears. However, neither of the subcutaneous challenge doses of P. acnes (20 μg or 100 μg – data not shown) caused augmented ear swelling in the P. acnes-sensitized group compared to the corresponding challenge in the vehicle-sensitized group.

A.3.3 Prior intradermal injection with P. acnes does not alter the proportions of CD4+ or CD8+ T cells in ears or draining lymph nodes in response to P. acnes challenge in C57BL/6 mice.

After sacrifice, ears and draining cervical lymph nodes were harvested and homogenized. Ear samples were pooled within each treatment group, and leukocytes were enriched from ear samples using Ficoll-Paque gradients. Leukocytes were then incubated with concanavalin A and brefeldin A for four hours at 37° C before fixing, staining for surface markers and intracellular cytokines, and analysis by flow cytometry. For analysis, T cells were first gated on forward scatter versus side scatter plots and then on CD3 expression. Proportions were calculated by dividing the number of cells expressing either CD4 or CD8 by the total number of CD3+ T cells. While the total numbers of T cells isolated from draining lymph nodes, spleen, and blood were relatively consistent between mice, the total numbers of T cells isolated from pooled ear tissue varied markedly. In both sensitization groups, ear tissue that had been injected with live P. acnes and the corresponding draining lymph nodes contained higher absolute numbers of CD4+ and CD8+ lymphocytes at 2 and 4 days after challenge compared to ears injected with saline vehicle. However, the proportions of total lymphocytes in the ear-draining lymph nodes that were CD4+ or CD8+ did not differ between P. acnes-challenged ears and saline-challenged ears, nor did they differ between sensitization groups at any time point (Figure A.4). Additionally, no differences were observed in CD4 and CD8 proportions in the spleen or blood between sensitization groups (data not shown).

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106 ______

Figure A.4: Proportions of CD4+ and CD8+ T cells in ears and draining lymph nodes in P. acnes-sensitized mice in response to P. acnes challenge. Mice were sensitized with three intradermal injections of heat-killed P. acnes or vehicle and three weeks after the last sensitization injection were challenged with subcutaneous injections of live P. acnes („P.acnes Chal.‟) or saline vehicle („Saline Chal.‟) as described. Mean (± SEM) percentages of CD4+ and CD8+ T cells observed in each of the P. acnes-sensitized (dark-blue bars) and vehicle-sensitized (light- blue bars) groups are shown for the ears and their corresponding draining cervical lymph nodes (CerLN).

______

107 ______

Figure A-5: Proportions of Treg in ears and draining lymph nodes in P. acnes-sensitized mice in response to P. acnes challenge. Mean (± SEM) percentages of CD4+ T cells that expressed Foxp3+ in each of the P. acnes- sensitized (dark-green bars) and vehicle-sensitized (light-green bars) groups are shown for ears and their corresponding draining cervical lymph nodes (CerLN).

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108 A.3.4 Prior intradermal injection with P. acnes does not alter the proportions of T cell subsets or activation markers in ears or draining lymph nodes in response to P. acnes challenge in C57BL/6 mice.

To assess lymphocyte activation and subtype proportions, ear and lymph node samples were stained with panels of fluorescently-conjugated antibodies against markers of activation or maturity (CD45RB, CD62L, CD69, CD25, and cutaneous lymphocyte antigen (CLA)) as well as subtype markers and intracellular cytokines (Foxp3, IFNγ, IL-4, and IL-17). T cell subtype proportions were calculated by dividing the number of cells expressing subtype markers by the total number of CD4+ or CD8+ T cells.

Proportions of Treg did not differ in draining lymph nodes between challenges or sensitization groups (Figure A.5). Additionally, percentages of CD4+ and CD8+ cells expressing CD45RB, CD62L, CD69, or CLA in draining lymph nodes did not differ between P. acnes-challenged ears or vehicle- challenged ears in any sensitization group, nor did they differ between sensitization groups (data not shown). The pooled ear samples did not contain enough total CD4+ or CD8+ lymphocytes in order to be able to make conclusions regarding subtypes. Samples that appeared to have differing proportions of Treg in fact only contained a few lymphocytes in the whole sample (such as seen in Figure A.5 in the 2-day P. acnes-challenged ear samples).

A.4 Discussion

This study attempted to create a mouse model of hypersensitivity to P. acnes for the purpose of using as a model for acne inflammation in humans. While this model was successful in eliciting a general inflammatory response to P. acnes, it was unsuccessful in inducing a hypersensitivity reaction to P. acnes. In these experiments, mice sensitized with P. acnes did not show a difference in ear swelling, CD4/CD8 proportions, T cell subtype proportions, or T cell activation markers in the ear or draining cervical lymph nodes compared to mice sensitized with saline vehicle. These results suggest that the particular sensitization time-lines and dosage protocols used in these experiments were ineffective at inducing a sensitization to P. acnes. A few different animal models have been used in other labs to mimic the interaction of P. acnes with the human sebaceous gland, however, there is no perfect animal model because only human sebaceous glands produce the right milieu of lipids to support P. acnes growth. Therefore, many animal models for P. acnes infection have used subcutaneous or intradermal injections of P. acnes preparations to mimic sebaceous gland colonization. Each animal model that has used this route of P. acnes infection

109 has elicited a different immunological response to the P. acnes. De Young et al. injected female Sprague- Dawley rats with 140 μg of formalin-killed P. acnes (in saline/0.1% Thimerosol vehicle) one time intradermally in the ear, producing an inflammatory reaction that caused a 2-3 fold-change increase in ear thickness compared to vehicle for up to 44 days after injection.176 This group also successfully created a hypersensitivity model where they injected rats with 35 or 140 μg P. acnes in one ear, and one week later they were challenged in the opposite ear with a 17.5 μg.177 Both groups of P. acnes-sensitized rats experienced more inflammation in response to challenge than non-sensitized rats. Rats remained sensitized for up to three weeks after the first injection, and the resultant inflammatory response from challenge lasted for up to two weeks. Interestingly, this group also found that different strains of P. acnes isolated from the skin of various acne patients and normal volunteers induced different inflammatory responses in rats, though the source of each P. acnes strain (acne patient versus normal volunteer) did not correlate with the amount of inflammation it induced. The experiments in this Thesis differed from De Young‟s in that they used heat-killed instead of formalin-killed P. acnes, and C57BL/6 mice instead of Sprague Dawley rats. Additionally, this Thesis did not use Thimerosal (an organomercury compound that is used as a vaccine preservative) in any of the vehicles. There are currently no other reports of successful hyper-sensitization of an animal to P. acnes in the literature. More recent studies in the literature have focused on creating a vaccine to protect against P. acnes-induced inflammation. Nakatsuji et al. successfully induced inflammation in their model with a one-time intradermal injection of 1x107 CFU live P. acnes into the ears of ICR mice.136 For vaccination experiments, they immunized mice intranasally with 1x108 CFU heat-killed P. acnes three times at three- week intervals and then challenged the mice subcutaneously with 1x107 CFU live P. acnes in 20 μL in ear. Mice that had been vaccinated showed reduced ear inflammation in response to challenge. In addition, they produced antibodies specific to P. acnes. This Thesis used the same P. acnes strain and growth/preparation protocols as this study; however, the difference in mouse strains and routes of administration differed. C57BL/6 mice were chosen for this study because of their common usage in the immunology literature. Additionally, our lab had used this strain previously for investigating skin changes with isotretinoin treatment in vivo, and it would have been ideal to be able to correlate those results with the studies in this Thesis. Preliminary experiments with these mice showed that a one-time subcutaneous injection in the ear with P. acnes produced an inflammatory response as measured by ear thickness. These findings were comparable to those seen in other studies in the literature. For example, De Young et al. detected a two-fold change in rat ear thickness (compared to a baseline thickness of 0.5 mm).177 However, C57BL/6 mice did not display a magnified inflammatory challenge response after administration of repeated sensitizing injections. In the high-dose P. acnes-challenged mice, the ears of

110 the P. acnes-sensitized mice were actually less swollen than those of the vehicle-sensitized mice at two and four days after challenge (data not shown). This data suggests that the intradermal injections with killed P. acnes were protective against a live P. acnes challenge. The mice sensitized with killed P. acnes did not experience a great deal of inflammation at the sensitization injection sites on the back, though each injection created a small palpable granuloma (about 5 mm in diameter) that lasted for a few days. The choice of the C57BL/6 strain may be responsible for the lack of hypersensitivity response to repeated P. acnes injections. C57BL/6 mice are known to be resistant to infection with certain pathogens. This may be an artifact of their unique HLA phenotype, H-2KbDb, making them more resistant to infections compared to strains with other HLA H-2K and H-2D alleles. If pursued, future attempts at creating a mouse model for P. acnes hypersensitivity could employ alternative mouse strains that may be more susceptible to infection. It is possible that the methods used in this study did not isolate the full repertoire of T cells and macrophages from skin samples, and as such, the data may inadvertently misrepresent the T cell numbers and/or proportions present in mouse skin in vivo. While total numbers and proportions of leukocytes in the LN samples within each treatment group showed high reproducibility, generally there were not high enough absolute numbers of CD4+ and CD8+ T cells in the ear samples to be able to make conclusions about subset proportions. Pooled ear samples contained anywhere from zero detectable isolated leukocytes to over 700. These experiments used enzymatic and mechanical digestion to isolate white blood cells from mouse ears. While this method has been used extensively in the literature, it is now known that enzymatic and mechanical digestion methods do not yield the full number of white blood cells from skin, either by incomplete freeing of cells from surrounding tissue or by degrading free cells. Newer techniques can be used to obtain large numbers of T cells from fresh human skin punch biopsies.85 In lymphocyte migration assays, fresh biopsies are placed in culture dishes that contain a chemokine gradient, and over the course of 9 to 15 days lymphocytes migrate towards the gradient out of the biopsy tissue. These methods are attractive for full yield of lymphocytes in terms of absolute numbers, but it is not yet clear as to whether or not migrating lymphocytes change their phenotype in any way in the span of 15 days that it takes for them to migrate into surrounding medium. There are many factors that must be considered when creating an animal model such as the one attempted here. The species and strain of animal, strain and preparation of P. acnes, route of sensitization, dose for sensitization, number and timing of sensitization injections, time before challenge, dose for challenge, and route of challenge all have numerous possibilities, making it implausible to try all possible combinations. Experiments in this Thesis tried sensitization time-lines and dosages based on reports in the literature using P. acnes as an adjuvant with other pathogens (such as M. tuberculosis and rabies virus) as well as hypersensitivity models with other types of bacteria. However, none of the chosen

111 procedures were successful in inducing hypersensitivity. Mice sensitized with P. acnes by intraperitoneal injection did not display increased inflammation upon challenge compared to vehicle-sensitized mice (data not shown), indicating that in this model the route of sensitization (intradermal versus intraperitoneal) did not affect results. Additionally, mice that were challenged two weeks, three weeks, or four weeks after the last intradermal sensitization injection all experienced the same amount of ear inflammation with challenge, none of which were significantly greater then vehicle-sensitized mice. While the particular procedures used in this Thesis did not create a successful model for P. acnes hypersensitivity, such a model may still be feasible. Due to the bias towards positive research findings in publication, it is possible that many other groups have also attempted and failed to create a similar mouse model for acne research. The limitations of animal models as well as the known differences in immune responses between humans and animals make research with human subjects more attractive. While it is difficult to convince many patients to give skin biopsies, information gained from just a few clinical samples may be more valuable than information from a flawed or irrelevant animal model.

A.5 Methods

A.5.1 Mice

Mouse experiments were done in accordance with the Penn State Hershey Institutional Animal Care and Use Committee (Penn State IACUC-approved protocol #2010-050) and the Federation of American Societies for Experimental Biology principles for animal research. Female C57BL/6 mice (Jackson Laboratories) were housed in an isolation cubicle in common housing conditions in the Penn State Hershey Central Animal Quarters. Mice were fed standard rodent diet with 18% protein and were exposed to a 12-hour light/dark cycle. All mice were six to eight weeks old at the start of P. acnes immunization. Ear thickness measurements were taken at baseline (before challenge) and immediately after sacrificing using a micrometer in 0.005 inch increments.

A.5.2 Sensitization and Challenge with P. acnes

112 Difco Reinforced Clostridal Broth (BD Biosciences), which was then overlaid with N2 gas and grown overnight at 37° C in a shaker until bacteria reached their log growth phase. Bacteria were then washed twice with PBS and resuspended in sterile 0.9% (w/v) NaCl saline solution. Heat-killed preparations were made by heating P. acnes suspensions to 100° C for 20 minutes and stored at -20° C. After heat inactivation, P. acnes was unable to grow on agar plates (data not shown). Live preparations were grown and prepared fresh before each use. For each sensitization and challenge injection, mice were briefly anesthetized for about 30 minutes with a 50 to 70 μL intraperitoneal injection of a 10 mg/mL ketamine/0.6 mg/mL xylazine solution. To sensitize them to P. acnes, mice were injected with 200 μL containing 450 μg of heat-killed P. acnes or saline vehicle intradermally in the center of the back on days 0, 3, and 6 using a 26 ½-gauge ⅝-inch intradermal-beveled needle (BD Biosciences). Three weeks after the last sensitization injection (on day 26), mice were challenged subcutaneously with 35 μL containing either 20 μg or 100 μg of live P. acnes in the right ear and saline vehicle in the left ear using a 1 cc 28-gauge ½-inch insulin syringe (BD Biosciences).

A.5.3 Tissue Harvest and Processing

Two, four, and seven days after challenge (days 22, 24, and 27) mice were sacrificed by CO2 narcosis using a compressed gas tank not in contact with the animal along with exsanguinations by heart stick. Blood was collected into 10 mL Cath-Loc Prefilled Syringes containing 50 units of sodium-heparin (Strategic Applications Incorporated) and then mixed with an equal volume of RPMI 1640 (made in the Penn State Department of Microbiology and Immunology central facility). The blood/RPMI mixture was overlaid onto an equal volume of Ficoll-Paque Premium (1.084 g/mL density, GE Healthcare) in 15 mL conical tubes and spun at 1000 x g for 20 minutes. The buffy coat was removed, washed twice with RPMI, and resuspended for culture. After heart stick, the spleen and draining lymph nodes from the challenge sites (superficial cervical lymph nodes, inguinal lymph nodes) were harvested from mice and stored in calcium and magnesium-free Hanks Balanced Salt Solution (HBSS) until processing. Spleens were rinsed with HBSS and chopped using a razor blade. To make single cell suspensions, spleen fragments and lymph nodes were pushed through 40 micron nylon mesh screens with the plunger of a 1 mL syringe. To lyse red blood cells, splenocytes were pelleted and resuspend in 1 mL ACK lysis buffer for five minutes. Splenocytes were then washed with HBSS and resuspended for cell culture.

113 After measurement with calipers, ears were cut off at the base for tissue processing. Ears within each treatment group (n = 3 or 4) were pooled in order to maximize lymphocyte recovery. Hair was removed by shaving and ears were chopped into pieces using surgical scissors. Pieces were pelleted and resuspended in 2 mL of 2 mg/mL Collagenase D (Sigma) in HBSS/0.1% (w/v) BSA. Collagenase suspensions were incubated in an 37° C water bath for 40 minutes, vortexing briefly every 10 minutes. Fragments were then ground through a metal 100 mesh strainer with the plunger of a 10 mL syringe. The resulting suspension was then strained using a 40 micron nylon mesh screen. White blood cells were enriched using a Ficoll-Paque gradient as described above and were then pelleted and resuspended for cell culture.

A.5.4 Cell Culture

All samples were resuspended in RPMI/10% (v/v) FBS/antibiotics for treatment. Ear samples from each sensitization group were pooled together for culture. Samples were plated at 1x105 cells in 200 μL/well in a round-bottom 96 well plate. Samples were treated with 5 μg/mL each concanavalin A plus 10 μg/mL brefeldin-A, 1 μg/mL of P. acnes sonicate plus brefeldin-A, or vehicle plus brefeldin-A for four hours. At the end of cell culture, cells were spun down, trypsinized, and washed twice with HBSS. Cells were resuspended in 1% (v/v) FBS/0.1% (w/v) NaN3/PBS for surface staining.

A.5.5 Antibodies and Flow Cytometry

Samples were stained with multiple panels of antibodies to detect monocytes and lymphocyte subsets. Monocyte antibody panels included the following: rat anti-mouse CD45 - V450 (clone 30-F11), rat anti-mouse CD14 - APC (clone rm-C5-3), rat anti-mouse TLR4 - PE (clone MTS510; all BD Biosciences) and mouse anti-mouse/human TLR2 - FITC (clone T2.5; Imgenex). To detect lymphocyte subsets, samples were first surface-stained with panels containing the following antibodies: hamster anti-mouse CD3(ε) - AlexaFluor700 (clone 500A2), rat anti-mouse CD4 - V450 (clone RM4-5), rat anti-mouse CD8a - V500 (clone 53-6.7), rat anti-mouse CD45RB - FITC (clone 16A), rat anti-mouse CD62L - APC-Cy7 (clone MEL-14), rat anti-mouse CD25 - PerCP-Cy5.5 (clone PC61), rat anti-mouse CD127 - PE (clone SB/199), hamster anti-mouse CD152 - PE (clone UC10-4F10- 11), rat anti-mouse CD162 (CLA) - PE (clone 2PH1), and hamster anti-mouse CD69 - biotin (clone H1.2F3), followed by secondary staining with Streptavidin - PE-TexasRed (all BD Biosciences). For

114 intracellular staining for cytokines, cells were fixed and permeabilized using Foxp3 Buffer Staining Kits (EBiosciences) according to manufacturer‟s directions. In brief, each sample was resuspended in 100 μL of Fix/Perm solution and incubated for 30 minutes at room temperature in the dark. Samples were washed twice with Permeabilization Buffer, resuspended in 100 μL of Permeabilization Buffer, and stained with the following antibodies: rat anti-mouse Foxp3 - AlexaFluor647 (clone MF23), rat anti- mouse IL-4 - APC (clone 11B11), rat anti-mouse IFN - PE-Cy7 (clone XMG1.2), and rat anti-mouse IL17A - PerCP-Cy5.5 (clone TC11-18H10; all BD Biosciences). Stained samples were resuspended in HBSS/0.1% (w/v) BSA for analysis by flow cytometry. Samples were run on a LSR II flow cytometer using FACSDiva software (BD Biosciences). Data was analyzed using FlowJo software (Tree Star).

A.5.6 Statistical Analysis of the Data

Ear thickness measurements before and after injections were compared using paired Student‟s t- tests. Fold-changes of ear thickness measurements and percentages of lymphocyte subpopulations and were compared between sensitization groups using unpaired Student‟s t-tests, with a P value < 0.05 being considered significant.

115 Appendix B

The Function of NGAL’s Glycan

B.1 Chapter Abstract

Neutrophil gelatinase-associated lipocalin (NGAL) is an antimicrobial protein that can exert its function through binding to siderophores. As an inflammatory response protein, NGAL has been detected in a variety of diseases and cancers. However, reports have shown that NGAL can both impair and promote tumorigenesis. The mechanism behind these conflicting functions is unknown. NGAL‟s crystal structure and modifications have been elucidated, but very little is known about the functional significance of each of its physical features, including its single glycan group located on asparagine 65. Our lab has previously shown that recombinant mammalian-expressed human NGAL (NGAL-(M)) can induce apoptosis in SEB-1, but recombinant bacterially-expressed NGAL (NGAL-(B)) cannot induce apoptosis in SEB-1. This observation led us to hypothesize that NGAL‟s glycan may be essential to its apoptotic action. In this Appendix, we confirm by glycan staining and mass spectrometry that NGAL- (M) is glycosylated on N65, and that NGAL-(B) is not. Tunicamycin treatment of SEB-1 prevented glycosylation of expressed NGAL, but did not prevent NGAL secretion out of the cell. NGAL-(M)‟s glycan was successfully removed by treatment with PNGase F enzyme without denaturing NGAL. Because attempts at purifying native NGAL from solutions by MagneHis beads or immunoprecipitation were unsuccessful, whole PNGase F reactions were used to treat SEB-1. Apoptotic studies using TUNEL assays and annexin V/PI staining were inconclusive due to the fact that previous data with NGAL-(M) could not be replicated. Additional troubleshooting needs to be performed on NGAL purification methods and apoptosis assays in order for these studies to move forward.

B.2 Introduction

NGAL (also called lipocalin 2 or siderocalin) is an antimicrobial peptide that was originally discovered bound to MMP-9 in neutrophil granules, but has since been shown to be expressed in a large variety of cell types.329-331 NGAL has antimicrobial activity against various Gram positive and negative bacteria including Escherichia coli, Klebsiella pneumoniae, and Bacillus subtilis, but NGAL expression has also been detected in a variety of aseptic inflammatory disease such as renal injury, vascular injury, cancer, inflammatory bowel disease, and type 2 diabetes.54,332-343 It has been named as a urinary

116 biomarker for various renal pathologies including renal ischemic injury, IgA nephropathy, lupus nephritis, and polycystic kidney disease.344-347 However, within each disease context it is not yet understood if NGAL is a protective response to injury or part of the pathogenesis. NGAL‟s role in cancer is one area in which such contradictory studies exist. NGAL expression is detected in a wide variety of cancers including renal, breast, pancreatic, colonic, cervical, ovarian, brain, thyroid, esophageal, and gastric cancers, but it is not known if its expression is pathogenic or a protective response to the tumor.333,336,348-352 Some studies have shown that NGAL can suppress invasion, angiogenesis, and metastasis of some cancers such as pancreatic and colonic, and induce apoptosis in some cancer cell lines.333,350,353-355 However, a number of reports have also shown that NGAL expression can protect A549 adenocarcinoma cells against apoptosis and promote the proliferation and metastasis of cancers of the thyroid, breast and ovary.352,356 In these cases, it appears that NGAL is acting as a response to, not a cause of, apoptosis. The mechanism for the discrepancies in NGAL‟s function is unknown. NGAL exerts much of its functions by sequestering and transporting iron in siderophores, which are small compounds that chelate ferric iron. Siderophores bind to Fe3+ at a higher affinity than mammalian iron-binding proteins such as ferritin, lactoferrin, and transferrin. Bacteria secrete siderophores to scavenge iron from the environment and then uptake the Fe:siderophore complexes by receptor-mediated endocytosis. NGAL can sequester iron from bacteria by binding to bacterial siderophores such as enterobactin from Gram-negative bacteria, bacillibactin from Gram-positive bacteria, and carboxymycobactins from mycobacteria.54,56 Recent reports have also identified putative mammalian siderophores 1,2-dihydroxybenzene (called catechol) and 2,5-dihydroxybenzoic acid, which are similar in structure to 2,3-dihydroxybenzoic acid, the iron-binding component of enterobactin.55,58 NGAL binding to mammalian siderophores is thought to be a means for iron-trafficking in tissues, parallel in function to transferrin. Accordingly, in the context of cancer, NGAL could bind to siderophores to either sequester iron away from cells to induce apoptosis, or deliver iron to cells by receptor-mediated endocytosis to promote growth and proliferation (Figure B.1). The question remains, then, as to what factors determine whether an NGAL-siderophore complex either sequesters iron or delivers iron to a cell. This could be dependent on microenvironment iron concentrations, siderophore concentrations, or whether a cell expresses the appropriate cell surface receptor necessary to engulf the NGAL-siderophore complex. Equally unknown are the factors that affect NGAL‟s interaction with siderophores and its receptor. NGAL‟s structure has been elucidated through crystallographic studies, but very little is known about the functional significance of each of its physical features.

117 ______

Figure B.1: Model for cellular iron-trafficking by NGAL. Siderophore-free NGAL (apo-NGAL) binds to intracellular siderophores to transport iron to the extracellular space. Siderophore-associated NGAL (holo-NGAL) binds iron in the extracellular space and delivers it into cells by means of receptor-mediated endocytosis. In a parallel pathway, iron can be transported into cells by binding to transferrin, which is then endocytosed using an iron transferrin receptor (FeTfR). Sid, siderophore; DMT, divalent metal ion transporter; FPN, ferroportin (iron efflux channel).

(J Am Coll Cardiol, 2010; 55:2024-2033) ______

Crystallographic and NMR studies have shown bacterially-expressed NGAL to be a β-barrel protein that contains eight β-pleated sheets and one α-helical region.357 Its pocket is lined with many polar and positively charged residues and is predicted to be able to bind to steroids, retinoids, and iron- binding bacterial siderophores such as enterobactin. NGAL is released from neutrophil granules in many different forms: monomer, disulphide-linked homodimer, and disulphide-linked heterodimers with MMP- 9.330,331 Studies of endogenous human NGAL have shown that NGAL contains one major N-linked glycan on N65 with a molecular weight of 1.3 to 2.2 kD, meaning that this glycan comprises roughly four to nine percent of NGAL‟s total molecular weight (Figure B.2).358 This glycan is reported to be a biantennary complex with or without outer arm fucose and sialic acid residues. One study reported the presence of two putative O-linked glycans on S5 and S14, but these have never been confirmed. Little is known about the potential function of NGAL‟s glycans.

118 ______

(Rudd et al. Biochemistry. 1999, 38, 13937-13950.) ______

Figure B.3: N-Linked Glycan Core Structure. All N-linked glycans contain a common Man3GlcNAc2 pentasaccharide core structure attached to an asparagine residue.

(Glycobiology Analysis Manual, 2nd Edition, Sigma-Aldrich) ______

119 Glycosylation of proteins is carried out by glycosyltransferases that attach glycans to asparagine (N-linked glycans) or serine/threonine (O-linked glycans) residues. While O-linked glycosylation is a post-translational modification, N-linked glycosylation actually occurs co-translationally in the endoplasmic reticulum. All N-linked glycans contain a common 3-mannose/2-N-acetylglucosamine pentasaccharide core attached to an asparagine residue in the consensus sequence Asp-X-Ser/Thr, where X is any amino acid except proline (Figure B.3). The GlcNAc phosphotransferase (GPT) enzyme catalyzes the transfer of N-actelyglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol phosphate in the first step of N-linked glycoprotein synthesis. Glycosylation can have pronounced effects on the function, structure, stability, and antigenicity of proteins. For example, the addition of extra N-linked glycans on recombinant erythropoietin increases its serum half-life by three-fold, whereas the addition of extra glycans to recombinant leptin enhances its hormonal activity.359 Deglycosylated fibronectin is more susceptible to proteolysis and deglycosylated IL-5 is more susceptible to heat denaturation than their glycosylated forms.360,361 For α1-microglobulin (another lipocalin similar to NGAL in molecular weight), glycosylation is essential for secretion out of the cell and localization to tissues, but not for its function.362 A classic example of the effect of glycosylation on antigenicity is the blood group antigens (A, B, and O), which are glycans found on the surface of erythrocytes that are responsible for blood transfusion reactions. Glycans can also comprise a large proportion of a protein‟s molecular weight, as in the case of mucin. Studies in our lab have shown that recombinant mammalian-expressed human NGAL (NGAL- (M)) can induce apoptosis in SEB-1, but neither recombinant bacterially-expressed NGAL with a bound siderophore (holo-NGAL-(B)) nor NGAL without a bound siderophore (apo-NGAL-(B)) cannot induce apoptosis in SEB-1.57,363 Knowing that bacterially-expressed recombinant human proteins are not glycosylated in the same way as proteins from mammalian cell expression systems, these observations led us to hypothesize that NGAL‟s glycan may be essential to its apoptotic action.

B.3 Results

B.3.1 NGAL runs as a double band in western blots.

During previous studies on NGAL‟s involvement in 13-cis RA‟s apoptotic actions, we observed that NGAL frequently ran as a double band on western blots with sizes of roughly 21 and 25 kD. This phenomenon was observed with multiple different treatments used to induce NGAL expression (Figure B.4). Reports in the literature also observed the same double band in NGAL westerns.364 We hypothesized that the presence or absence of glycans could be responsible for the double bands observed

120 on NGAL western blots and/or the differences in NGAL‟s ability to induce apoptosis. We therefore investigated the glycosylation status of NGAL-(M), apo-NGAL-(B), and holo-NGAL-(B).

B.3.2 Mammalian-expressed NGAL is glycosylated, while bacterially-expressed NGAL is not.

Samples of commercially-purchased recombinant NGAL-(M) and apo- and holo-NGAL-(B) made in our lab were run on denaturing gels and assayed for glycan content using an in-gel total glycan stain and then total protein stain. Only the NGAL-(M) stained positive with the glycan stain, whereas all of the recombinant NGALs became visible with the total protein stain (Figure B.5). This supported our hypothesis that NGAL-(M) is glycosylated, while neither apo- nor holo-NGAL-(B) is glycosylated. We next sought to cleave the glycan(s) from NGAL-(M) in order to investigate its functionality in its deglycosylated form. Peptide: N-Glycosidase F (PNGase F) is an endoglycosidase that removes all N- linked glycans at the base (Figure B.6) and is thus commonly used in glycobiology for assessing proteins‟ function with and without N-linked glycans. We utilized PNGase F to confirm that the NGAL- (M) glycans detected by the in-gel glycan stain were N-linked. NGAL-(M) was denatured at 100° C for 10 minutes and then treated with PNGase F for one hour at 37° C using the protein denaturing reagent NP-40 according to the enzyme manufacturer‟s directions. Reactions were then run on denaturing gels and stained for glycans and then total protein. Treatment of denatured NGAL-(M) with PNGase F caused a gel shift of about two to three kD with a near 100 percent efficiency of deglycosylation (Figure B.5). Leftover PNGase F enzyme from the deglycosylation reaction was also detected in the “Deglycosylated NGAL-(M)” lane as expected. Additionally, the lower molecular weight NGAL observed in the deglycosylation reactions did not stain positively for glycans as the normal molecular weight NGAL did, suggesting that PNGase F can successfully deglycosylate denatured NGAL and that NGAL-(M)‟s glycans are all N-linked. ______

______

121 ______

Figure B.5: Detection of glycosylation in recombinant NGAL. Samples of NGAL-(M), apo-NGAL-(B), and holo-NGAL-(B) were run on gels and stained for glycans and then for total protein. Additionally, a sample of NGAL-(M) was denatured and deglycosylated with PNGase F enzyme as described. Horseradish peroxidase (HP; a glycosylated protein) and soy trypsin inhibitor (TI; an unglycosylated protein) were run as controls for the glycan stain.

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Glycobiology Analysis Manual, 2nd Edition, Sigma-Aldrich ______

122

B.3.3 NGAL’s glycan is successfully removed by PNGase F

PNGase F was then used to treat non-denatured NGAL-(M) to see if this enzyme could remove the glycan(s) from NGAL in its native conformation. NGAL-(M) was treated with PNGase F without heat denaturing or the use of denaturing buffers for one hour at 37° C, and the reactions were then analyzed using SDS-PAGE as above. When native NGAL-(M) was treated with PNGase F, the same gel shift of 2 kD was observed, but the efficiency of deglycosylation was significantly less than was observed with the denatured NGAL (Figure B.7). Overnight incubation of native NGAL-(M) with the PNGase F enzyme produced a much higher efficiency of deglycosylation compared to the one hour incubation (close to 80%; data not shown). ______

______

123

B.3.4 PNGase F removal of NGAL’s glycan is confirmed by mass spectrometry

PNGase F+NGAL reactions were run on denaturing gels and detected with total protein stain to label total protein (Figure B.11). Untreated and deglycosylated NGAL bands were cut out of the gel and analyzed by mass spectrometry (MS). Untreated NGAL-(M) (Sample 1 on the gel in Figure B.11) was confirmed to have a glycan on N65 and no other modifications. PNGase F-treated NGAL-(M) (Sample 2) was shown to have no modifications on N65, confirming the conclusion that PNGase F can successfully cleave the N-linked glycan from NGAL‟s N65 residue, and that NGAL-(M) does not have any other glycans.

B.3.5 Deglycosylated NGAL can be detected by western blot

To confirm that deglycosylated NGAL could be detected by western blotting with the same antibody as we had previously used for normal NGAL-(M), whole PNGase F/NGAL deglycosylation reactions were run on denaturing gels and blots were incubated with anti-NGAL monoclonal antibody. Deglycosylated NGAL in the PNGase F reactions was easily detected on western blots (Figure B.8).

______

Figure B.8: Western blot for deglycosylated NGAL. Whole PNGase F/NGAL deglycosylation reactions were analyzed by SDS-PAGE and western blotting for NGAL. NGAL-(M) incubated with the same buffers but without enzyme (“NGAL-(M) control rxn”) was included as a control for the deglycosylation reactions. Untreated NGAL- (M) and apo-NGAL-(B) was included on the blot as controls for NGAL detection with western blotting.

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124 B.3.6 Deglycosylated NGAL cannot be purified from solutions using MagneHis beads

We next sought to purify native deglycosylated NGAL-(M) from PNGase F reactions for use in treating cells. The NGAL-(M) that we purchase commercially from R&D Systems has a 10x histidine tag (His tag) on the C-terminal end. Therefore, we first attempted to purify deglycosylated NGAL-(M) using MagneHis beads (Promega), which are used to isolate His-tagged proteins from solutions. MagneHis beads were used according to manufacturer‟s directions to purify NGAL from PNGase F reactions, and both whole unpurified reactions and MagneHis purified samples were analyzed by SDS-PAGE and staining for total protein (Figure B.9). The unpurified PNGase reactions showed two bands around 22 and 25 kD, while the MagneHis purified reactions showed only one band at 25 kD. The absence of the lower molecular weight band in the MagneHis purifications suggested that the MagneHis system failed to purify deglycosylated NGAL from solution along with the glycosylated form.

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125 B.3.7 NGAL can be successfully isolated from solutions using Dynabeads

An attempt at purification of NGAL from SEB-1 lysates and media was made by immunoprecipitation (IP) using Dynabeads (Invitrogen). Anti-NGAL monoclonal antibody was cross- linked to the beads, and bead/antibody complexes were used to precipitate NGAL from total cell lysates and media samples from SEB-1 treated with either 1 μM 13-cis RA or 10 ng/mL IL-1β. Immunoprecipitated protein was run on denaturing gels and stained with glycan and total protein stains. As controls, the supernatants from the sample/bead binding reaction were saved and run on the gel to detect any NGAL that had not bound to the beads. Additionally, a sample of the anti-NGAL antibody alone was denatured and run on the gel as a comparison for detecting any eluted antibody fragments in the IP reactions. The media samples from 13-cis RA and IL-1β-treated SEB-1 showed major bands at 25, 55, 60, 62, and 100 kD (Figure B.10). The antibody control lane had strong bands at 24, 55, 62, and 100 kD, and the 55, 62, and 100 kD bands matched well to the corresponding bands in the IP reaction lanes. The 24 kD band in the antibody control lane migrated slightly less than the 25 kD band in the NGAL IP lanes, suggesting that the 25 kD band in the IP lanes may not be Ab fragment and is in fact NGAL. The SEB-1 lysates were not as concentrated as the media samples, and no detectable bands were observed in these lanes on the gel. ______

Figure B.10: Purification of NGAL by immunoprecipitation with Dynabeads. Mouse anti-NGAL antibody was cross-linked to Dynabeads, and complexes were used to purify NGAL from PNGase F reactions. IP eluents and supernatants were analyzed by SDS-PAGE and stained for total protein. A sample of anti-NGAL antibody was included as a control.

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126 To confirm the identity of the immunoprecipitated products, major bands were cut out and analyzed by MS (Figure B.11). MS results confirmed that the 24 kD band (Sample 4) in the antibody control did contain mouse IgG fragments (a γ heavy chain and κ light chain, which correspond to the isotype listed on the antibody product sheet). The 25 kD band in the NGAL IP reactions (Sample 3) was confirmed to contain NGAL, though it also contained some antibody fragments that were seen in the antibody control as well as some bovine apolipoprotein A1, which was likely a contamination from the sebocyte media (which contains FBS). MS analysis was unsuccessful in determining the presence or absence of the glycan on NGAL immunoprecipitated from SEB-1 media.

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Figure B.11: MS analysis of NGAL glycosylation. a) Recombinant NGAL-(M) was treated with PNGase F as described. Reactions were analyzed by SDS-PAGE, and the indicated bands were cut out and sent for analysis by mass spectrometry. b) NGAL was immunoprecipitated from treated SEB-1 lysates and media samples, and the eluted product was analyzed by SDS-PAGE. The indicated bands in the 13-cis RA-treated media IP and the anti- NGAL antibody control lanes were excised and sent for analysis by mass spectrometry.

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127 B.3.8 NGAL cannot be detected with glycan staining of total cell lysates

To determine if glycosylated NGAL would be detectable in total cell lysates or media, SEB-1 cells were treated with 13-cis RA or IL-1β as above, and cell lysates and media samples analyzed by SDS-PAGE and stained for glycans and total protein. No clear bands around NGAL‟s molecular weight (21-25 kD) were observed in either the glycan stain or the total protein stain for either the lysates or media samples (Figure B.12).

B.3.9 Tunicamycin can inhibit glycosylation of NGAL produced in SEB-1

The inability to detect glycans on NGAL produced by SEB-1 using MS or glycan staining of total cell protein prompted us to use tunicamycin to confirm NGAL‟s endogenous glycosylation. Tunicamycin is a membrane-permeable, small molecule compound that inhibits N-linked glycan synthesis in eukaryotic cells by inhibiting the GPT enzyme in the first step of N-linked glycoprotein synthesis. At high concentrations, tunicamycin can induce cell cycle arrest in G1 phase by globally inhibiting tyrosine incorporation into proteins. To first determine the toxicity of tunicamycin in SEB-1, dose-response curves were performed for cell death assays using Cell Titer Blue kits (Promega). Tunicamycin concentrations of 100 μg/mL significantly induced death in SEB-1 at all time points, with a roughly 10- fold increase in cell death at 3 hours compared to vehicle, 20-fold increase at six hours, and 40-fold increase at 24 hours (Figure B.13). A 48-hour time-point was not done for this concentration. Tunicamycin concentrations ranging from 10 μg/mL to 10 pg/mL did not induce cell death at any time point up to 48 hours in culture. Western blots showed that 24 hour treatment with tunicamycin concentrations of 10 μg/mL and above significantly induced expression of cleaved caspase 3 over vehicle (data not shown). Additionally, to ensure that tunicamycin did not inhibit total protein synthesis in SEB- 1, total protein content per cell was calculated for treated cells. SEB-1 were plated in 100 mm plates and treated with doses ranging from 10 ng/mL to 10 μg/mL tunicamycin for 24 hours. At the end of the treatment, cells were trypsinized and counted, and total cell lysates were made from the counted cells and assayed for protein concentration using BCA assays. 24-hour treatment with tunicamycin at concentrations 10 μg/mL and below did not significantly affect the overall protein content per cell in SEB-1 (data not shown).

128 ______

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129 To assess the overall effect of tunicamycin treatment on the glycosylation of total protein in SEB- 1, cells were treated with 10 pg/mL, 1 ng/mL, 100 ng/mL, or 10 μg/mL of tunicamycin for 24 hours, and total protein lysates and media supernatants were analyzed by SDS-PAGE and stained for glycans and then total protein. Tunicamycin treatment did not appear to have an effect on total protein glycan staining (Figure B.14). To determine if tunicamycin could inhibit the glycosylation of NGAL produced in SEB-1, SEB-1 were treated with 1 μM 13-cis RA alone, 1 μM 13-cis RA plus 10 ng/mL tunicamycin, 1 μM 13-cis RA plus 10 μg/mL tunicamycin, or 10 μg/mL tunicamycin alone for 24 hours. Western blots of total cell lysates showed that 13-cis RA treatment alone induced an NGAL band around 21 kD, and the addition of 10 ng/mL tunicamycin partially shifted the NGAL band down to 19 kD, suggesting that this dose partially inhibits glycosylation of NGAL (Figure B.15). The addition of 10 μg/mL tunicamycin completely shifted the NGAL band to 19 kD, suggesting that this dose is sufficient to completely inhibit the glycosylation of NGAL in SEB-1 cells. Interestingly, tunicamycin treatment alone induced NGAL expression in SEB-1, though the NGAL that was expressed was still non-glycosylated. These data confirm that the NGAL produced endogenously in SEB-1 in response to 13-cis RA is glycosylated, and that the glycan is N-linked.

B.3.10 NGAL’s N-linked glycan is not essential for its secretion from SEB-1

In order to determine if NGAL‟s N-linked glycan is necessary for its secretion out of the cell, SEB-1 were treated with 1 μM 13-cis RA alone, 1 μM 13-cis RA plus 10 ng/mL tunicamycin, 1 μM 13- cis RA plus 10 μg/mL tunicamycin, or 10 μg/mL tunicamycin alone for 24 hours. Media supernatants were analyzed for NGAL content by western blot. Identical patterns of NGAL deglycosylation were detected in both cell lysates and media supernatants of tunicamycin-treated SEB-1 (Figure B.15). The presence of deglycosylated NGAL in the media samples indicates that NGAL‟s N-linked glycan is not essential for its secretion out of cells.

B.3.11 Apoptosis studies with deglycosylated NGAL were inconclusive

Because of the obstacles we encountered in purifying NGAL from deglycosylation reactions, we attempted to bypass the need for this purification by using whole PNGase F reactions to treat SEB-1 for apoptosis studies. SEB-1 were treated with 1 ng/mL normal NGAL-(M), deglycosylated NGAL-(M) (whole PNGase F reaction), or vehicle for 24 hours. Treatment controls for the deglycosylation reactions included NGAL-(M) that was incubated in the same buffers as the deglycosylation reactions but without

130 PNGase F enzyme, and PNGase F and its reaction buffers without NGAL. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to detect apoptotic cells, and three fields in each well were photographed for green fluorescence and brightfield. The percent of dying cells was calculated by dividing the number of cells staining positive by the total number of cells in the brightfield. Figure B.16 shows representative fields from each treatment group. The percent of positive cells was not significantly different in any of the treatment groups compared to the others, including the NGAL-(M) group (data not shown), which was puzzling since we have previously used TUNEL assays to show that NGAL-(M) induces apoptosis in SEB-1.

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131 ______

Figure B.15: SEB-1 secretion of NGAL. SEB-1 were treated for 24 hours with 1 μM 13-cis RA alone, 1 μM 13- cis RA plus 10 ng/mL tunicamycin, 1 μM 13-cis RA plus 10 μg/mL tunicamycin, or 10 μg/mL tunicamycin alone. Media supernatants (top row) and cell lysates (bottom two rows) were analyzed by western blot for NGAL expression. NGAL-(M) (labeled „rhNGAL”) was included on each gel as a positive control, and β-actin expression was used as a loading control for the cell lysate samples.

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The difficulty in distinguishing the validity on the TUNEL assays prompted us to try annexin V and propidium iodide (PI) staining as an alternative method for detecting apoptosis. SEB-1 were treated as above, trypsinized, stained using PI and anti-annexin antibodies, and analyzed by flow cytometry. The percentages of early apoptotic cells, late apoptotic cells, and necrotic cells were not significantly different in any of the treatment groups compared to the others (Figure B.17). Again, the normal NGAL-(M) failed to induce apoptosis compared to vehicle. However, the staurosporine positive control did significantly induce higher percentages of cells in early apoptosis, late apoptosis, and necrosis compared to the other treatment groups, indicating that the assay itself was working.

132

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133 ______

Figure B.17: Annexin V/PI assays. SEB-1 were treated with 1 μM normal NGAL-(M), the whole NGAL-(M) deglycosylation reaction, or vehicle („H2O vehicle‟) for 24 hours. Cell were then trypsinized, stained with PI and antibodies for annexin V, and analyzed by flow cytometry. Mean (± SEM) percentages of apoptotic cells are displayed for each treatment group. Controls included cells treated with 0.1 μM staurosporine as a positive control for apoptosis, as well as controls for the deglycosylation reactions: „NGAL-(M) control rxn‟ is NGAL that was incubated in the same buffers as the deglycosylation reactions but without PNGase F enzyme, and „Deglyc control rxn‟ is PNGase F and its reaction buffers without NGAL added. Data is representative of four separate experiments.

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B.4 Discussion

The experiments in this thesis confirmed that the commercial NGAL-(M) that we use in our lab contains one N-linked glycan on N65 and that the apo- and holo-NGAL-(B) that we produced in our lab are not glycosylated. We successfully cleaved the glycan off of native NGAL using PNGase F without prior denaturation, and also prevented the glycosylation of NGAL expressed by SEB-1 using tunicamycin treatment. We have shown here that NGAL produced by SEB-1 is glycosylated, and that NGAL‟s glycan is not essential for its secretion out of SEB-1 cells. However, attempts at purification of NGAL from solutions and assessing its apoptotic ability without its glycan were unsuccessful.

134 For these studies, the inability to purify NGAL from solutions presents an insurmountable problem. The intent of purification was to isolate non-denatured deglycosylated NGAL from the PNGase F reactions or tunicamycin-treated SEB-1 for use in treating cells to see if the glycan is essential for its apoptotic function. Because the commercial NGAL-(M) that we use still has a 10x His tag attached to the C-terminal end, we tried using MagneHis beads to purify NGAL from PNGase F reactions; however, this system was unable to isolate the deglycosylated form of NGAL. It is likely that either the deglycosylated NGAL could not bind to the MagneHis beads, or it bound too tightly and did not elute with the normal glycosylated form. Purification of his-tagged apo- and holo-NGAL-(B) from E. coli expression systems using his tag-specific nickel NTA columns has been performed successfully in our lab in the past, but those methods are expensive, time-consuming, and unsuitable for very small quantities of protein such as those found in the PNGase F reactions. Direct IP with anti-NGAL antibody did enrich NGAL from lysates, but the eluents were not pure and contained contaminating proteins such as antibody fragments and apolipoprotein A-1. Finally, while PNGase F reactions were successfully and cleanly separated by electrophoresis, the NGAL was then denatured and no longer active. Native polyacrylamide gel electrophoresis (PAGE) was also attempted (data not shown), but was unsuccessful because NGAL is cationic. Future studies will need to first solve the problem of purifying native deglycosylated NGAL from solutions in order to investigate its apoptotic ability. An option for future attempts is the use of quantitative preparative native continuous PAGE, which is a specific method of native PAGE where metalloproteins can be separated by use of a running buffer with pH 10.00 and an eluent of pH 8.0. Given the difficulties in purifying NGAL, an attempt was made to bypass the need for purification by directly treating SEB-1 with PNGase F-treated native NGAL-(M) whole reactions. PNGase F enzyme alone as well as NGAL-(M) incubated with the kit buffers but without enzyme were used to treat SEB-1 as controls in these experiments. This approach is not ideal, because the deglycosylation reactions still contain active PNGase F when they are used to treat SEB-1, so it is possible that leftover PNGase F could cleave N-linked glycans on SEB-1 surface proteins or on proteins in the media that would then have downstream consequences. Regardless of this risk, apoptotic assays using this approach were inconclusive. We observed a very high level of background staining with the TUNEL assays, which could either have been a result of high fluorescent background staining or a high baseline level of apoptosis. If the high background indicated a high baseline level of apoptosis, then nearly all of the cells (even in the vehicle treatment group) were apoptotic. Alternatively, if the high background was merely background fluorescence, then there were no detectable positive cells in any of the treatment groups, including the normal NGAL-(M), which we have successfully used to induce apoptosis in SEB-1 in previous experiments.57 Additional attempts at assaying apoptosis using annexin V/PI staining were also unsuccessful. While the staurosporine positive control treatment group did

135 confirm that this assay was working, NGAL-(M) was still unable to induce apoptosis in SEB-1 as it has in previous experiments in our lab. The reasons for this are still unknown. We considered the possibility that the SEB-1 cells had spontaneously changed, which is always a possibility in immortalized cell lines. We recovered new batches of SEB-1 from low passages, but still could not induce apoptosis in them with NGAL treatment. To rule out the possibility that our NGAL stocks were expired or damaged, we purchased new vials of NGAL of the same lot number as well as new lots of NGAL-(M) from R&D Systems, but we could still not replicate our published data. This problem was never solved, and thus it was never determined if the whole NGAL deglycosylation reactions could induce apoptosis in SEB-1 or not. We successfully inhibited the glycosylation of NGAL expressed by SEB-1 using tunicamycin treatment. Partial inhibition of NGAL‟s glycosylation was achieved at 10 ng/mL tunicamycin, and complete inhibition was achieved at 10 μg/mL, which is below the toxic concentration as determined by cell death assays. The efficiency of glycosylation inhibition was not examined at intermediate doses between 10 ng/mL and 10 μg/mL, so it is conceivable that a dose lower than 10 μg/mL can still completely inhibit glycosylation of NGAL. Tunicamycin treatment for 24 hours did not appear to affect total protein glycan staining in SEB-1 lysates or media. This could be because it only inhibits N-linked glycans, and these were too few to be visible with the GelCode glycan stain which detects both N-linked and O-linked glycans. Alternatively, many cellular glycoproteins may have half lives longer than 48 hours because glycosylation can improve the stability of proteins. For these experiments, cells were treated with tunicamycin for only 24 hours, during which time many glycoproteins may not turn over significantly. NGAL‟s half-life in cells has not yet been determined, though one study has reported NGAL‟s half-life in plasma to be 10 to 20 minutes.365 Experiments in this Thesis co-treated cells with tunicamycin and 13-cis RA so that all NGAL induced by 13-cis RA was not glycosylated. Future experiments could determine approximate half-lives for both glycosylated and deglycosylated intracellular NGAL by pre-treating cells with 13-cis RA before tunicamycin treatment to determine the time needed for complete turnover of normal NGAL to deglycosylated NGAL, or by analyzing NGAL glycosylation status after the removal of tunicamycin treatment. The experiments presented here relied on a protein gel glycan staining kit to assess the glycan content of NGAL and other proteins in cell lysates and media. Glycan staining of proteins in a gel is not as sensitive as other methods for detecting glycans. The sensitivity of gel stains depends on the amount of protein present as well as the extent of its glycosylation; for example, the GelCode Glycoprotein Stain product sheet says that it can detect glycosylation of 0.16 μg of HP and 0.625 ng of avidin per band on a gel. We found that this glycan stain could only detect glycosylation of NGAL-(M) when 2.5 μg or more of pure recombinant protein had been loaded on the gel, which is likely due to the fact that NGAL is only

136 4 to 9% carbohydrate compared to other proteins such as HP, which is 21.8% carbohydrate.366 Knowing this, the fact that the stain did not pick up a clear glycosylated band around NGAL‟s size in non-purified SEB-1 lysates or media supernatants is not surprising. Previous data from our lab found that treatment of SEB-1 with 1 μM 13-cis RA induced NGAL expression at roughly 25 ng per μg of total cell protein at 48 hours of treatment (and it would be expected that this concentration would be less at just 24 hours of treatment as was done in this Thesis).363 Adding more than 100 μg of lysate or media protein in one lane overloaded the gel, but 100 μg protein per lane was not enough for the GelCode kit to detect glycosylated NGAL in a total cell lysate. It is also possible that the kit did not detect NGAL in total lysates or media because NGAL made by SEB-1 is not glycosylated. Whereas MS analysis was able to detect the presence or absence of the glycan on recombinant NGAL-(M) from PNGase F reactions, it was inconclusive in detecting the N-linked glycan on NGAL immunoprecipitated from SEB-1 media, probably because there was not enough NGAL present (and too much contaminating proteins) in the band sent for analysis. However, NGAL‟s gel shift that is observed with tunicamycin treatment of SEB-1 argues that NGAL produced by SEB-1 is in fact glycosylated. Though we have confirmed that NGAL-(M) has a glycan, it is unlikely that the presence or absence of the glycan would cause the 21 and 25 kD double bands seen on western blot. Treatment of SEB-1 with tunicamycin shifted the 21 kD NGAL band down to 19 kD, indicating that the 21 kD band seen in SEB-1 lysates does not represent deglycosylated NGAL, but is actually something different. NGAL does have a 20 amino acid secretory sequence that is thought to be cleaved before secretion, which may account for the 21 and 25 kD bands. Additionally, NGAL in the media samples consistently ran one or two kD larger than NGAL in the lysates, which could represent another post-translational modification. Phosphorylation is a possibility; one paper showed that the mouse homolog 24p3 is phosphorylated, though there are no reports in the literature that shown that human NGAL is phosphorylated.367 Usually only NGAL monomers are observed on western blots, though occasionally we have seen dimers around 50 kD in SEB-1 lysates and pure recombinant NGALs like in Figure B.8 (probably due to incomplete denaturing of samples before running the gel). It is interesting to note, though, that NGAL has been found as a monomer, homodimer, and heterodimer with MMP-9 in neutrophils. We have never investigated if the NGAL secreted by SEB-1 is a dimer or not. While the experiments in this Appendix did not determine if NGAL‟s glycan is essential for its apoptotic action, they did imply that the presence of the glycan affects NGAL‟s conformation. The MagneHis kit, which purifies His-tagged proteins from solution, could not purify deglycosylated NGAL- (M) that has a His tag on the C-terminal end. This suggests that the glycan could affect NGAL‟s tertiary structure, at least at the C-terminal end. From experiments in our lab as well as others, it is now known that both non-glycosylated and glycosylated NGAL can bind to bacterial siderophores. Additionally, it

137 has since been shown in other labs that NGAL-(B) can induce apoptosis in mammalian cells, allegedly via binding to siderophores. It would therefore be interesting to investigate if the glycan‟s effect on NGAL‟s confirmation then influences NGAL‟s affinity for siderophores, or binding to its receptor. Additionally, it is currently unknown if NGAL‟s homodimerization or heterodimerization with MMP-9 affects its function through confirmation changes. Elucidating the function of NGAL‟s structural variations may give insight into its dual nature in both inducing and preventing apoptosis.

B.5 Methods

B.5.1 SEB-1 Culture and Treatments

SEB-1 sebocytes were cultured in standard sebocyte media containing: 5.5 mM low glucose DMEM 3:1 Ham‟s F12, 2.5% (v/v) FBS, 0.4 mg/ml hydrocortisone, 1.8x10-4 M adenine, 10 ng/ml insulin, 3 ng/ml epidermal growth factor, 1.2x10-10 M cholera toxin, and antibiotics as previously described.14 Recombinant mammalian-expressed human NGAL (amino acids 21-198) was purchased from R&D Systems. Recombinant bacterially-expressed human apo- and holo-NGAL were produced in our lab as previously described.363 In brief, the NGAL cDNA construct (provided by Jack Cowland and Niels Borregaard, University of Copenhagen, Copenhagen, Denmark) was cloned into a pBB131 vector and transformed into XL-1 Blue E. coli to produce holo-NGAL (which is bound to the siderophore enterobactin), or cloned into a pET28a vector and transformed into BL21 E. coli to produce apo-NGAL (which does not have a siderophore bound to it). Bacterial cultures were grown to log phase in ZYP5020 auto-induction media, pelleted, and lysed. Apo- and holo-NGAL were purified from bacterial lysates using a nickel NTA column and then concentrated using a Sephadex G-100 column (both GE Healthcare).

B.5.2 Western Blots

70 μg of total protein for each lysate and 100 μg of total protein or 70 μg of total protein for each sample was run on a 10-well 12% Bis-Tris gel with MES at 200 V for 35 minutes. Protein was transferred to PVDF membrane (Millipore) at 30 V for one hour. The membrane was blocked with 5% (w/v) milk/TBS-T for one hour at room temperature on a shaker, and then incubated in mouse anti-NGAL mAb (Abcam, clone 5G5) in 5% (w/v) milk/TST-T overnight at 4° C on a shaker. Blots were then

138 washed three times with TBS-T for five minutes each, and incubated with rabbit anti-mouse polyclonal secondary antibody (Abcam) in 5% (w/v) milk/TBS-T for two hours at room temperature. Blots were developed using the SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce) according to manufacturer‟s directions, exposed to HyBloy CL Autoradiography Film (Denville Scientific), and developed with a Konica SRX-101 film processor (Konica Corporation).

B.5.3 Glycan Staining of Protein Gels

SEB-1 were treated in triplicate with 1 μM 13-cis RA, 10 ng/mL IL-1β, or vehicle for 72 hours. Media was collected and pooled for each treatment, then concentrated using centrifugation devices. 70 μg of total protein for each lysate and 100 μg of total protein or 70 μg of total protein for each sample was run on a denaturing 10-well 12% Bis-Tris gel with MES. Gels were stained with the GelCode Glycoprotein Staining Kit (Pierce, Thermo Scientific), photographed, and then stained with SimplyBlue Coomassie-G250 SafeStain total protein stain (Invitrogen) according to manufacturer‟s directions. Glycan staining controls of horseradish peroxidase (a glycosylated protein) and soy trypsin inhibitor (an unglycosylated protein, both included with GelCode kit) were also run on each gel. For tunicamycin assays, SEB-1 were treated in triplicate with varying doses of tunicamycin (Sigma-Aldrich) (10 μg/mL, 100 ng/mL, 1 ng/mL, 10 pg/mL, or vehicle) for 24 hours. Media was collected and pooled for each treatment, then concentrated using Amicon Ultra Ultracel 10 kD Centrifugal Filters (Millipore). Gels were run and stained as described above. A sample of untreated media was also concentrated and run on the media gel as a control to determine if the glycoproteins in the media samples are made by SEB-1 or are already found in the sebocyte media.

B.5.4 PNGase F Treatment of NGAL

N-linked glycans were removed from recombinant NGAL using PNGase F enzyme (New England Biolabs) according to manufacturer‟s directions. A test was first done on denatured NGAL. 5 μg of recombinant mammalian-expressed human NGAL (R&D Systems, amino acids 21 through 198) was added to G7 Reaction Buffer, NP-40, and water to a total volume of 20 μL. Reactions were heated to 100° C for 10 minutes and then cooled to room temperature. 500 units of PNGase F enzyme (New England Biolabs) was added to each reaction, and reactions were incubated for one hour at 37° C. Reactions using RNaseB protein (provided with the enzyme) as a substrate were used as controls for

139 PNGase F‟s activity. To determine the efficacy of PNGase F‟s removal of N-linked glycans, samples were analyzed by SDS-PAGE and stained with glycan stains. Once it was determined that PNGase F could remove N-linked glycans from denatured NGAL, reactions were then carried out using native NGAL (without including denaturing NP-40 buffer) and incubated at 37° C for one to 16 hours. Reactions were again checked on denaturing gels to determine the efficiency of glycan removal.

B.5.5 Immunoprecipitation

NGAL was immunoprecipitated from PNGase F reactions using Dynabeads Protein G IP Kit (Invitrogen) with mouse anti-NGAL mAb (Abcam, clone 5G5) according to kit manufacturer‟s directions. 100 μL of beads were used for each reaction with 100 μL of sample, and a DynaMag magnet (Invitrogen) was used for bead sequestering steps. Beads were first washed three times with 0.5 mL of 0.1 M sodium citrate buffer (pH 5.0). Then 10 μg of anti-NGAL antibody in 100 μL of PBS was added to the beads and incubated with gentle mixing for 40 minutes at room temperature. Beads were again washed three times with 0.1 M sodium citrate buffer and then twice with 1 ml of 0.2 M triethanolamine (pH 8.2). To prevent co-elution of antibody with precipitated NGAL, anti-NGAL antibody was cross-linked to the beads. Beads were resuspended in 1 ml of freshly made 20 mM dimethyl pimelimidate in 0.2 M triethanolamine (pH 8.2) and incubated with gentle mixing for 30 minutes at room temperature. The reaction was stopped by resuspending the beads in 1 ml of 50 mM Tris (pH 7.5), and incubated for 15 min with gentle mixing and then washing three times with 1 ml of PBS/0.1% (v/v) Tween-20. 100 μL of sample was added to the beads and incubated overnight at 4° C. The beads were washed three times with PBS and protein was eluted in two batches of 30 μL of 0.1 M citrate (pH 2.5) each. Beads were rinsed in 50 μL Na-citrate (pH 8.0) and then PBS for reuse later. The cross-linked bead/antibody complexes were reused up to five times before discarding. To determine the purity of immunoprecipitation samples, eluents were analyzed by SDS-PAGE. Supernatants from the samples after incubation with the beads were also analyzed by SDS-PAGE to check for any residual NGAL not bound to beads. Additionally, 3 μg of anti-NGAL Ab was run by itself as a control to check if any Ab fragments had not eluted with the NGAL.

140 B.5.6 Cell Death Assays

For tunicamycin toxicity assays, SEB-1 were seeded at 2x104 cells/well in 96-well plate were treated with serial dilutions of tunicamycin for 3 hours, 6 hours, 24 hours, or 48 hours in a 96-well plate. The CellTiter-Blue kit (Promega) was used to detect cell viability. Cell death was measured by recording the fluorescence at 560 nM excitation/590 nM emission. The background average of fluorescence values of the culture medium was subtracted from the average fluorescence values of experimental wells.

B.5.7 Mass Spectrometry

Immunoprecipitation reactions run on denaturing gels were stained with SimplyBlue SafeStain to label total protein. Protein bands were excised from the gel using a razor blade, briefly stored at -20° C, and then analyzed for glycan content by mass spectrometry (services provided by Midwest Bio Services; Overland Park, KS).

B.5.8 Annexin/PI Staining and Flow Cytometry

SEB-1 were plated in 35 mm plates, grown for 24 hours, and then treated with 1μM normal NGAL-(M), deglycosylated NGAL-(M), or vehicle for 24 hours. Cell were then trypsinized and stained with PI and FITC-conjugated antibodies for annexin V using ApoAlert FITC-Annexin V Kit (Clontech) kit according to manufacturer‟s directions. Stained cells were analyzed by flow cytometry using a BD Scan 2 cytometer with Cell Quest software (BD Biosciences). At least 10,000 events were collected for each sample. Cells were first gated by scatter properties to exclude debris and were then plotted for FITC versus APC. The percent of cells in early apoptosis (FITC-positive), late apoptosis (PI-positive FITC- positive), and necrosis (PI-positive) were calculated for each treatment group and compared using Student‟s t-tests, with P < 0.05 considered as significant. Controls included cells treated with 0.1 μM staurosporine as a positive control for apoptosis, as well as controls for the deglycosylation reactions: „NGAL control‟ is NGAL that was incubated in the same buffers as the deglycosylation reactions but without PNGase F enzyme, and „PNGaseF control‟ is PNGase F and its reaction buffers without NGAL.

141 B.5.9 TUNEL Assays

SEB-1 were treated with 1 μM normal mammalian-expressed NGAL, deglycosylated NGAL, or vehicle for 24 hours. Treatment controls for the deglycosylation reactions included NGAL that was incubated in the same buffers as the deglycosylation reactions but without PNGase F enzyme, and PNGase F and its reaction buffers without NGAL. After the treatment period, cells were fixed with 4% (w/v) paraformaldehyde/PBS (pH 7.4) for one hour at room temperature and permeabilized in 0.1% (v/v) Triton/0.1% (w/v) sodium citrate/PBS for 10 minutes on ice. For TUNEL staining, fluorescein In Situ Cell Death Detection Kits (Roche) were used, and three fields in each well were photographed for green fluorescence and brightfield. The percent of apoptotic cells in each field was calculated by dividing the number of green fluorescence-positive cells by the total number of cells. The percentages of apoptotic cells in each treatment group were compared using unpaired Student‟s t-tests, with P < 0.05 being considered significant.

142 Appendix C

NF-κB Studies

C.1 Chapter Abstract

Our lab has previously shown that NGAL is an important mediator of 13-cis RA-induced apoptosis in sebocytes, but this effect may not be entirely retinoic acid receptor (RAR)-mediated57. Because retinoids have been shown to be able to activate the NF-κB pathway in multiple cell types, we hypothesized that 13-cis RA may be able to induce NGAL expression in sebocytes by both direct RAR- mediated and indirect mechanisms. This Appendix shows that 13-cis RA may be able to activate NF-κB in SEB-1 in a delayed time-course, and that various NF-κB stimuli also induce NGAL expression in sebocytes. The delayed activation of NF-κB in sebocytes may represent an indirect mechanism which contributes to 13-cis RA‟s induction of NGAL.

C.2 Introduction

Our lab has previously shown that NGAL is an important mediator of 13-cis RA-induced apoptosis in sebocytes.57 We also found that blocking RAR transcription factors did not completely ablate the induction of NGAL (unpublished data), suggesting that 13-cis RA could be working through both direct RAR-mediated and indirect mechanisms. The human gene that encodes NGAL, lipocalin 2 (LCN2), contains multiple transcriptional elements in its promoter, including multiple RAREs and an NF- B binding site (Figure C.1). Other labs have demonstrated that NGAL can be induced by NF- B stimuli in multiple cell lines, with a focus on cells of an epithelial lineage.368-373 Our own lab has found that P. acnes, which is thought to stimulate cells through TLR-2/1, can induce NGAL expression in SEB- 1.363 However, the regulatory mechanisms of NGAL expression in sebocytes have not been fully characterized. The NF-κB signaling system is a complex network that controls the expression of inflammatory response genes including cytokines, immune modulators, and antimicrobial peptides. The NF-κB transcriptional dimer typically consists of the subunits p50 and p65 (RelA), though other subunits can predominate in specific sub-pathways. When inactive, these dimeric complexes are bound to inhibitory IκB proteins in the cytoplasm. In the canonical NF-κB signaling pathway, upstream stimuli activate IκB kinase (IKK), which phosphorylates IκB to promote its degradation, thus releasing the transcriptional

143 subunits and allowing them to translocate to the nucleus. Once in the nucleus, activated p50/p65 dimers recruit histone acetylases and transcription cofactors that allow NF-κB to gain access to κB binding sequences in the promoters of target genes.

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Figure C.1: The LCN2 gene promoter. The LCN2 gene promoter contains many different transcription factor binding elements, including a κB binding site and retinoic acid response elements.

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More than 150 different stimuli can affect NF-κB activation, including cytokines, pathogens, and stresses such as UV light or oxidative stress.374 Oxidative stress resulting from free radicals or other reactive oxygen species (ROS) directly activates NF-κB signaling by causing phosphorylation and degradation of IκB, leading to activation of inflammatory or protective stress genes.375 While environmental ROS (such as ozone) can be harmful, ROS such as O2•- and •OH are used endogenously by immune cells to attack pathogens. The presence of ROS in cells activates stress responses that initiate either inflammatory or apoptotic pathways. Because the skin functions as the first barrier to pathogens and environmental stress, it must be able to activate specific immunologic defenses. Fittingly, NF-κB dysregulation is implicated in many inflammatory skin diseases including psoriasis, dermatitis, skin cancer, and reaction to sunburn.374 In these contexts, it is disputed as to whether inflammation is protective, damaging, or both. Furthermore, NF-κB-dependent inflammatory genes and cytokines (TNFα, IL-1β, IL-8, IL-10) are up-regulated in acne lesions compared to normal skin, but the role of inflammation in acne pathogenesis versus resolution has not yet been resolved.118 Though inflammation has historically been regarded as pathogenic in acne, oral 13-cis RA treatment induces a temporary inflammatory „flare‟ of acne before remission begins. Because we have shown that the effects of 13-cis RA on sebocytes are not entirely retinoid receptor-mediated, it is possible that retinoids can alter specific immune responses in skin, an effect that has been demonstrated in

144 other contexts.376 Retinoids can activate NF- B in multiple cell types through physical receptor interactions or the creation of ROS.288,377-379 Other studies have shown that NGAL expression can be induced by ROS and that the induction of NGAL by retinoids is mediated by ROS in other cell lines.267,380 This data indicates that it may be possible for retinoids to induce NGAL expression by multiple mechanisms, one of which may be through either direct (via gene transcription) or indirect (via ROS production) action on NF-κB pathways. The experiments in this Appendix tested the hypothesis that 13- cis RA induces NGAL in sebocytes partially through activation of NF-κB.

C.3 Results

C.3.1 The NF-κB pathway is intact in SEB-1

To confirm that SEB-1 cells have an intact NF-κB signaling pathway, immunofluorescence for p65 translocation was performed. SEB-1 cells were treated with 10 ng/mL IL-1β for one hour in 8-well culture slides. At the end of treatment, cells were fixed, permeabilized, and incubated with DAPI, primary antibodies to p65, and fluorescently-conjugated secondary antibodies. Fields in each treatment group were photographed and the percent of cells with p65 translocation was calculated. One hour of IL- 1β induced p65 translocation from the cytoplasm to the nucleus in most cells (Figure C.2). ______

Figure C.2: NF- B translocation in SEB-1 sebocytes. Immunofluorescence staining was performed on cultures treated with IL-1β for one hour using primary antibodies to p65 and DAPI.

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145 ______

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146 C.3.2 13-cis RA activates NF-κB in a delayed time-course in SEB-1

To investigate whether 13-cis RA can activate NF-κB in SEB-1, SEB-1 cells were treated for various times (0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 18 hours) with 10 ng/mL IL-1β or 1 μM 13-cis RA. „0 minute‟ incubations were performed by aspirating old media from the cells, adding fresh media containing IL-1β or 13-cis RA, and then immediately aspirating the fresh media and fixing the cells. Cells were fixed and stained as described above. IL-1β treatment significantly induced p65 translocation from the cytoplasm to the nucleus at 0, 15, 30, and 60 minutes of treatment compared to vehicle. 13-cis RA induced p65 translocation at 18 hours of treatment, though this effect was not significant (Figure C.3). NF-κB activation was also confirmed by western blot for phospho-p65. SEB-1 cells were treated with IL-1β or 13-cis RA as above, and total cell lysates were prepared and probed for phospho-p65 by western blot (Figure C.4). Levels of phospho-p65 were increased with 15 minutes, 30 minutes, and 1 hour of IL-1β treatment as well as 30 minutes and one hour of 13-cis RA treatment, confirming the immunofluorescence assays for p65 translocation.

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147 C.3.3 NF-κB stimuli induce NGAL expression in SEB-1

To determine whether NF-κB activation could induce NGAL expression in SEB-1, SEB-1 were treated with cytokines and various TLR ligands and assayed for NGAL protein by western blot. To test the ability of inflammatory cytokines IL-1β and TNFα to induce NGAL expression in SEB-1, SEB-1 were treated with various doses of each for 72 hours. Total cell lysates were subjected to SDS-PAGE and blots were incubated with primary antibodies to NGAL as well as β-actin as a loading control. Both IL- 1β and TNFα increased NGAL expression over vehicle in SEB-1 (Figure C.5). Our lab has previously shown that P. acnes sonicate, which stimulates cells through TLR2/1, can induce NGAL expression in sebocytes. To determine if TLR-2 signaling could induce NGAL via dimerization with either TLR-1 or TLR-6, SEB-1 were treated with 10 μg/mL zymosan (a TLR-2/6 heterodimer ligand) for 72 hours or 25 μg/mL LTA (a TLR-2/1 heterodimer ligand) for six hours. Total cell protein was subjected to SDS-PAGE and blots were incubated with primary antibodies to NGAL. LTA increased NGAL expression, but zymosan treatment did not, indicating that the TLR-2/6 heterodimer may not be able to regulate NGAL expression in SEB-1 (Figure C.6). To investigate whether signaling through other TLRs could also induce NGAL expression, SEB-1 sebocytes were treated for 24 hours with, 20 μg/mL poly(I:C) (a TLR-3 ligand), 1 μg/mL LPS (a TLR-4 ligand), or vehicle controls for 24 hours. NGAL was increased with poly(I:C) treatment, but not LPS, indicating that stimulation of TLR3 and TLR2/1 pathways can induce NGAL expression in SEB-1, but that TLR-4 and TLR-2/6 stimulation cannot.

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148 ______

Figure C.6: NGAL induction by TLR ligands. SEB-1 sebocytes were treated with 20 μg/mL poly(I:C), 10 μg/mL zymosan, 25 μg/mL LTA, 1 μg/mL LPS, or vehicle controls for 24 hours. Total cell protein was subjected to SDS- PAGE. Blots were incubated with primary antibodies to NGAL as well as β-actin as a loading control.

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C.4 Discussion

Experiments in this Appendix demonstrate that the NF-κB signaling pathway is intact in SEB-1 sebocytes, and that various NF-κB stimuli can induce NGAL expression. The timeline of NF-κB activation in SEB-1 shows that even in vehicle-treated cultures, p65 translocated to the nucleus in up to 45% of cells between 15 minutes and one hour after treatment. This may be because the cells were responding to changes in temperature (being removed from the incubator). In the case of cells treated with the vehicle for 13-cis RA (which is 100% ethanol), the vehicle itself could have activated stress response pathways that converge on NF-κB. Even with a high baseline of p65 translocation, IL-1β significantly increased p65 translocation over vehicle at 15 minutes, 30 minutes, and one hour of treatment, indicating that the NF-κB pathway is functional in SEB-1. We have shown here that NGAL protein can be increased by TLR-2/1 or TLR-3 stimulation, but not by TLR-2/6 or TLR-4 stimulation. Additionally, both IL-1β and TNFα can increase NGAL expression in SEB-1. This observation represents a novel finding since TNFα has not been shown to induce NGAL expression in other cell types.369 This is because NGAL expression is thought to be controlled by IκB-δ, a specific NF-κB binding cofactor that is activated by IL-1β but not TNFα, even though both treatments promote NF-κB binding to the LCN2 gene promoter.370 The mechanisms behind NGAL induction in SEB-1 by TNFα are unknown. It is possible that TNFα treatment can induce NGAL indirectly by promoting the expression of IL-1β which would then act on SEB-1 cells in an autocrine

149 manner. This would explain why TNFα was not as potent as IL-1β in inducing NGAL at comparable doses. Another possibility is that NGAL is regulated by alternative IκB proteins other than IκB-δ in SEB- 1. The presence of surface receptor-specific IκB proteins in SEB-1 could also elucidate why stimulation of some TLRs can induce NGAL, but not others. IκB-δ expression has not been investigated in SEB-1. The experiments in this Appendix show that 13-cis RA may be able to activate NF-κB in a delayed time-course compared to IL-1β. Though not significant, 13-cis RA induced p65 translocation over vehicle at 18 hours of treatment. Additional experiments are needed in order to confirm this pattern. Time-points beyond 18 hours were not tested; future studies could also investigate a longer time-course to determine the peak time of NF-κB activation. Because reports in the literature demonstrate that NGAL expression can be induced by ROS, and that the induction of NGAL by retinoids is mediated by ROS in other cell lines, it is possible that 13-cis RA has a similar effect in sebocytes. To investigate this possibility, future experiments could quantify the amount of ROS in SEB-1 produced by 13-cis RA treatment or co-treat cells with 13-cis RA and antioxidants to determine if the blockage of ROS formation or action could also prevent NGAL induction. Because NGAL induction by 13-cis RA may not be entirely RAR-dependent, determining if 13-cis RA can also create ROS may shed light on alternative pathways by which 13-cis RA can act in sebocytes.

C.5 Methods

C.5.1 SEB-1 Cell Culture and Treatments

SEB-1 sebocytes were culture in standard sebocyte media containing: 5.5 mM low glucose DMEM 3:1 Ham‟s F12, 2.5% (v/v) FBS, 0.4 mg/ml hydrocortisone, 1.8x10-4 M adenine, 10 ng/ml insulin, 3 ng/ml epidermal growth factor, 1.2x10-10 M cholera toxin, and antibiotics as previously described.14 All 13-cis RA (Sigma-Aldrich) stocks and solutions were used in a sterile culture hood under yellow light. 13-cis RA was resuspended in 100% ethanol at 50 μM. The solution was aliquotted, overlaid with N2 gas, and stored at -20° C until use. IL-1β and TNFα (Sigma-Aldrich and R&D Systems, respectively) were reconstituted in sterile water to a dilution of 1 mg/mL and then diluted to 100 μg/mL in PBS/0.1% (w/v) BSA. Solutions were aliquotted and stored at -20° C until use. Thawed aliquots were not reused. Escherichia coli LPS (Sigma), Bacillus subtillus LTA (Sigma), Saccharomyces cerevisiae

150 cell wall zymosan (Invitrogen), and poly I:C (InvivoGen) were resuspended in sterile nanopure water and stored in aliquots at -20° C.

C.5.2 p65 Translocation Assays

SEB-1 were seeded in 8-well chamber slides at 1x104 cells in 500 μL media per well. Cells were incubated at 37° C for 24 hours and then treated with 1 μM 13-cis RA or 10 ng/mL IL-1β for 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours. At the end of the treatment time, media was aspirated and wells were washed twice with 500 μL of PBS and fixed with cold methanol for 10 minutes at -20° C. Cells were washed three times with PBS and permeabilized with PBS/0.1% (v/v) Triton/0.1% (w/v) sodium citrate on ice for 15 minutes. After three more washes with PBS, slides were blocked with PBS/1% (w/v) BSA for 30 minutes at room temperature. Cells were incubated in monoclonal mouse anti-p65 antibody (BD Biosciences) or mouse IgG1 isotype control (BD Biosciences) diluted in PBS/1% (w/v) BSA overnight at 4 C. At the end of the incubation, cells were washed three times with PBS/0.3% (v/v) Tween 20 and incubated in a solution of PBS/1% (w/v) BSA with anti-mouse fluorescin antibody and DAPI for two hours at room temperature. Slides were lastly washed three times with PBS/0.3% (v/v) Tween 20 and mounted onto a coverslip with mounting medium. Three fields of green fluorescence, blue fluorescence, and brightfield pictures were obtained for each well of the slides.

C.5.3 Western Blots

For phospho-p65 western blots, SEB-1 were plated in 100 mm culture plates and grown for 24 hours before treating with 1 μM 13-cis RA or 10 ng/mL IL-1β for 15 minutes, 30 minutes, 1 hour, 2 hours, or 6 hours. At the end of the incubation period total cell lysates were made by scraping cells in 200 μL lysis buffer with a rubber policeman, flash freezing the lysates in liquid nitrogen, and shearing DNA with a 22-gauge needle. Lysates were run on 4-12% Bis-Tris gels (Invitrogen) at 200 V for 35 minutes in MES running buffer (Invitrogen). Protein was transferred to PVDF membranes at 30 V for one hour at room temperature. Blots were blocked in 5% (w/v) milk/TBS-T for one hour at room temperature and then incubated in polyclonal rabbit anti-phospho-p65 (Ser529) antibody (Abcam) in 5% (w/v) milk/TBS-T overnight at 4° C. Blots were washed three times for five minutes in TBS-T at room temperature and then incubated in goat anti-rabbit HRP-conjugated antibody in 5% (w/v) milk/TBS-T for one hour at room temperature. Blots were finally washed twice with TBS-T, once with TBS, and then

151 developed using the West Pico kit (Thermo Scientific). After developing, blots were stripped for 10 minutes at room temperature and washed once with nanopure water before incubation in rabbit anti- - actin in 5% (w/v) milk/TBS-T for two hours at room temperature. Blots were again washed three times in TBS-T, incubated in goat anti-rabbit-HRP antibody in 5% (w/v) milk/TBS-T for one hour at room temperature, and developed as above. For NGAL western blots, SEB-1 were treated with 10 μg/mL zymosan (a TLR-2/6 heterodimer ligand) for 72 hours or 25 μg/mL LTA (a TLR-2/1 heterodimer ligand) for 6 hours. Total cell protein was subjected to SDS-PAGE and western blotting was performed as above. Blots were incubated with monoclonal mouse anti-human NGAL antibody (Abcam) and then goat anti-mouse HRP-conjugated secondary antibody (Santa Cruz) and developed as above.

152 References

1. Chuong, C.M., et al. What is the 'true' function of skin? Exp Dermatol 11, 159-187 (2002). 2. James, A.T. & Wheatley, V.R. Studies of sebum. 6. The determination of the component fatty acids of human forearm sebum by gas-liquid chromatography. Biochem J 63, 269-273 (1956). 3. Haahti, E., Horning, E.C. & Castren, O. Microanalysis of "sebum" and sebum-like materials by temperature programmed gas chromatography. Scand J Clin Lab Invest 14, 368-372 (1962). 4. Cassidy, D., Lee, C., Laker, M. & Kealey, T. Lipogenesis in isolated human sebaceous glands. FEBS Letters 200, 173-176 (1986). 5. Proksch, E., Feingold, K.R. & Elias, P.M. Epidermal HMG CoA reductase activity in essential fatty acid deficiency: barrier requirements rather than eicosanoid generation regulate cholesterol synthesis. The Journal of investigative dermatology 99, 216-220 (1992). 6. Smythe, C.D., Greenall, M. & Kealey, T. The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol. Journal of Investigative Dermatology 111, 139- 148 (1998). 7. Kligman, A.M. The Uses of Sebum. The British journal of dermatology 75, 307-319 (1963). 8. Flurh, J.W., et al. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice. Journal of Investigative Dermatology 120, 728-737 (2003). 9. Thiele, J.J., Weber, S.U. & Packer, L. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin. The Journal of investigative dermatology 113, 1006-1010 (1999). 10. Gebhart, W., Metze, D. & Jurecka, W. Identification of secretory immunoglobulin A in human sweat and sweat glands. The Journal of investigative dermatology 92, 648 (1989). 11. Stewart, M.E., Steele, W.A. & Downing, D.T. Changes in the relative amounts of endogenous and exogenous fatty acids in sebaceous lipids during early adolescence. The Journal of investigative dermatology 92, 371-378 (1989). 12. Cooper, M.F., McGrath, H. & Shuster, S. Sebaceous lipogenesis in human skin. Variability with age and with severity of acne. The British journal of dermatology 94, 165-172 (1976). 13. Jacobsen, E., et al. Age-related changes in sebaceous wax ester secretion rates in men and women. The Journal of investigative dermatology 85, 483-485 (1985). 14. Thiboutot, D., et al. Human skin is a steroidogenic tissue: Steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1). The Journal of investigative dermatology 120, 905-914 (2003). 15. Rosignoli, C., Nicholas, J., Jomard, A. & Michel, S. Involvement of the SREBP pathway in the mode of action of androgens in sebaceous glands in vivo. Exp Dermatol 12, 480-489 (2003). 16. Smith, T.M., Cong, Z., Gilliland, K.L., Clawson, G.A. & Thiboutot, D.M. Insulin-like growth factor-1 induces lipid production in human SEB-1 sebocytes via sterol response element-binding protein-1. The Journal of investigative dermatology 126, 1226-1232 (2006). 17. Smith, T.M., Gilliland, K., Clawson, G.A. & Thiboutot, D. IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway. The Journal of investigative dermatology 128, 1286-1293 (2008). 18. Smith, R.N., Braue, A., Varigos, G.A. & Mann, N.J. The effect of a low glycemic load diet on acne vulgaris and the fatty acid composition of skin surface triglycerides. J Dermatol Sci 50, 41- 52 (2008). 19. Smith, R., et al. A pilot study to determine the short-term effects of a low glycemic load diet on hormonal markers of acne: a nonrandomized, parallel, controlled feeding trial. Mol Nutr Food Res 52, 718-726 (2008). 20. Melnik, B.C. & Schmitz, G. Role of insulin, insulin-like growth factor-1, hyperglycaemic food and milk consumption in the pathogenesis of acne vulgaris. Exp Dermatol 18, 833-841 (2009).

153 21. Berra, B. & Rizzo, A.M. Glycemic index, glycemic load: new evidence for a link with acne. J Am Coll Nutr 28 Suppl, 450S-454S (2009). 22. Thody, A.J., Cooper, M.F., Bowden, P.E., Meddis, D. & Shuster, S. Effect of alpha-melanocyte- stimulating hormone and testosterone on cutaneous and modified sebaceous glands in the rat. Journal of Endocrinology 71, 279-288 (1976). 23. Thiboutot, D., Sivarajah, A., Gilliland, K., Cong, Z. & Clawson, G. The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells. The Journal of investigative dermatology 115, 614-619 (2000). 24. Zhang, L., Li, W.H., Anthonavage, M. & Eisinger, M. Melanocortin-5 receptor: a marker of human sebocyte differentiation. Peptides 27, 413-420 (2006). 25. Rosen, E.D., et al. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Molecular Cell 4, 611-617 (1999). 26. Trivedi, N.R., et al. Peroxisome proliferator-activated receptors increase human sebum production. The Journal of investigative dermatology 126, 2002-2009 (2006). 27. Downie, M.M., Sanders, D., Maier, L., Stock, D. & Kealey, T. Peroxisome proliferator-activated receptor and farnesoid X receptor ligands differentially regulate sebaceous differentiation in human sebaeous gland organ cultures in vitro. The British journal of dermatology 151, 766-775 (2004). 28. Chen, W., Yang, C., Sheu, H., Seltmann, H. & Zouboulis, C. Expression of peroxisome proliferator-activated receptor and CCAAT/enhancer binding protein transcription factors in cultured human sebocytes. The Journal of investigative dermatology 121, 441-447 (2003). 29. Pivarcsi, A., et al. Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes. Arch Dermatol Res 296, 120-124 (2004). 30. Baker, B.S., Ovigne, J.M., Powles, A.V., Corcoran, S. & Fry, L. Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis. The British journal of dermatology 148, 670-679 (2003). 31. Kawai, K., et al. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci 30, 185-194 (2002). 32. Kollisch, G., et al. Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology 114, 531-541 (2005). 33. McInturff, J.E., Modlin, R.L. & Kim, J. The role of toll-like receptors in the pathogenesis and treatment of dermatological disease. The Journal of investigative dermatology 125, 1-8 (2005). 34. Matsuguchi, T., Takagi, K., Musikacharoen, T. & Yoshikai, Y. Gene expressions of lipopolysaccharide receptors, toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes. Blood 95, 1378-1385 (2000). 35. Musikacharoen, T., Matsuguchi, T., Kikuchi, T. & Yoshikai, Y. NF-kappa B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression. J Immunol 166, 4516-4524 (2001). 36. Haehnel, V., Schwarzfischer, L., Fenton, M.J. & Rehli, M. Transcriptional regulation of the human toll-like receptor 2 gene in monocytes and macrophages. J Immunol 168, 5629-5637 (2002). 37. Johnson, C.M. & Tapping, R.I. Microbial products stimulate human Toll-like receptor 2 expression through histone modification surrounding a proximal NF-kappaB-binding site. The Journal of biological chemistry 282, 31197-31205 (2007). 38. Kutukculer, N., Yeniay, B.S., Aksu, G. & Berdeli, A. Arg753Gln polymorphism of the human toll-like receptor-2 gene in children with recurrent febrile infections. Biochem Genet 45, 507-514 (2007). 39. Lorenz, E., Mira, J.P., Cornish, K.L., Arbour, N.C. & Schwartz, D.A. A novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun 68, 6398-6401 (2000).

154 40. Kijpittayarit, S., Eid, A.J., Brown, R.A., Paya, C.V. & Razonable, R.R. Relationship between Toll-like receptor 2 polymorphism and cytomegalovirus disease after liver transplantation. Clin Infect Dis 44, 1315-1320 (2007). 41. Ogus, A.C., et al. The Arg753GLn polymorphism of the human toll-like receptor 2 gene in tuberculosis disease. Eur Respir J 23, 219-223 (2004). 42. Ahmad-Nejad, P., et al. The toll-like receptor 2 R753Q polymorphism defines a subgroup of patients with atopic dermatitis having severe phenotype. The Journal of allergy and clinical immunology 113, 565-567 (2004). 43. Niebuhr, M., Langnickel, J., Sigel, S. & Werfel, T. Dysregulation of CD36 upon TLR-2 stimulation in monocytes from patients with atopic dermatitis and the TLR2 R753Q polymorphism. Exp Dermatol 19, e296-298 (2010). 44. Schroder, N.W., et al. High frequency of polymorphism Arg753Gln of the Toll-like receptor-2 gene detected by a novel allele-specific PCR. J Mol Med 81, 368-372 (2003). 45. Kang, T.J., Yeum, C.E., Kim, B.C., You, E.Y. & Chae, G.T. Differential production of interleukin-10 and interleukin-12 in mononuclear cells from leprosy patients with a Toll-like receptor 2 mutation. Immunology 112, 674-680 (2004). 46. Ben-Ali, M., Barbouche, M.R., Bousnina, S., Chabbou, A. & Dellagi, K. Toll-like receptor 2 Arg677Trp polymorphism is associated with susceptibility to tuberculosis in Tunisian patients. Clin Diagn Lab Immunol 11, 625-626 (2004). 47. Candia, L., Marquez, J., Hernandez, C., Zea, A.H. & Espinoza, L.R. Toll-like receptor-2 expression is upregulated in antigen-presenting cells from patients with psoriatic arthritis: a pathogenic role for innate immunity? The Journal of rheumatology 34, 374-379 (2007). 48. Do, J.E., Kwon, S.Y., Park, S. & Lee, E.S. Effects of vitamin D on expression of Toll-like receptors of monocytes from patients with Behcet's disease. Rheumatology (Oxford, England) 47, 840-848 (2008). 49. Sumegi, A., et al. Analysis of components of the CD14/TLR system on leukocytes of patients with atopic dermatitis. Int Arch Allergy Immunol 143, 177-184 (2007). 50. Iwahashi, M., et al. Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis. Arthritis and rheumatism 50, 1457-1467 (2004). 51. Fathy, A., Mohamed, R.W., Ismael, N.A. & El-Akhras, M.A. Expression of toll-like receptor 2 on peripheral blood monocytes of patients with inflammatory and noninflammatory acne vulgaris. Egypt J of Immun 16, 127-134 (2009). 52. Hunger, R.E., Surovy, A.M., Hassan, A.S., Braathen, L.R. & Yawalkar, N. Toll-like receptor 2 is highly expressed in lesions of acne inversa and colocalizes with C-type lectin receptor. The British journal of dermatology 158, 691-697 (2008). 53. Chen, X., et al. Synergistic effect of antibacterial agents human beta-defensins, cathelicidin LL- 37 and lysozyme against Staphylococcus aureus and Escherichia coli. J Dermatol Sci 40, 123-132 (2005). 54. Goetz, D.H., et al. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10, 1033-1043 (2002). 55. Bao, G., et al. Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex. Nat Chem Biol 6, 602-609 (2010). 56. Holmes, M.A., Paulsene, W., Jide, X., Ratledge, C. & Strong, R.K. Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure 13, 29-41 (2005). 57. Nelson, A.M., et al. Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid- induced apoptosis of human sebaceous gland cells. The Journal of clinical investigation 118, 1468-1478 (2008).

155 58. Devireddy, L.R., Hart, D.O., Goetz, D.H. & Green, M.R. A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell 141, 1006-1017 (2010). 59. Chen, X., et al. Antimicrobial peptides human beta-defensin (hBD)-3 and hBD-4 activate mast cells and increase skin vascular permeability. European journal of immunology 37, 434-444 (2007). 60. Sampanthanarak, P., et al. The effect of antibacterial peptide human beta-defensin-2 on interleukin-18 secretion by keratinocytes. J Dermatol Sci 37, 188-191 (2005). 61. Niyonsaba, F., et al. Antimicrobial peptides human beta-defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines. The Journal of investigative dermatology 127, 594-604 (2007). 62. Niyonsaba, F., Ushio, H., Nagaoka, I., Okumura, K. & Ogawa, H. The human beta-defensins (-1, -2, -3, -4) and cathelicidin LL-37 induce IL-18 secretion through p38 and ERK MAPK activation in primary human keratinocytes. J Immunol 175, 1776-1784 (2005). 63. Hollox, E.J., et al. Psoriasis is associated with increased beta-defensin genomic copy number. Nat Genet 40, 23-25 (2008). 64. Ong, P.Y., et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. The New England journal of medicine 347, 1151-1160 (2002). 65. Trivedi, N.R., Gilliland, K.L., Zhao, W., Liu, W. & Thiboutot, D.M. Gene array expression profiling in acne lesions reveals marked upregulation of genes involved in inflammation and matrix remodeling. The Journal of investigative dermatology 126, 1071-1079 (2006). 66. Braff, M.H., Zaiou, M., Fierer, J., Nizet, V. & Gallo, R.L. Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens. Infect Immun 73, 6771-6781 (2005). 67. Lee, D.Y., et al. Sebocytes express functional cathelicidin antimicrobial peptides and can act to kill propionibacterium acnes. The Journal of investigative dermatology 128, 1863-1866 (2008). 68. van der Does, A.M., et al. LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature. J Immunol 185, 1442-1449 (2010). 69. Alalwani, S.M., et al. The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils. European journal of immunology 40, 1118-1126 (2010). 70. Yamasaki, K., et al. Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nature medicine 13, 975-980 (2007). 71. Burtenshaw, J.M. The mechanism of self-disinfection of the human skin and its appendages. J Hyg (Lond) 42, 184-210 (1942). 72. Nakatsuji, T., et al. Sebum Free Fatty Acids Enhance the Innate Immune Defense of Human Sebocytes by Upregulating [beta]-Defensin-2 Expression. The Journal of investigative dermatology 130, 985-994 (2010). 73. Miller, S.J., Aly, R., Shinefeld, H.R. & Elias, P.M. In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids. Archives of dermatology 124, 209-215 (1988). 74. Nakatsuji, T., et al. Antimicrobial Property of Lauric Acid Against Propionibacterium Acnes: Its Therapeutic Potential for Inflammatory Acne Vulgaris. The Journal of investigative dermatology 129, 2480-2488 (2009). 75. Wille, J.J. & Kydonieus, A. Palmitoleic acid isomer (C16:1delta6) in human skin sebum is effective against gram-positive bacteria. Skin Pharmacol Appl Skin Physiol 16, 176-187 (2003). 76. Drake, D.R., Brogden, K.A., Dawson, D.V. & Wertz, P.W. Thematic review series: skin lipids. Antimicrobial lipids at the skin surface. J Lipid Res 49, 4-11 (2008). 77. Georgel, P., et al. A toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with gram-positive bacteria. Infect Immun 73, 4512-4521 (2005). 78. Skrivanova, E., Savka, O.G. & Marounek, M. In vitro effect of C2-C18 fatty acids on Salmonellas. Folia Microbiol (Praha) 49, 199-202 (2004).

156 79. Yamasaki, K., et al. TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes. The Journal of investigative dermatology 131, 688-697 (2011). 80. Gallo, R.L. & Nakatsuji, T. Microbial Symbiosis with the Innate Immune Defense System of the Skin [Published online ahead of print June 23, 2011]. The Journal of investigative dermatology, doi: 10.1038/jid.2011.1182. 81. Zarember, K.A. & Godowski, P.J. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 168, 554-561 (2002). 82. Clark, R.A., et al. The vast majority of CLA+ T cells are resident in normal skin. J Immunol 176, 4431-4439 (2006). 83. Nestle, F.O., et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. The Journal of experimental medicine 202, 135-143 (2005). 84. Zhou, L., Chong, M.M. & Littman, D.R. Plasticity of CD4+ T cell lineage differentiation. Immunity 30, 646-655 (2009). 85. Clark, R.A., et al. A novel method for the isolation of skin resident T cells from normal and diseased human skin. The Journal of investigative dermatology 126, 1059-1070 (2006). 86. Wang, J., Huizinga, T.W.J. & Toes, R.E.M. De Novo Generation and Enhanced Suppression of Human CD4+CD25+ Regulatory T Cells by Retinoic Acid. Vol. 183 4119-4126 (2009). 87. Nolting, J., et al. Retinoic acid can enhance conversion of naive into regulatory T cells independently of secreted cytokines. The Journal of experimental medicine 206, 2131-2139 (2009). 88. Mucida, D., et al. Reciprocal TH17 and Regulatory T Cell Differentiation Mediated by Retinoic Acid. Vol. 317 256-260 (2007). 89. Vukmanovic-Stejic, M., et al. The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo. The Journal of clinical investigation 118, 3639-3650 (2008). 90. Lee, J.H., et al. The levels of CD4+CD25+ regulatory T cells in paediatric patients with allergic rhinitis and bronchial asthma. Clinical and experimental immunology 148, 53-63 (2007). 91. Ehrenstein, M.R., et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy. The Journal of experimental medicine 200, 277-285 (2004). 92. Nadkarni, S., Mauri, C. & Ehrenstein, M.R. Anti-TNF-alpha therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-beta. The Journal of experimental medicine 204, 33-39 (2007). 93. Flores-Borja, F., Jury, E.C., Mauri, C. & Ehrenstein, M.R. Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis. Proceedings of the National Academy of Sciences of the United States of America 105, 19396-19401 (2008). 94. Flores-Borja, F., Mauri, C. & Ehrenstein, M.R. Restoring the balance: harnessing regulatory T cells for therapy in rheumatoid arthritis. European journal of immunology 38, 934-937 (2008). 95. Izcue, A., Coombes, J.L. & Powrie, F. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol Rev 212, 256-271 (2006). 96. Eastaff-Leung, N., Mabarrack, N., Barbour, A., Cummins, A. & Barry, S. Foxp3+ regulatory T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease. Journal of clinical immunology 30, 80-89 (2010). 97. Denning, T.L., Kim, G. & Kronenberg, M. Cutting edge: CD4+CD25+ regulatory T cells impaired for intestinal homing can prevent colitis. J Immunol 174, 7487-7491 (2005). 98. Kristensen, N.N., Olsen, J., Gad, M. & Claesson, M.H. Genome-wide expression profiling during protection from colitis by regulatory T cells. Inflamm Bowel Dis 14, 75-87 (2008). 99. Yan, A.C. Current concepts in acne management. Adolesc Med Clin 17, 613-637; abstract x-xi (2006).

157 100. White, G.M. Recent findings in the epidemiologic evidence, classification, and subtypes of acne vulgaris. Journal of the American Academy of Dermatology 39, S34-37 (1998). 101. Bickers, D.R., et al. The burden of skin diseases: 2004: A joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. Journal of the American Academy of Dermatology 55, 490-500 (2006). 102. Baldwin, H.E. The interaction between acne vulgaris and the psyche. Cutis 70, 133-139 (2002). 103. Cunliffe, W.J. Acne and unemployment. The British journal of dermatology 115, 386 (1986). 104. Gupta, M.A. & Gupta, A.K. Depression and suicidal ideation in dermatology patients with acne, alopecia areata, atopic dermatitis and psoriasis. The British journal of dermatology 139, 846-850 (1998). 105. Cotterill, J.A. & Cunliffe, W.J. Suicide in dermatological patients. British Journal of Dermatology 137, 246-250 (1997). 106. Bataille, V., Snieder, H., MacGregor, A.J., Sasieni, P. & Spector, T.D. The influence of genetics and environmental factors in the pathogenesis of acne: a twin study of acne in women. The Journal of investigative dermatology 119, 1317-1322 (2002). 107. Pappas, A., Johnsen, S., Liu, J.C. & Eisinger, M. Sebum analysis of individuals with and without acne. Dermatoendocrinol 1, 157-161 (2009). 108. Cotterill, J., Cunliffe, W. & Williamson, B. Severity of acne and sebum excretion rate. British Journal of Dermatology 85, 93-94 (1971). 109. Thiboutot, D., Gilliland, K., Light, J. & Lookingbill, D. Androgen metabolism in sebaceous glands from subjects with and without acne [see comments]. Archives of dermatology 135, 1041- 1045 (1999). 110. Aizawa, H. & Niimura, M. Elevated serum insulin-like growth factor-1 (IGF-1) levels in women with postadolescent acne. The Journal of dermatology 22, 249-252 (1995). 111. Thiboutot, D., Knaggs, H., Gilliland, K. & Lin, G. Activity of 5-alpha-reductase and 17-beta- hydroxysteroid dehydrogenase in the infrainfundibulum of subjects with and without acne vulgaris. Dermatology 196, 38-42 (1998). 112. Thiboutot, D., et al. Activity of the type 1 5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin. The Journal of investigative dermatology 105, 209-214 (1995). 113. Leyden, J., et al. A systemic type I 5 alpha-reductase inhibitor is ineffective in the treatment of acne vulgaris. Journal of the American Academy of Dermatology 50, 443-447 (2004). 114. Seiffert, K., Seltmann, H., Fritsch, M. & Zouboulis, C.C. Inhibition of 5alpha-reductase activity in SZ95 sebocytes and HaCaT keratinocytes in vitro. Horm Metab Res 39, 141-148 (2007). 115. Ottaviani, M., et al. Peroxidated squalene induces the production of inflammatory mediators in HaCaT keratinocytes: a possible role in acne vulgaris. The Journal of investigative dermatology 126, 2430-2437 (2006). 116. Kligman, A.M. An overview of acne. The Journal of investigative dermatology 62, pp.268-287 (1974). 117. Norris, J.F. & Cunliffe, W.J. A histological and immunocytochemical study of early acne lesions. The British journal of dermatology 118, 651-659 (1988). 118. Kang, S., et al. Inflammation and extracellular matrix degradation mediated by activated transcription factors nuclear factor-kappaB and activator protein-1 in inflammatory acne lesions in vivo. Am J Pathol 166, 1691-1699 (2005). 119. Hengge, U.R., Ruzicka, T., Schwartz, R.A. & Cork, M.J. Adverse effects of topical glucocorticosteroids. Journal of the American Academy of Dermatology 54, 1-15 (2006). 120. Brook, I. & Frazier, E.H. Infections caused by Propionibacterium species. Rev Infect Dis 13, 819- 822 (1991). 121. Perry, A.L. & Lambert, P.A. Propionibacterium acnes. Lett Appl Microbiol 42, 185-188 (2006).

158 122. Warr, G.W. & James, K. Effect of Corynebacterium parvum on the class and subclass of antibody produced in the response of different strains of mice to sheep erythrocytes. Immunology 28, 431- 442 (1975). 123. Halpern, B.N., Biozzi, G., Stiffel, C. & Mouton, D. Inhibition of tumour growth by administration of killed corynebacterium parvum. Nature 212, 853-854 (1966). 124. Ghaffar, A., Cullen, R.T., Dunbar, N. & Woodruff, M.F. Anti-tumour effect in vitro of lymphocytes and macrophages from mice treated with Corynebacterium parvum. British journal of cancer 29, 199-205 (1974). 125. Woodruff, M.F., McBride, W.H. & Dunbar, N. Tumour growth, phagocytic activity and antibody response in Corynebacterium parvum-treated mice. Clinical and experimental immunology 17, 509-518 (1974). 126. Ananias, R.Z., et al. Modulatory effect of killed Propionibacterium acnes and its purified soluble polysaccharide on peritoneal exudate cells from C57Bl/6 mice: major NKT cell recruitment and increased cytotoxicity. Scand J Immunol 65, 538-548 (2007). 127. Ha, D.K., Lawton, J.W. & Gardner, I.D. The effect of in vivo modulation of macrophage activities on Mycobacterium lepraemurium infection. J Comp Pathol 96, 565-573 (1986). 128. Nussenzweig, R.S. Increased nonspecific resstance to malaria produced by administration of killed Corynebacterium parvum. Exp Parasitol 21, 224-231 (1967). 129. Mussalem, J.S., et al. Adjuvant effect of the Propionibacterium acnes and its purified soluble polysaccharide on the immunization with plasmidial DNA containing a Trypanosoma cruzi gene. Microbiol Immunol 50, 253-263 (2006). 130. Krahenbuhl, J.L., Humphres, R.C. & Henika, P.C. Effects of Propionibacterium acnes treatment on the course of Mycobacterium leprae infection in mice. Infect Immun 37, 183-188 (1982). 131. Brener, Z. & Cardoso, J.E. Nonspecific resistance against Trypanosoma cruzi enhanced by Corynebacterium parvum. J Parasitol 62, 645-646 (1976). 132. Mochizuki, H., Nomura, T., Kawamura, I. & Mitsuyama, M. Enhanced resistance to Gram- positive bacterium and increased susceptibility to bacterial endotoxin in mice sensitized with Propionibacterium acnes: involvement of Toll-like receptor. FEMS Immunol Med Microbiol 43, 287-293 (2005). 133. Green, S., et al. Corynebacterium parvum as the priming agent in the production of tumor necrosis factor in the mouse. J Natl Cancer Inst 59, 1519-1522 (1977). 134. Michalak-Stoma, A., et al. The effect of Propionibacterium acnes on maturation of dendritic cells derived from acne patients' peripherial blood mononuclear cells. Folia Histochemica et Cytobiologica 46, 535-539 (2008). 135. Nakatsuji, T., et al. Bioengineering a humanized acne microenvironment model: proteomics analysis of host responses to Propionibacterium acnes infection in vivo. Proteomics 8, 3406-3415 (2008). 136. Nakatsuji, T., et al. Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris. PloS one 3, e1551 (2008). 137. Squaiella, C.C., et al. In vivo and in vitro effect of killed Propionibacterium acnes and its purified soluble polysaccharide on mouse bone marrow stem cells and dendritic cell differentiation. Immunobiology 211, 105-116 (2006). 138. Squaiella, C.C., et al. Modulation of the type I hypersensitivity late phase reaction to OVA by Propionibacterium acnes-soluble polysaccharide. Immunol Lett 121, 157-166 (2008). 139. Bruggeman, H., et al. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science (New York, N.Y 305, 671-673 (2004). 140. Bruggemann, H. Insights in the pathogenic potential of Propionibacterium acnes from its complete genome. Semin Cutan Med Surg 24, 67-72 (2005). 141. Jugeau, S., et al. Induction of toll-like receptors by Propionibacterium acnes. The British journal of dermatology 153, 1105-1113 (2005).

159 142. Kim, J., et al. Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses. J Immunol 169, 1535-1541 (2002). 143. Graham, G.M., Farrar, M.D., Cruse-Sawyer, J.E., Holland, K.T. & Ingham, E. Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL. The British journal of dermatology 150, 421-428 (2004). 144. Lyte, P., Sur, R., Nigam, A. & Southall, M.D. Heat-killed Propionibacterium acnes is capable of inducing inflammatory responses in skin. Exp Dermatol 18, 1070-1072 (2009). 145. Nagy, I., et al. Distinct strains of Propionibacterium acnes induce selective human beta-defensin- 2 and interleukin-8 expression in human keratinocytes through toll-like receptors. The Journal of investigative dermatology 124, 931-938 (2005). 146. Lumsden, K.R., et al. Isotretinoin increases skin-surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne. The British journal of dermatology 165, 302-310 (2011). 147. Jappe, U., Ingham, E., Henwood, J. & Holland, K.T. Propionibacterium acnes and inflammation in acne; P. acnes has T-cell mitogenic activity. The British journal of dermatology 146, 202-209 (2002). 148. Leyden, J.J. The evolving role of Propionibacterium acnes in acne. Semin Cutan Med Surg 20, 139-143 (2001). 149. Higaki, S., Kitagawa, T., Kagoura, M., Morohashi, M. & Yamagishi, T. Correlation between Propionibacterium acnes biotypes, lipase activity and rash degree in acne patients. The Journal of dermatology 27, 519-522 (2000). 150. Jappe, U., et al. Evidence for diversity within Propionibacterium acnes: a comparison of the T- cell stimulatory activity of isolates from inflammatory acne, endocarditis and the laboratory. J Eur Acad Dermatol Venereol 18, 450-454 (2004). 151. Leyden, J.J. Propionibacterium levels in patients with and without acne vulgaris. The Journal of investigative dermatology 65, 382 (1975). 152. Brook, I. Pathogenicity of Propionibacterium acnes in mixed infections with facultative bacteria. J Med Microbiol 34, 249-252 (1991). 153. Gowland, G., Ward, R.M., Holland, K.T. & Cunliffe, W.J. Cellular immunity to P. acnes in the normal population and patients with acne vulgaris. The British journal of dermatology 99, 43-47 (1978). 154. Sugisaki, H., et al. Increased interferon-gamma, interleukin-12p40 and IL-8 production in Propionibacterium acnes-treated peripheral blood mononuclear cells from patient with acne vulgaris: host response but not bacterial species is the determinant factor of the disease. J Dermatol Sci 55, 47-52 (2009). 155. Puhvel, S.M. Study of antibody levels to Corynebacterium acnes. Archives of dermatology 99, 421 (1964). 156. Puhvel, S.M., Warnick, M.A. & Sternberg, T.H. Levels of antibody to Staphylococcus epidermidis in patients with acne vulgaris. Archives of dermatology 92, 88-90 (1965). 157. Tian, L.M., et al. Association Study of Tumor Necrosis Factor Receptor Type 2 M196R and Toll- Like Receptor 2 Arg753Gln Polymorphisms with Acne Vulgaris in a Chinese Han Ethnic Group. Dermatology (2010). 158. Koreck, A., et al. TLR2 and TLR4 polymorphisms are not associated with acne vulgaris. Dermatology 213, 267-269 (2006). 159. Inui, S., Nakao, T. & Itami, S. Modulation of androgen receptor transcriptional activity by anti- acne reagents. J Dermatol Sci 36, 97-101 (2004). 160. Cooper, A.J. Systematic review of Propionibacterium acnes resistance to systemic antibiotics. Med J Aust 169, 259-261 (1998). 161. Strauss, J.S. & Klingman, A.M. Effect of cyclic progestin-estrogen therapy on sebum and acne in women. JAMA 190, 815 (1964).

160 162. Goodfellow, A., et al. Oral spironolactone improves acne vulgaris and reduces sebum excretion. The British journal of dermatology 111, 209-214 (1984). 163. Peck, G.L. & Yoder, F.W. Treatment of lamellar ichthyosis and other keratinising dermatoses with an oral synthetic retinoid. Lancet 2, 1172-1174 (1976). 164. Peck, G.L. Prolonged remissions of cystic acne with 13-cis retinoic acid. The New England journal of medicine 300, 329 (1979). 165. Garattini, E., Gianni, M. & Terao, M. Retinoid related molecules an emerging class of apoptotic agents with promising therapeutic potential in oncology: pharmacological activity and mechanisms of action. Curr Pharm Des 10, 433-448 (2004). 166. Liu, A., Yang, D.J., Gerhardstein, P.C. & Hsu, S. Relapse of acne following isotretinoin treatment: a retrospective study of 405 patients. J Drugs Dermatol 7, 963-966 (2008). 167. Azoulay, L., Oraichi, D. & Berard, A. Isotretinoin therapy and the incidence of acne relapse: a nested case-control study. The British journal of dermatology 157, 1240-1248 (2007). 168. Lee, J.W., et al. Effectiveness of Conventional, Low-dose and Intermittent Oral Isotretinoin in the Treatment of Acne: A Randomized, Controlled Comparative study. The British journal of dermatology (2010). 169. Zane, L.T., Leyden, W.A., Marqueling, A.L. & Manos, M.M. A population-based analysis of laboratory abnormalities during isotretinoin therapy for acne vulgaris. Archives of dermatology 142, 1016-1022 (2006). 170. FDA orders new Accutane warning label. Dermatology World 8, 6 (1998). 171. Ahuja, D., Saenz-Robles, M.T. & Pipas, J.M. SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 24, 7729-7745 (2005). 172. Zouboulis, C.C., Seltmann, H., Neitzel, H. & Orfanos, C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). The Journal of investigative dermatology 113, 1011-1020 (1999). 173. Webster, G.F., Ruggieri, M.R. & McGinley, K.J. Correlation of Propionibacterium acnes populations with the presence of triglycerides on nonhuman skin. Appl Environ Microbiol 41, 1269-1270 (1981). 174. Xie, J., et al. Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391, 90- 92 (1998). 175. Fraser, F.C. The effect of oral administration of vitamin A on the expression of the recessive gene rhino in the mouse. Anat Rec 97, 415 (1947). 176. De Young, L.M., Young, J.M., Ballaron, S.J., Spires, D.A. & Puhvel, S.M. Intradermal injection of Propionibacterium acnes: a model of inflammation relevant to acne. The Journal of investigative dermatology 83, 394-398 (1984). 177. De Young, L.M., et al. Acne-like chronic inflammatory activity of Propionibacterium acnes preparations in an animal model: correlation with ability to stimulate the reticuloendothelial system. The Journal of investigative dermatology 85, 255-258 (1985). 178. Simon, G.A. & Maibach, H.I. Relevance of hairless mouse as an experimental model of percutaneous penetration in man. Skin Pharmacol Appl Skin Physiol 11, 80-86 (1998). 179. Serup, J. Formation of oiliness and sebum output--comparison of a lipid-absorbant and occlusive- tape method with photometry. Clin Exp Dermatol 16, 258-263 (1991). 180. Saint-Leger, D. & Cohen, E. Practical study of qualitative and quantitative sebum excretion on the human forehead. The British journal of dermatology 113, 551-557 (1985). 181. Dietary Supplement Factsheet: Vitamin A and Carotenoids. (Office of Dietary Supplements, National Institutes of Health, 2006). 182. Sommer, A., Tarwotjo, I., Hussaini, G. & Susanto, D. Increased mortality in children with mild vitamin A deficiency. Lancet 2, 585-588 (1983). 183. Kark, J.D., Smith, A.H., Switzer, B.R. & Hames, C.G. Serum vitamin A (retinol) and cancer incidence in Evans County, Georgia. J Natl Cancer Inst 66, 7-16 (1981).

161 184. Shekelle, R.B., et al. Dietary vitamin A and risk of cancer in the Western Electric study. Lancet 2, 1185-1190 (1981). 185. Fiorella, P.D. & Napoli, J.L. Microsomal retinoic acid metabolism. Effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step. The Journal of biological chemistry 269, 10538-10544 (1994). 186. Napoli, J.L. Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin Immunol Immunopathol 80, S52-62 (1996). 187. Takase, S., Ong, D.E. & Chytil, F. Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus. Arch Biochem Biophys 247, 328-334 (1986). 188. Tzimas, G. & Nau, H. The role of metabolism and toxicokinetics in retinoid teratogenesis. Curr Pharm Des 7, 803-831 (2001). 189. White, J.A., et al. Identification of the human cytochrome P450, P450RAI-2, which is predominantly expressed in the adult cerebellum and is responsible for all-trans-retinoic acid metabolism. Proceedings of the National Academy of Sciences of the United States of America 97, 6403-6408 (2000). 190. Nau, H. Teratogenicity of isotretinoin revisited: species variation and the role of all-trans-retinoic acid. Journal of the American Academy of Dermatology 45, S183-187 (2001). 191. de Lera, A.R., Bourguet, W., Altucci, L. & Gronemeyer, H. Design of selective nuclear receptor modulators: RAR and RXR as a case study. Nat Rev Drug Discov 6, 811-820 (2007). 192. Germain, P., et al. International Union of Pharmacology. LXIII. Retinoid X receptors. Pharmacological reviews 58, 760-772 (2006). 193. Schug, T.T., Berry, D.C., Shaw, N.S., Travis, S.N. & Noy, N. Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell 129, 723- 733 (2007). 194. Wiesenberg, I., Missbach, M., Kahlen, J.P., Schrader, M. & Carlberg, C. Transcriptional activation of the nuclear receptor RZR alpha by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand. Nucleic Acids Res 23, 327-333 (1995). 195. Stehlin-Gaon, C., et al. All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR beta. Nat Struct Biol 10, 820-825 (2003). 196. Kallen, J.A., et al. X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Structure 10, 1697-1707 (2002). 197. Bastien, J. & Rochette-Egly, C. Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 328, 1-16 (2004). 198. Eckhoff, C. & Nau, H. Identification and quantitation of all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma. J Lipid Res 31, 1445-1454 (1990). 199. Eckhoff, C. & Nau, H. Vitamin A supplementation increases levels of retinoic acid compounds in human plasma: possible implications for teratogenesis. Arch Toxicol 64, 502-503 (1990). 200. Boehm, N., Samama, B., Cribier, B. & Rochette-Egly, C. Retinoic-acid receptor beta expression in melanocytes. Eur J Dermatol 14, 19-23 (2004). 201. Chakravarti, N., et al. Expression of retinoid receptors in sebaceous cell carcinoma. Journal of cutaneous pathology 33, 10-17 (2006). 202. Roos, T.C., Jugert, F.K., Merk, H.F. & Bickers, D.R. Retinoid metabolism in the skin. Pharmacological reviews 50, 315-333 (1998). 203. Elder, J.T., et al. Retinoic acid receptors and binding proteins in human skin. The Journal of investigative dermatology 98, 36S-41S (1992). 204. Straumfjord, J.V. Vitamin A: Its Effects on Acne. Northwest Med 42, 219-225 (1943). 205. Anderson, J.A.D., and Stokoe, I.H. Vitamin A in Acne Vulgaris. Brit Med J 2, 294-296 (1963). 206. Lynch, F.W. & Cook, C.D. Acne vulgaris treated with vitamin A. Arch Dermatol Syph 55, 355- 357 (1947).

162 207. Harms, M., Philippe, I., Ceyrac, D. & Saurat, J.H. Isotretinoin ineffective topically. Lancet 1, 398 (1985). 208. Ellis, C.N., et al. Comparison of adapalene 0.1% solution and tretinoin 0.025% gel in the topical treatment of acne vulgaris. British Journal of Dermatology 139, 41-47 (1998). 209. Dunlap, F.E., Baker, M.D., Plott, R.T. & Verschoore, M. Adapalene 0.1% gel has low skin irritation potential even when applied immediately after washing. British Journal of Dermatology 139, 23-25 (1998). 210. Rigas, J.R., et al. Constitutive variability in the pharmacokinetics of the natural retinoid, all-trans- retinoic acid, and its modulation by ketoconazole. J Natl Cancer Inst 85, 1921-1926 (1993). 211. Muindi, J.R., et al. Clinical pharmacology of oral all-trans retinoic acid in patients with acute promyelocytic leukemia. Cancer research 52, 2138-2142 (1992). 212. Muindi, J., et al. Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia. Blood 79, 299-303 (1992). 213. Muindi, J.F., Scher, H.I., Rigas, J.R., Warrell, R.P., Jr. & Young, C.W. Elevated plasma lipid peroxide content correlates with rapid plasma clearance of all-trans-retinoic acid in patients with advanced cancer. Cancer research 54, 2125-2128 (1994). 214. Reynolds, C.P., Matthay, K.K., Villablanca, J.G. & Maurer, B.J. Retinoid therapy of high-risk neuroblastoma. Cancer Lett 197, 185-192 (2003). 215. Tsukada, M., et al. 13-cis retinoic acid exerts its specific activity on human sebocytes through selective intracellular isomerization to all-trans retinoic acid and binding to retinoid acid receptors. The Journal of investigative dermatology 115, 321-327 (2000). 216. Astrom, A., et al. Molecular cloning of two human cellular retinoic acid-binding proteins (CRABP). Retinoic acid-induced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro. The Journal of biological chemistry 266, 17662-17666 (1991). 217. Elder, J.T., Cromie, M.A., Griffiths, C.E., Chambon, P. & Voorhees, J.J. Stimulus-selective induction of CRABP-II mRNA: a marker for retinoic acid action in human skin. The Journal of investigative dermatology 100, 356-359 (1993). 218. Griffiths, C.E., et al. Comparison of CD271 (adapalene) and all-trans retinoic acid in human skin: dissociation of epidermal effects and CRABP-II mRNA expression. The Journal of investigative dermatology 101, 325-328 (1993). 219. Hirschel-Scholz, S., Siegenthaler, G. & Saurat, J.H. Isotretinoin differs from other synthetic retinoids in its modulation of human cellular retinoic acid binding protein (CRABP). The British journal of dermatology 120, 639-644 (1989). 220. Hirschel-Scholz, S., Siegenthaler, G. & Saurat, J.H. Ligand-specific and non-specific in vivo modulation of human epidermal cellular retinoic acid binding protein (CRABP). Eur J Clin Invest 19, 220-227 (1989). 221. Rollman, O. & Vahlquist, A. Oral isotretinoin (13-cis-retinoic acid) therapy in severe acne: drug and vitamin A concentrations in serum and skin. The Journal of investigative dermatology 86, 384-389 (1986). 222. Sitzmann, J.H., Bauer, F.W., Cunliffe, W.J., Holland, D.B. & Lemotte, P.K. In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients. The British journal of dermatology 133, 241-248 (1995). 223. Saurer, L., McCullough, K.C. & Summerfield, A. In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid. J Immunol 179, 3504-3514 (2007). 224. Mora, J.R., Iwata, M. & von Andrian, U.H. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 8, 685-698 (2008).

163 225. Iwata, M., Eshima, Y. & Kagechika, H. Retinoic acids exert direct effects on T cells to suppress Th1 development and enhance Th2 development via retinoic acid receptors. Int Immunol 15, 1017-1025 (2003). 226. Dennert, G. & Lotan, R. Effects of retinoic acid on the immune system: stimulation of T killer cell induction. European journal of immunology 8, 23-29 (1978). 227. Fox, F.E., et al. Retinoids synergize with interleukin-2 to augment IFN-gamma and interleukin- 12 production by human peripheral blood mononuclear cells. J Interferon Cytokine Res 19, 407- 415 (1999). 228. Mucida, D., et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science (New York, N.Y 317, 256-260 (2007). 229. Elias, K.M., et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Vol. 111 1013-1020 (2008). 230. Xiao, S., et al. Retinoic Acid Increases Foxp3+ Regulatory T Cells and Inhibits Development of Th17 Cells by Enhancing TGF-{beta}-Driven Smad3 Signaling and Inhibiting IL-6 and IL-23 Receptor Expression. Vol. 181 2277-2284 (2008). 231. Klemann, C., et al. Synthetic retinoid AM80 inhibits Th17 cells and ameliorates experimental autoimmune encephalomyelitis. Am J Pathol 174, 2234-2245 (2009). 232. Ivanov, II, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121-1133 (2006). 233. Szeles, L., et al. Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells. Mol Endocrinol 24, 2218-2231 (2010). 234. Geissmann, F., et al. Retinoids regulate survival and antigen presentation by immature dendritic cells. The Journal of experimental medicine 198, 623-634 (2003). 235. Wang, X., Allen, C. & Ballow, M. Retinoic acid enhances the production of IL-10 while reducing the synthesis of IL-12 and TNF-alpha from LPS-stimulated monocytes/macrophages. Journal of clinical immunology 27, 193-200 (2007). 236. Tsai, Y.C., et al. Effects of all-trans retinoic acid on Th1- and Th2-related chemokines production in monocytes. Inflammation 31, 428-433 (2008). 237. Liu, P.T., Krutzik, S.R., Kim, J. & Modlin, R.L. Cutting edge: all-trans retinoic acid down- regulates TLR2 expression and function. J Immunol 174, 2467-2470 (2005). 238. Zhang, Y., et al. Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4(+)CD25(+) regulatory T cells in chronic hepatitis B virus infection. Virology 397, 34-42 (2010). 239. Kang, B.Y., et al. Retinoid-mediated inhibition of interleukin-12 production in mouse macrophages suppresses Th1 cytokine profile in CD4(+) T cells. Br J Pharmacol 130, 581-586 (2000). 240. Adelman, D.C., Yen, T.Y., Cumberland, W.G., Sidell, N. & Saxon, A. 13-cis retinoic acid enhances in vivo B-lymphocyte differentiation in patients with common variable immunodeficiency. The Journal of allergy and clinical immunology 88, 705-712 (1991). 241. Wiedermann, U., Hanson, L.A., Holmgren, J., Kahu, H. & Dahlgren, U.I. Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine. Infect Immun 61, 3952-3957 (1993). 242. Wiedermann, U., Hanson, L.A., Kahu, H. & Dahlgren, U.I. Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats. Immunology 80, 581-586 (1993). 243. McDermott, M.R., et al. Impaired intestinal localization of mesenteric lymphoblasts associated with vitamin A deficiency and protein-calorie malnutrition. Immunology 45, 1-5 (1982).

164 244. Bjersing, J.L., Telemo, E., Dahlgren, U. & Hanson, L.A. Loss of ileal IgA+ plasma cells and of CD4+ lymphocytes in ileal Peyer's patches of vitamin A deficient rats. Clinical and experimental immunology 130, 404-408 (2002). 245. Yamanaka, K., et al. Vitamins A and D are potent inhibitors of cutaneous lymphocyte-associated antigen expression. The Journal of allergy and clinical immunology 121, 148-157 e143 (2008). 246. Moore, C., et al. Transforming growth factor-beta and all-trans retinoic acid generate ex vivo transgenic regulatory T cells with intestinal homing receptors. Transplant Proc 41, 2670-2672 (2009). 247. Kang, S.G., Wang, C., Matsumoto, S. & Kim, C.H. High and low vitamin A therapies induce distinct FoxP3+ T-cell subsets and effectively control intestinal inflammation. Gastroenterology 137, 1391-1402 e1391-1396 (2009). 248. Iwata, M., et al. Retinoic acid imprints gut-homing specificity on T cells. Immunity 21, 527-538 (2004). 249. Benson, M.J., Pino-Lagos, K., Rosemblatt, M. & Noelle, R.J. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co- stimulation. The Journal of experimental medicine 204, 1765-1774 (2007). 250. Coombes, J.L., et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. The Journal of experimental medicine 204, 1757-1764 (2007). 251. Sun, C.M., et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. The Journal of experimental medicine 204, 1775-1785 (2007). 252. Guilliams, M., et al. Skin-draining lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells. Blood 115, 1958-1968 (2010). 253. Edele, F., et al. Cutting edge: instructive role of peripheral tissue cells in the imprinting of T cell homing receptor patterns. J Immunol 181, 3745-3749 (2008). 254. Molenaar, R., et al. Expression of Retinaldehyde Dehydrogenase Enzymes in Mucosal Dendritic Cells and Gut-Draining Lymph Node Stromal Cells Is Controlled by Dietary Vitamin A. J Immunol. 255. Bai, A., et al. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. Journal of leukocyte biology 86, 959- 969 (2009). 256. Crockett, S.D., Gulati, A., Sandler, R.S. & Kappelman, M.D. A causal association between isotretinoin and inflammatory bowel disease has yet to be established. Am J Gastroenterol 104, 2387-2393 (2009). 257. Reddy, D., Siegel, C.A., Sands, B.E. & Kane, S. Possible association between isotretinoin and inflammatory bowel disease. Am J Gastroenterol 101, 1569-1573 (2006). 258. Borobio, E., et al. [Isotretinoin and ulcerous colitis]. An Sist Sanit Navar 27, 241-243 (2004). 259. Reniers, D.E. & Howard, J.M. Isotretinoin-induced inflammatory bowel disease in an adolescent. Ann Pharmacother 35, 1214-1216 (2001). 260. Crockett, S.D., Porter, C.Q., Martin, C.F., Sandler, R.S. & Kappelman, M.D. Isotretinoin use and the risk of inflammatory bowel disease: a case-control study. Am J Gastroenterol 105, 1986-1993 (2010). 261. Bernstein, C.N., Nugent, Z., Longobardi, T. & Blanchard, J.F. Isotretinoin is not associated with inflammatory bowel disease: a population-based case-control study. Am J Gastroenterol 104, 2774-2778 (2009). 262. Obermeier, F., Hofmann, C. & Falk, W. Inflammatory bowel diseases: when natural friends turn into enemies-the importance of CpG motifs of bacterial DNA in intestinal homeostasis and chronic intestinal inflammation. Int J Inflam 2010, 641910 (2010).

165 263. Roselli, M., et al. Prevention of TNBS-induced colitis by different Lactobacillus and Bifidobacterium strains is associated with an expansion of gammadeltaT and regulatory T cells of intestinal intraepithelial lymphocytes. Inflamm Bowel Dis 15, 1526-1536 (2009). 264. Margolis, D.J., Fanelli, M., Hoffstad, O. & Lewis, J.D. Potential association between the oral tetracycline class of antimicrobials used to treat acne and inflammatory bowel disease. Am J Gastroenterol 105, 2610-2616 (2010). 265. Morizane, S., Yamasaki, K., Kabigting, F.D. & Gallo, R.L. Kallikrein expression and cathelicidin processing are independently controlled in keratinocytes by calcium, vitamin D(3), and retinoic acid. The Journal of investigative dermatology 130, 1297-1306 (2010). 266. Wu, H., Zhang, G., Minton, J.E., Ross, C.R. & Blecha, F. Regulation of cathelicidin gene expression: induction by lipopolysaccharide, interleukin-6, retinoic acid, and Salmonella enterica serovar typhimurium infection. Infect Immun 68, 5552-5558 (2000). 267. Caramuta, S., et al. Regulation of lipocalin-2 gene by the cancer chemopreventive retinoid 4- HPR. Int J Cancer 119, 1599-1606 (2006). 268. Nagpal, S., et al. Negative regulation of two hyperproliferative keratinocyte differentiation markers by a retinoic acid receptor-specific retinoid: insight into the mechanism of retinoid action in psoriasis. Cell Growth Differ 7, 1783-1791 (1996). 269. Zelickson, A.S., Strauss, J.S. & Mottaz, J. Ultrastructural changes in open comedones following treatment of cystic acne with isotretinoin. Am J Dermatopathol 7, 241-244 (1985). 270. Landthaler, M., Kummermehr, J., Wagner, A. & Plewig, G. Inhibitory effects of 13-cis reinoic acid on human sebaceous glands. Archives of dermatology 269, 297-3069 (1980). 271. Hommel, L., Geiger, J.M., Harms, M. & Saurat, J.H. Sebum excretion rate in subjects treated with oral all-trans-retinoic acid. Dermatology 193, 127-130 (1996). 272. Zelickson, A.S., Strauss, J.S. & Mottaz, J. Ultrastructural changes in sebaceous glands following treatment of cystic acne with isotretinoin. Am J Dermatopathol 8, 139-143 (1986). 273. Nelson, A.M., Cong, Z., Gilliland, K.L. & Thiboutot, D.M. TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells. (2011). 274. Strauss, J.S. & Stranieri, A.M. Changes in long-term sebum production from isotretinoin therapy. Journal of the American Academy of Dermatology 6, 751 (1982). 275. Layton, A.M., Knaggs, H., Taylor, J. & Cunliffe, W.J. Isotretinoin for acne vulgaris--10 years later: a safe and successful treatment. The British journal of dermatology 129, 292-296 (1993). 276. Tenaud, I., Khammari, A. & Dreno, B. In vitro modulation of TLR-2, CD1d and IL-10 by adapalene on normal human skin and acne inflammatory lesions. Exp Dermatol 16, 500-506 (2007). 277. Rollman, O. & Vahlquist, A. Vitamin A in skin and serum--studies of acne vulgaris, atopic dermatitis, ichthyosis vulgaris and lichen planus. The British journal of dermatology 113, 405- 413 (1985). 278. Mier, P.D. & van den Hurk, J.J. Plasma vitamin A levels in the common dermatoses. The British journal of dermatology 91, 155-159 (1974). 279. Muindi, J.R., Young, C.W. & Warrell, R.P., Jr. Clinical pharmacology of all-trans retinoic acid. Leukemia 8, 1807-1812 (1994). 280. Wang, F., et al. Retinoic acid 4-hydroxylase inducibility and clinical response to isotretinoin in patients with acne. Journal of the American Academy of Dermatology 61, 252-258 (2009). 281. Michaelsson, G., Vahlquist, A. & Juhlin, L. Serum zinc and retinol-binding protein in acne. The British journal of dermatology 96, 283-286 (1977). 282. Phillips, W.E., Murray, T.K. & Campbell, J.S. Serum vitamin A and carotenoids of Canadians. Can Med Assoc J 102, 1085-1086 (1970). 283. Tang, G.W. & Russell, R.M. 13-cis-retinoic acid is an endogenous compound in human serum. J Lipid Res 31, 175-182 (1990).

166 284. Chen, K., White, T.J., Juzba, M. & Chang, E. Oral isotretinoin: an analysis of its utilization in a managed care organization. J Manag Care Pharm 8, 272-277 (2002). 285. Abdel-Hamid, M.F., Aly, D.G., Saad, N.E., Emam, H.M. & Ayoub, D.F. Serum levels of interleukin-8, tumor necrosis factor-alpha and gamma-interferon in Egyptian psoriatic patients and correlation with disease severity. The Journal of dermatology (2010). 286. Nelson, A.M., Gilliland, K.L., Cong, Z. & Thiboutot, D.M. 13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes. The Journal of investigative dermatology 126, 2178-2189 (2006). 287. Motomura, K., Ohata, M., Satre, M. & Tsukamoto, H. Destabilization of TNF-alpha mRNA by retinoic acid in hepatic macrophages: implications for alcoholic liver disease. American journal of physiology 281, E420-429 (2001). 288. Na, S.Y., et al. Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NFkappaB. The Journal of biological chemistry 274, 7674-7680 (1999). 289. Caillon, F., et al. Interleukin-10 secretion from CD14+ peripheral blood mononuclear cells is downregulated in patients with acne vulgaris. The British journal of dermatology 162, 296-303 (2009). 290. Wolk, K., et al. Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa. J Immunol 186, 1228-1239 (2011). 291. van der Zee, H.H., et al. Elevated levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)- 1beta and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-alpha and IL- 1beta. The British journal of dermatology 164, 1292-1298 (2011). 292. Wang, J., Huizinga, T.W. & Toes, R.E. De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol 183, 4119-4126 (2009). 293. Yokokawa, J., et al. Enhanced functionality of CD4+CD25(high)FoxP3+ regulatory T cells in the peripheral blood of patients with prostate cancer. Clin Cancer Res 14, 1032-1040 (2008). 294. Motta, M., et al. Increased expression of CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell chronic lymphocytic leukemia. Leukemia 19, 1788-1793 (2005). 295. Woo, E.Y., et al. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer research 61, 4766-4772 (2001). 296. Sasada, T., Kimura, M., Yoshida, Y., Kanai, M. & Takabayashi, A. CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression. Cancer 98, 1089-1099 (2003). 297. Curiel, T.J., et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nature medicine 10, 942-949 (2004). 298. Liyanage, U.K., et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169, 2756- 2761 (2002). 299. Gunaydin, R.O., Kesikli, S.A., Kansu, E. & Hosal, A.S. Identification of the peripheral blood levels of interleukin-12, interleukin-10, and transforming growth factor-beta in patients with laryngeal squamous cell carcinoma. Head & neck ([published online ahead of print April 5, 2011]). 300. Aravalli, R.N., Hu, S., Rowen, T.N., Palmquist, J.M. & Lokensgard, J.R. Cutting edge: TLR2- mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus. J Immunol 175, 4189-4193 (2005). 301. Kurt-Jones, E.A., et al. Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis. Proceedings of the National Academy of Sciences of the United States of America 101, 1315-1320 (2004). 302. Wang, J.P., et al. Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2. Journal of virology 79, 12658-12666 (2005).

167 303. Prescott, S.L., et al. Presymptomatic differences in Toll-like receptor function in infants who have allergy. The Journal of allergy and clinical immunology 122, 391-399, 399 e391-395 (2008). 304. Jones, B.M., Nicholson, J.K.A., Holman, R.C. & Hubbard, M. Comparison of monocyte separation methods using flow cytometric analysis. J of Immun Meth 125, 41-47 (1989). 305. Kane, M.A., Chen, N., Sparks, S. & Napoli, J.L. Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS. Biochem J 388, 363-369 (2005). 306. Qu, J., Qu, Y. & Straubinger, R.M. Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry. Analytical chemistry 79, 3786-3793 (2007). 307. Shi, J. & Wei, L. Rho kinase in the regulation of cell death and survival. Archivum immunologiae et therapiae experimentalis 55, 61-75 (2007). 308. Narumiya, S., Ishizaki, T. & Uehata, M. Use and properties of ROCK-specific inhibitor Y-27632. Methods in enzymology 325, 273-284 (2000). 309. Ishizaki, T., et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Molecular pharmacology 57, 976-983 (2000). 310. Chapman, S., Liu, X., Meyers, C., Schlegel, R. & McBride, A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. The Journal of clinical investigation 120, 2619-2626 (2010). 311. Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature biotechnology 25, 681-686 (2007). 312. Terunuma, A., Limgala, R.P., Park, C.J., Choudhary, I. & Vogel, J.C. Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632. Tissue engineering 16, 1363-1368 (2010). 313. Choudhry, R., Hodgins, M.B., Van der Kwast, T.H., Brinkmann, A.O. & Boersma, W.J. Localization of androgen receptors in human skin by immunohistochemistry: implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands. J Endocrinol 133, 467- 475 (1992). 314. Rossol-Allison, J., et al. Rho GTPase activity modulates Wnt3a/beta-catenin signaling. Cellular signalling 21, 1559-1568 (2009). 315. Horsley, V., et al. Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell 126, 597-609 (2006). 316. Micalizzi, D.S., Farabaugh, S.M. & Ford, H.L. Epithelial-mesenchymal transition in cancer: parallels between normal development and tumor progression. Journal of mammary gland biology and neoplasia 15, 117-134 (2010). 317. Grunert, S., Jechlinger, M. & Beug, H. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol 4, 657-665 (2003). 318. Krawetz, R. & Kelly, G.M. Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm. Cellular signalling 20, 506-517 (2008). 319. Masson-Gadais, B., et al. The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3. Journal of cellular physiology 206, 831-842 (2006). 320. Balasubramanian, S., Jasty, S., Sitalakshmi, G., Madhavan, H.N. & Krishnakumar, S. Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells. The Indian journal of medical research 128, 616-622 (2008). 321. Khoo, A.L., et al. 1,25-dihydroxyvitamin D3 modulates cytokine production induced by Candida albicans: impact of seasonal variation of immune responses. J Infect Dis 203, 122-130 (2011). 322. Cunliffe, W.J. & Norris, J.F. Isotretinoin--an explanation for its long-term benefit. Dermatologica 175 Suppl 1, 133-137 (1987).

168 323. Kerr, I.G., Lippman, M.E., Jenkins, J. & Myers, C.E. Pharmacology of 13-cis-retinoic acid in humans. Cancer research 42, 2069-2073 (1982). 324. Myles, A. & Aggarwal, A. Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis-related arthritis subtype of juvenile idiopathic arthritis. Rheumatology (Oxford, England) 50, 481-488 (2011). 325. Hurtado-Nedelec, M., et al. Characterization of the immune response in the synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome. Rheumatology (Oxford, England) 47, 1160- 1167 (2008). 326. Govoni, M., Colina, M., Massara, A. & Trotta, F. "SAPHO syndrome and infections". Autoimmunity reviews 8, 256-259 (2009). 327. Assmann, G., et al. Efficacy of antibiotic therapy for SAPHO syndrome is lost after its discontinuation: an interventional study. Arthritis research & therapy 11, R140 (2009). 328. Galadari, H., et al. Synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome treated with a combination of isotretinoin and pamidronate. Journal of the American Academy of Dermatology 61, 123-125 (2009). 329. Kjeldsen, L., Johnsen, A.H., Sengelov, H. & Borregaard, N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. The Journal of biological chemistry 268, 10425-10432 (1993). 330. Kjeldsen, L., Bainton, D.F., Sengelov, H. & Borregaard, N. Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils. Blood 83, 799-807 (1994). 331. Triebel, S., Blaser, J., Reinke, H. & Tschesche, H. A 25 kDa alpha 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase. FEBS Lett 314, 386-388 (1992). 332. Stoesz, S.P., et al. Heterogeneous expression of the lipocalin NGAL in primary breast cancers. Int J Cancer 79, 565-572 (1998). 333. Lim, R., et al. Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer: NGAL is associated with epidermal growth factor-induced epithelio- mesenchymal transition. Int J Cancer 120, 2426-2434 (2007). 334. Gwira, J.A., et al. Expression of Neutrophil Gelatinase-associated Lipocalin Regulates Epithelial Morphogenesis in Vitro. Vol. 280 7875-7882 (2005). 335. Devarajan, P. Neutrophil gelatinase-associated lipocalin: new paths for an old shuttle. Cancer therapy 5, 463-470 (2007). 336. Fang, W.K., et al. A novel alternative spliced variant of neutrophil gelatinase-associated lipocalin receptor in oesophageal carcinoma cells. Biochem J 403, 297-303 (2007). 337. Zhang, J., et al. The Role of Lipocalin 2 in the Regulation of Inflammation in Adipocytes and Macrophages. Vol. 22 1416-1426 (2008). 338. Alpizar-Alpizar, W., et al. Neutrophil gelatinase-associated lipocalin (NGAL/Lcn2) is upregulated in gastric mucosa infected with Helicobacter pylori. Virchows Arch 455, 225-233 (2009). 339. Syrjanen, S., et al. Up-regulation of lipocalin 2 is associated with high-risk human papillomavirus and grade of cervical lesion at baseline but does not predict outcomes of infections or incident cervical intraepithelial neoplasia. Am J Clin Pathol 134, 50-59 (2010). 340. Manfredi, M.A., et al. Increased incidence of urinary matrix metalloproteinases as predictors of disease in pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 14, 1091-1096 (2008). 341. Nielsen, B.S., et al. Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 38, 414-420 (1996). 342. Miethke, M. & Skerra, A. Neutrophil gelatinase-associated lipocalin expresses antimicrobial activity by interfering with L-norepinephrine-mediated bacterial iron acquisition. Antimicrob Agents Chemother 54, 1580-1589 (2010).

169 343. Bachman, M.A., Miller, V.L. & Weiser, J.N. Mucosal lipocalin 2 has pro-inflammatory and iron- sequestering effects in response to bacterial enterobactin. PLoS Pathog 5, e1000622 (2009). 344. Ding, H., et al. Urinary neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for renal tubulointerstitial injury in IgA nephropathy. Clinical immunology (Orlando, Fla 123, 227-234 (2007). 345. Suzuki, M., et al. Neutrophil gelatinase-associated lipocalin as a biomarker of disease activity in pediatric lupus nephritis. Pediatr Nephrol 23, 403-412 (2008). 346. Mishra, J., et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14, 2534-2543 (2003). 347. Bolignano, D., et al. Neutrophil gelatinase-associated lipocalin in patients with autosomal- dominant polycystic kidney disease. Am J Nephrol 27, 373-378 (2007). 348. Barresi, V., Tuccari, G. & Barresi, G. NGAL immunohistochemical expression in brain primary and metastatic tumors. Clin Neuropathol 29, 317-322. 349. Barresi, V., et al. Neutrophil gelatinase-associated lipocalin immunoexpression in renal tumors: correlation with histotype and histological grade. Oncol Rep 24, 305-310. 350. Tong, Z., et al. Neutrophil gelatinase-associated lipocalin: a novel suppressor of invasion and angiogenesis in pancreatic cancer. Cancer research 68, 6100-6108 (2008). 351. Wang, H.J., et al. Expressions of neutrophil gelatinase-associated lipocalin in gastric cancer: a potential biomarker for prognosis and an ancillary diagnostic test. Anat Rec (Hoboken) 293, 1855-1863. 352. Iannetti, A., et al. The neutrophil gelatinase-associated lipocalin (NGAL), a NF-kappaB-regulated gene, is a survival factor for thyroid neoplastic cells. Proceedings of the National Academy of Sciences of the United States of America 105, 14058-14063 (2008). 353. Hanai, J., et al. Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells. The Journal of biological chemistry 280, 13641-13647 (2005). 354. Lee, H.J., et al. Ectopic expression of neutrophil gelatinase-associated lipocalin suppresses the invasion and liver metastasis of colon cancer cells. Int J Cancer 118, 2490-2497 (2006). 355. Venkatesha, S., Hanai, J., Seth, P., Karumanchi, S.A. & Sukhatme, V.P. Lipocalin 2 antagonizes the proangiogenic action of ras in transformed cells. Mol Cancer Res 4, 821-829 (2006). 356. Tong, Z., et al. Neutrophil gelatinase-associated lipocalin as a survival factor. Biochem J 391, 441-448 (2005). 357. Coles, M., et al. The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin. J Mol Biol 289, 139-157 (1999). 358. Rudd, P.M., et al. Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin. Biochemistry 38, 13937-13950 (1999). 359. Elliott, S., et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nature biotechnology 21, 414-421 (2003). 360. Bernard, B.A., Yamada, K.M. & Olden, K. Carbohydrates selectively protect a specific domain of fibronectin against proteases. The Journal of biological chemistry 257, 8549-8554 (1982). 361. Kodama, S., et al. Role of sugar chains in the in-vitro activity of recombinant human interleukin 5. Eur J Biochem 211, 903-908 (1993). 362. Wester, L., et al. Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties. Glycobiology 10, 891-900 (2000). 363. Lumsden, K.R. Pennsylvania State University (2009). 364. Leng, X., et al. Lipocalin 2 is required for BCR-ABL-induced tumorigenesis. Oncogene 27, 6110-6119 (2008). 365. Axelsson, L., Bergenfeldt, M. & Ohlsson, K. Studies of the release and turnover of a human neutrophil lipocalin. Scand J Clin Lab Invest 55, 577-588 (1995). 366. Yang, B.Y., Gray, J.S. & Montgomery, R. The glycans of horseradish peroxidase. Carbohydr Res 287, 203-212 (1996).

170 367. Lee, Y.C., Lin, S.D., Yu, H.M., Chen, S.T. & Chu, S.T. Phosphorylation of the 24p3 protein secreted from mouse uterus in vitro and in vivo. J Protein Chem 20, 563-569 (2001). 368. Bu, D.X., et al. Induction of neutrophil gelatinase-associated lipocalin in vascular injury via activation of nuclear factor-kappaB. Am J Pathol 169, 2245-2253 (2006). 369. Cowland, J.B., Sorensen, O.E., Sehested, M. & Borregaard, N. Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha. J Immunol 171, 6630-6639 (2003). 370. Cowland, J.B., Muta, T. & Borregaard, N. IL-1beta-Specific Up-Regulation of Neutrophil Gelatinase-Associated Lipocalin Is Controlled by I{kappa}B-{zeta}. 176, 5559-5566 (2006). 371. Matsuo, S., Yamazaki, S., Takeshige, K. & Muta, T. Crucial roles of binding sites for NF-kB and C/EBPs in IkB-z-mediated transcriptional activation. 405, 605-615 (2007). 372. Fujino, R.S., et al. Spermatogonial cell-mediated activation of an IkappaBzeta-independent nuclear factor-kappaB pathway in Sertoli cells induces transcription of the lipocalin-2 gene. Mol Endocrinol 20, 904-915 (2006). 373. Sorensen, O.E., et al. Wound Healing and Expression of Antimicrobial Peptides/Polypeptides in Human Keratinocytes, a Consequence of Common Growth Factors. Vol. 170 5583-5589 (2003). 374. Bell, S., et al. Involvement of NF-[kappa]B signalling in skin physiology and disease. Cellular signalling 15, 1-7 (2003). 375. Rahman, I., Marwick, J. & Kirkham, P. Redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation, NF-[kappa]B and pro-inflammatory gene expression. Biochemical Pharmacology 68, 1255-1267 (2004). 376. Ross, A.C., Chen, Q. & Ma, Y. Augmentation of antibody responses by retinoic acid and costimulatory molecules. Semin Immunol 21, 42-50 (2009). 377. Nawata, H., Maeda, Y., Sumimoto, Y., Miyatake, J. & Kanamaru, A. A mechanism of apoptosis induced by all-trans retinoic acid on adult T-cell leukemia cells: a possible involvement of the Tax/NF-kappaB signaling pathway. Leuk Res 25, 323-331 (2001). 378. Wu, J.M., DiPietrantonio, A.M. & Hsieh, T.C. Mechanism of fenretinide (4-HPR)-induced cell death. Apoptosis 6, 377-388 (2001). 379. Klamt, F., et al. Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway. Cell Biol Int 32, 100-106 (2008). 380. Roudkenar, M.H., et al. Oxidative Stress Induced Lipocalin 2 Gene Expression: Addressing its Expression under the Harmful Conditions. Journal of Radiation Research 48, 39-44 (2007).

VITA

Melanie Claire Dispenza

Address: 600 N. Hartley Street, Apt. 215 York, PA 17404 [email protected]

Education: 2013 M.D. The Pennsylvania State University College of Medicine, Hershey, PA 2011 Ph.D., Physiology, The Pennsylvania State University, Hershey, PA 2005 B.S., Biochemistry/B.A., Biology, University of Virginia, Charlottesville, VA

Professional Experience: 2007 – 2011 Graduate Fellow, Department of Cellular and Molecular Physiology, The Pennsylvania State University, Hershey, PA 2004 Research Assistant, Dr. Victor H. Engelhard, University of Virginia, Charlottesville, VA 2002 – 2003 Research Assistant, Dr. Michael Wormington, University of Virginia, Charlottesville, VA 2002 Research Intern, Neuralstem Inc., Gaithersburg, MD 2000 – 2002 Research Intern, Dr. Jonathan M. Auerbach, The National Institutes of Health, Bethesda, MD

Grant Support: 2010 The American Acne and Rosacea Society Clinical Research Grant. PI: MC Dispenza. “Modulation of the Immune System by Isotretinoin in Acne Patients.” 2010 Finkelstein Memorial Student Research Award. PI: MC Dispenza. “Modulation of the Immune System by Isotretinoin in Acne Patients.

Publications: MC Dispenza, EB Wolpert, KL Gilliland, Z Cong, AM Nelson, and DM Thiboutot. Systemic isotretinoin therapy normalizes exaggerated TLR-2-mediated innate immune responses in acne patients. Submitted.

MC Dispenza and TJ Craig. Discrepancies Between Guidelines and International Practice in Treatment of Hereditary Angioedema. In preparation.

KR Lumsden, AM Nelson, MC Dispenza, KL Gilliland, Z Cong, AL Zaenglein, and DM Thiboutot. Isotretinoin increases skin surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne. British Journal of Dermatology. 2011. Aug; 165(2): 302-10.

SL Sheasley-O'Neill, CC Brinkman, AR Ferguson, MC Dispenza, and VH Engelhard. Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and nonlymphoid tissue distribution of CD8 T cells. Journal of Immunology. 2007. Feb; 178(3): 1512-22.