REGULATION OF SIGNALING IN AIRWAY

AND REMODELING DURING

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Nosayba Al-Azzam

August, 2017 REGULATION OF EICOSANOID SIGNALING IN AIRWAY INFLAMMATION

AND REMODELING DURING ASTHMA

Nosayba Al-Azzam

Dissertation

Approved: Accepted:

Advisor Department Chair Dr. Sailaja Paruchuri Dr. Christopher J. Ziegler

Committee Member Dean of the College Dr. Adam Smith Dr. John Green

Committee Member. Dean of the Graduate School Dr. Leah Shriver Dr. Chand K. Midha

Committee Member Date Dr. Michael Konopka

Committee Member Dr. Richard Londraville

ii ABSTRACT

Asthma is an allergic disease that is caused by activation of several inflammatory and structural cells. These cells orchestrate together to release inflammatory mediators, resulting in the pathophysiological effects seen in asthma. The released mediators can synergize to enhance each other’s response and they can modify the secretion or the effect of other mediators. Mast cells (MCs) are one of the important effector cells in asthma. MCs synthesize and secrete many inflammatory mediators upon activation including , tryptase, and . Eicosanoids, which include (PGs) and cysteinyl (cys-LTs), are bioactive lipid mediators that are implicated in many pathological conditions including asthma. Cys-LTs consisting of LTC4, LTD4, LTE4, are potent inflammatory mediators that act through two main G- coupled receptors

(GPCRs), CysLT1R and CysLT2R. PGE2 induces its effects through four different GPCRs;

EP1-4. The role of PGE2 in asthma is controversial and its effect is mainly dependent on the type and the dominant EP through which the signal is transduced. Although both eicosanoid mediators, cys-LTs and PGs, play a prominent role in the pathogenesis of asthma and other inflammatory diseases, it is not known if there is a crosstalk between these two eicosanoid mediators. In Chapter III of this dissertation, we addressed this gap in literature and determined that LTD4 and PGE2 synergize to potentiate peripheral vascular edema and lung inflammation in vivo and MC inflammatory response in vitro.

MCs are terminally differentiated cells, and they usually halt their proliferation after differentiation. However, inflammatory conditions such as asthma are associated with high MC proliferation rate as well as reactivity (mastocytosis). MC proliferation, differentiation, and survival are regulated by the growth factor (SCF)

iii through its action on the c-Kit receptor. However, it is not yet known if inflammatory mediators such as cys-LTs can synergize with SCF to induce MC proliferation or if SCF can enhance cys-LT-mediated inflammatory responses. We demonstrate in Chapter IV of this dissertation that a potential cross-talk exists between LTD4 and SCF in enhancing both

SCF-mediated MC proliferation, as well as, LTD4-mediated inflammation.

Recurring inflammation in asthma results in structural changes in the lung airways which includes goblet cell metaplasia, mucus hypersecretion, and fibrosis. Lung fibrosis is mainly mediated through transforming growth factor-β (TGF-β) via inducing to myofibroblast differentiation. Apart from TGF-β, oxidative stress also plays a role in fibroblast differentiation. One of the players in inducing oxidative stress is NADPH oxidase 4 (NOX4), an that activates NADPH oxidation and hydrogen peroxide production. NOX4 expression is upregulated in patients with lung fibrosis; nevertheless, its role in asthma and its related airway remodeling have not been explored. Conversely, the eicosanoid PGE2 is shown to have a protective role in airway remodeling, but the underlying mechanism is not fully understood. In the last part of this dissertation, we demonstrate that NOX4 is an important effector molecule in TGF-β1 mediated fibroblast differentiation in vitro and its level is upregulated in Dermatophagoides farinae (Der. f) allergen – induced airway remodeling. More interestingly, PGE2 attenuates TGF-β1- mediated NOX4 expression and fibroblast differentiation.

In conclusion, we propose that several inflammatory mediators are up-regulated during asthma and their cross-talk determines the outcome response. Therefore, understanding the role of relevant receptors and combinational therapies that target specific

iv eicosanoid receptors might be a better therapeutic option for asthma, at least in the subset of asthmatics that are resistant to conventional steroids and related therapies.

v Dedication

THIS DISSERTATION IS DEDICATED TO THE SOUL OF MY MOM.

vi ACKNOWLEDGEMENT

I would never have been able to finish my Ph.D. degree without the guidance of my supervisor and committee members, help from friends, and support from my husband and family. I would like to thank the University of Akron and the department of chemistry for granting me the scholarship to pursue my Ph.D. I want to express my deepest appreciation to my great advisor, Dr, Sailaja Paruchuri whose door was always open to me and who taught me all the research skills and also supported me as a researcher and a mom through all the five years. I am also grateful to my committee members, Dr. Adam Smith, Dr. Richard Londraville, Dr. Leah Shriver, and Dr. Michael Konopka for all their help and guidance. Many thanks to my lab mates, Dr. Vinay Kondeti, Dr. Ernest Duah, Farai Gombedza, Prachi Patil, Matthew Snyderman, Samrawit Ghebreigziabher, Christina Budda and Bethany Hochstetler for all their help, patience, support, and encouragement. I would like also to express my warm thanks to my dad, brothers, sisters, uncles, aunts and all my family in law for their prayers, support, and encouragements. And Many thanks for my friends Dana Malkawi and Dima Malkawi who supported and encouraged me and they were always ready to help me in taking care of my kids during the hard days that we faced, Ala’a Alhamwi who listened always to my complaints and filled me always with positive energy to move ahead, Safa’a Aldamiri and Shatha Gharaibeh for their support and follow-up, and for Prachi Patil again for the jokes and nice time we spent together in Dr. Paruchuri’s lab for two years. Thanks to all the medicine and dentistry colleges faculty members at Jordan University of Science and Technology who were great teachers to me and for whom who provided the recommendation letters for my Ph.D. admission.

Finally, I would like to thank my great husband, Dr. Ibrahem Shatnawi. He has been supporting and standing by me in all sad, happy good and bad times. Also, I am grateful to my kids, Rama and Adam who tolerate my mood swinging during my busy times and for making my happier with all fun they do and smiles they draw on my face.

vii TABLE OF CONTENTS

LIST OF TABLES ...... xiv

LIST OF FIGURES ...... xv

LIST OF ABBREVIATIONS ...... xviii

CHAPTER

I. GENERAL INTRODUCTION ...... 1

Asthma ...... 1

Mast Cells ...... 3

Stem cell factor and its receptor, c-Kit ...... 5

Eicosanoids ...... 7

Leukotrienes ...... 8

Biosynthesis of Leukotrienes ...... 8

Biological and pathological effects of cysteinyl leukotrienes ...... 10

Prostaglandins ...... 11

Biosynthesis of Prostaglandins ...... 11

Biological and pathological effects of prostaglandins ...... 13

G-protein coupled receptors ...... 14

Cysteinyl receptors ...... 16

Prostaglandin E2 receptors (E-) ...... 18

PKG ...... 20

viii Proliferative and inflammatory ...... 21

Erk ...... 21

c-fos ...... 22

MIP-1β ...... 23

Tumor factor (TNF) ...... 25

IL-8 ...... 25

IL-5 ...... 26

IL-13 ...... 27

MUC5AC ...... 28

Gob-5 ...... 29

Fibroblast to myofibroblast differentiation ...... 29

TGF-β1 ...... 31

Fibrotic genes or ...... 34

α-SMA ...... 34

SM22 ...... 35

Rho GTPases and MRTF-A ...... 35

Fibronectin ...... 37

Plasminogen activator inhibitor-1 (PAI-1) ...... 38

Oxidative stress and antioxidants and their role in lung remodeling ...... 39

NOX4 ...... 41

ix II. MATERIALS AND METHODS ...... 44

Reagents ...... 44

Animals ...... 46

Intradermal injection of agonists and assessment of ear edema ...... 47

Der. f induced airway remodeling ...... 47

BAL Fluid Collection ...... 48

Tissue processing ...... 49

Tissue embedding ...... 49

Tissue sectioning ...... 49

Tissue staining ...... 50

Slides deparaffinization and rehydration ...... 50

H&E staining ...... 50

Toluidine blue staining ...... 51

Cell culture and activation ...... 52

The LAD2 MC line ...... 52

Human cord blood–derived mast cells ...... 52

Human Lung ...... 53

siRNA treatment ...... 54

Cell lysates and Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-

PAGE) ...... 54

x Western blotting ...... 55

ELISA ...... 57

qPCR ...... 59

RNA extraction from stimulated cells ...... 59

RNA extraction from lung tissues ...... 60

Reverse transcription ...... 60

Calcium flux measurement ...... 64

XTT Proliferation Assay ...... 64

Statistical Analysis ...... 65

III. LTD4 AND PGE2 SYNERGIZE TO ENHANCE INFLAMMATORY

RESPONSES IN VITRO AND VASCULAR EDEMA AND DER. F INDUCED

INFLAMMATION IN VIVO ...... 66

Introduction: ...... 66

Results...... 68

Combined treatment with PGE2 and LTD4 synergistically potentiates peripheral

vascular inflammation in mice ...... 68

2+ LTD4 primes PGE2-dependent Ca flux, and enhances c-fos and

expression and MIP-1β secretion in MCs ...... 70

CysLTRs and EPs expression in LAD2 cells ...... 71

Blocking CysLT1R partially attenuate the LTD4 and PGE2 synergism ...... 72

EP3 receptor partially mediates PGE2 and LTD4 enhanced effects ...... 73

xi

LTD4 and PGE2 synergistic responses were completely blocked by antagonizing

both CysLT1R and EP3 at once ...... 76

LTD4 and PGE2 enhanced inflammatory expression ...... 77

LTD4 and PGE2 enhanced PGD2 secretion and Erk phosphorylation...... 78

LTD4 plus PGE2 synergistic effects were mediated through PKG and Erk-

dependent pathways ...... 79

CysLT1R plus EP3 or PKG knockdown resulted in complete attenuation of LTD4

and PGE2 synergistic effects ...... 82

CysLT1R and EP3 antagonists together attenuates vascular inflammation induced

by LTD4 plus PGE2 ...... 83

Combined treatment with PGE2 and LTD4 synergistically potentiates Der. f–

induced airway remodeling in mice ...... 84

Discussion ...... 86

IV. MODULATION OF MAST CELL PROLIFERATIVE AND INFLAMMATORY

RESPONSES BY AND STEM CELL FACTOR SIGNALING

INTERACTIONS ...... 90

Introduction ...... 90

Results...... 91

SCF induces c-Kit phosphorylation in a concentration dependent manner ...... 91

SCF-mediated c-Kit phosphorylation is enhanced by LTD4 ...... 92

LTD4 enhances SCF-mediated proliferation ...... 93

xii

SCF pretreatment augmented LTD4-induced calcium flux ...... 94

SCF enhances LTD4 induced c-fos phosphorylation and expression ...... 95

SCF enhances LTD4-induced inflammatory signals ...... 96

Discussion ...... 97

V. PGE2 ROLE IN NAD(P)H OXIDASE 4 (NOX4)- REGULATED ASTHMATIC

AIRWAY REMODELING ...... 100

Introduction: ...... 100

Results...... 102

NOX4 expression and activity is enhanced by TGF-β1 stimulation ...... 102

NOX4 is crucial for TGF-β1-mediated fibroblast differentiation ...... 102

NOX4 mediates TGF-β1-induced MRTF-A and SM22 expression...... 104

TGF-β1-mediated PAI-1 expression is regulated by NOX4 ...... 106

PGE2 attenuates TGF-β1 mediated αSMA, FN, and NOX4 expression ...... 107

NOX4 is upregulated in Der. f–induced airway remodeling in vivo ...... 108

Discussion ...... 109

VI. SUMMARY ...... 111

REFERENCES ...... 113

APPENDICIES ...... 135

xiii

LIST OF TABLES

Table Page

1.1. The main bioactive mediators released from MC granules ...... 4

1.2. synthases and receptors ...... 13

2.1. Antagonists/inhibitors ...... 45

2.2. Buffers used for western blotting ...... 55

2.3. Primary for western blotting ...... 56

2.4. PGD2-ELISA reagents...... 58

2.5. Reverse transcription reagents...... 61

2.6. Primer sequences for target genes...... 62

xiv

LIST OF FIGURES

Figure Page 1.1. Schematic diagram of leukotriene biosynthesis...... 9

1.2. Schematic diagram of synthesis pathway...... 12

1.3. Schematic model of G-protein signaling cycle...... 15

1.4: Cysteinyl leukotrienes receptors and their antagonists...... 17

1.5. Differentiation of fibroblasts to myofibroblasts...... 30

1.6. The chemical structure of antioxidants used in this dissertation...... 40

1.7. NOX4 structure and function. Adapted from (Chen et al., 2012)...... 42

2.1. Structure of the most used inhibitors...... 46

3.1. Ear thickness in mice treated with LTD4 and PGE2...... 69

3.2. PGE2 and LTD4 synergistic effects in MCs...... 71

3.3. PGE2 synergism with LTD4 in dose-dependent manner and LAD2 expression levels of CysLTR and EP receptors...... 72

3.4. LTD4 and PGE2 synergistic effects are partly sensitive to CysLT1R antagonism. ...73

Figure 3.5. EP3 plays a partial role in PGE2 and LTD4 synergistic responses...... 74

3.6. Effect of Gαi, EP1 and EP4 inhibition on PGE2 and LTD4 synergism in MCs...... 75

3.7. PGE2 and LTD4 synergistic responses were partially blocked by the EP3 antagonist...... 75

3.8. LTD4 and PGE2 synergistic responses were completely attenuated by the combined effect of CysLT1R and EP3 antagonists...... 77

3.9. Inflammatory gene transcripts and COX-2 protein were induced by LTD4 and

PGE2...... 78

xv 3.10. LTD4 and PGE2 synergize in PGD2 secretion and Erk phosphorylation...... 79

3.11. Effect of PKC and PKA inhibition on LTD4 and PGE2 synergistic effects...... 80

3.12. LTD4 and PGE2 enhanced effects are mediated through PKG and Erk...... 81

3.13. Inhibition of LTD4 and PGE2 synergistic effects by knocking down CysLT1R and/or

EP3 and PKG...... 83

3.14. Ear thickness in BALB/c mice treated with LTD4+PGE2 and/or MK571+L-798. .84

3.15. PGE2 and LTD4 synergize in potentiating Der. f–induced airway remodeling in mice.

...... 86

3.16. Schematic model of PGE2 and LTD4 synergism in MCs...... 88

4.1. Dose-dependent phosphorylation of c-Kit receptor by SCF...... 92

4.2. LTD4 and SCF synergistically phosphorylate c-Kit...... 93

4.3. LTD4 enhances SCF-induced cell proliferation...... 94

4.4. SCF primes LTD4 -induced calcium flux ...... 95

4.5. SCF and LTD4 synergize to potentiate c-fos phosphorylation and expression...... 96

4.6. SCF enhances LTD4-induced inflammatory signals...... 97

4.7. A schematic model of SCF and LTD4 synergism in MCs...... 98

5.1. NOXs expression levels in hLF...... 102

5.2. NOX4 role in fibroblast to myofibroblast differentiation...... 103

5.3. NOX4 inhibition attenuates TGF-β1 mediated MRTF-A and SM22 expression. ....105

5.4. NOX4 mediates TGF-β1 enhanced PAI-1 expression...... 106

5.5. PGE2 attenuates TGF-β1-induced αSMA, FN and NOX4 expression...... 107

5.6. NOX4 expression is enhanced in the lungs of mice that were challenged with Der. f allergen...... 108

xvi 5.7. Shematic diagram of PGE2 role in NOX4-regulated fibroblast differentition ...... 110

xvii

LIST OF ABBREVIATIONS

BAL Bronchoalveolar lavage bFGF basic

COX

CysLT1R Cysteinyl leukotriene 1 receptor

CysLT2R Cysteinyl leukotriene 2 receptor

Der. f Dermatophagoides farinae

DMEM Dulbecco's Modified Eagle Medium

DPI Diphenyleneiodonium

ECM

ELISA Enzyme-linked immunosorbent assay

EP E-prostanoid

Erk Extracellular signal–regulated kinase

FcɛRI The high-affinity IgE receptor

FGFR Fibroblast growth factor receptor

FN Fibronectin

xviii GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GPCR G-protein coupled receptor

H&E Hematoxylin and Eosin hLF human lung fibroblasts hMC Human cord blood–derived mast cell

IgE Immunoglobulin E

IL- iNOS inducible nitric oxide synthase

JNK c-jun N-terminal kinase

LTC4S synthase

LOX Lipooxegenase

MAPK Mitogen-activated protein kinases

MC Mast cell

MEK MAPK/Erk Kinase

NAC N-Acetyl

NOS Nitric oxide synthase

NOX NADPH oxidase

PAI-1 Plasminogen activator inhibitor-1

xix PBS Phosphate buffered saline

PG Prostaglandin

PK Protein kinase qPCR Quantitative polymerase chain reaction

ROS Reactive species

RGS Regulator of G-protein signaling

PVDF polyvinylidene difluoride

RPMI Roswell Park Memorial Institute medium

SCF Stem cell factor siRNA Small interfering RNA

SMA Smooth Muscle

SRF serum responsive element

TGF-β Transforming growth factor beta

Th2 Type 2 T helper cells

TNF Tumor necrosis factor alpha

TRPV Transient receptor potential vanilloid

WT Wild type

xx

CHAPTER I GENERAL INTRODUCTION

Asthma

Asthma is a chronic inflammatory and allergic disease that affects over 300 million people throughout the world and is characterized by recurrent episodes of airway obstruction, which usually coincides with inflammation in the airways and bronchial hyper-responsiveness (Martinez et al. (2013); Sutcliffe et al., 2012). In addition to chronic inflammation, acute inflammatory episodes also occur upon allergen exposure and result in acute exacerbations of asthma (Barnes, 1996). Asthmatic patients usually experience the first symptoms in their childhood which continue through their . The airways’ smooth muscle contraction and inflammation caused mainly by and eicosanoids contribute to asthmatic clinical symptoms. In addition, recurrent and persistent inflammation leads to changes in the airway structure such as subepithelial fibrosis, and smooth muscle hyperplasia (Ishmael, 2011).

Asthma cannot be cured, but different therapies are administered to reduce the exacerbations and recurrence of the disease. Long-acting beta agonists alleviate asthmatic symptoms through its action on smooth muscle relaxation and it is usually used to induce bronchodilation. Another palliative medication is , which counteracts the cysteinyl 1 (CysLT1R)-mediated inflammatory response (Laidlaw et al., 2012; Martinez et al., 2013). However, neither long-acting beta agonists nor montelukast has a profound effect by themselves on asthmatic attacks treatment. Inhaled

1

corticosteroids showed a better therapeutic option to reduce inflammatory responses by binding to their cytoplasmic receptors which is followed by their translocation to the nucleus, where dimerization of the receptors occurs. The resultant complex binds to DNA and suppresses the expression of immune-inflammatory genes (Makinde et al., 2007;

Martinez et al., 2011; Poon et al., 2012). Corticosteroids block the synthesis of and all the inflammatory mediators downstream of it (Hong et al., 1976). However, low adherence of inhaled corticosteroids (Martinez et al., 2013) and the presence of many severe cases that are resistant to corticosteroid therapy limited its use as a therapeutic option (Poon et al., 2012). Therefore, identifying the exact inflammatory mediators that contribute to asthma inflammatory responses and targeting them specifically will be a better anti-inflammatory therapy. Different mediators can cross-talk to each other to amplify each other’s response and complicate the inflammatory outcome (Barnes et al.,

1998); therefore, a deeper understanding of the interplay between different mediators is needed and blocking the synergized molecules/receptors might be a better therapeutic option, especially for patients who are resistant to the current therapy.

A combination of environmental and genetic factors cause irregular and increased patient response to allergen and other environmental triggers. This response is mediated through an interplay between different cell types including respiratory epithelium, fibroblast and immune cells such as T-helper 2 (Th-2) cells, mast cells (MCs) and (Ishmael, 2011; Martinez et al., 2013). MCs are essential players in asthmatic acute inflammatory responses. In allergic reactions such as asthma, during the first exposure to the allergen (sensitization), the allergen is presented to the Th-2 cells, which in turn, activate B cells to manufacture and secrete the antigen-specific antibodies, 2

immunoglobulin E (IgE). After secretion, IgE binds to the high-affinity Fc epsilon receptor

(FcɛRI) present on the MC surface. The allergen binds and crosslinks the IgE on the sensitized MC surface upon a second exposure, leading to MC degranulation and secretion of and other inflammatory mediators; this causes an acute inflammatory response

(Holgate et al., 2008).

Mast Cells

The human is made up of many interconnected elements which constitute a dynamic and complex barrier to infection and disease (Chaplin, 2010). MCs are immune cells originally known for their immunoglobulin E (IgE)-mediated response; nevertheless, they also play many significant roles in both the innate and the adaptive immune responses (Wedemeyer et al., 2000).

MCs are granular hematopoietic cells that have cytoplasmic granules filled with pre- synthesized histamine, proteases and other pharmacologically active substances. MCs are derived from bone marrow myeloid progenitor cells and migrate while undifferentiated through the blood stream. When they reach the designated peripheral tissues, MCs differentiate and mature in response to tissue microenvironmental factors, mainly stem cell factor (SCF) (Bischoff, 2007; Cardamone et al., 2016; Gilfillan et al., 2006).

MCs are mainly located at the external-internal body barriers such as the lung, the gastrointestinal tract, and the skin. In addition, MCs are also present around blood vessels and in lymph nodes which assists them in performing their function (Cardamone et al.,

2016). MCs act as one of the most important cells in the first line of defense for the body

(Bischoff, 2007). They help the immune cells to fight the directly and also communicate with other cells to influence both innate and adaptive immune responses (Urb 3

et al., 2012). Through and secretion of reactive oxygen species, MCs can directly degrade the . MCs can modulate T cell function through the secretion of cytokines and which enhance T cell ; they can also act as antigen presenting cells for T cells (Urb et al., 2012). Furthermore, MCs release vasoactive substances such as histamine and eicosanoids that enhance vascular permeability and epithelial cell mucus secretion; resulting in pathogen halt. Other MC chemotactic factors include inflammatory protein 1 beta (MIP-1β) and chemoattractant protein-1 (MCP-1) that can attract more inflammatory cells to the site of inflammation

(Kondeti et al., 2013; Taub et al., 1995; Urb et al., 2012).

MCs are one of the main cells involved in inflammation and allergic reactions such as asthma (Bischoff, 2007; Wedemeyer et al., 2000). They express the FcɛRI receptors which bind to the IgE antibodies and cause a downstream signaling cascade that results in

MC release of inflammatory mediators in a process called degranulation. In degranulation, the cell’s cytoplasmic granules fuse with the plasma membrane which leads to exocytosis of the pre-stored contents, as well as the de novo synthesized ones (Table 1.1) causing an inflammatory response (Wedemeyer et al., 2000).

Table 1.1. The main bioactive mediators released from MC granules. (Gri et al.,

2012; Marshall, 2004)

Mediator Mediator examples Main physiological effects Group Biogenic Histamine , angiogenesis. amines Serotonin Vasoconstriction.

4

Enzymes Chymase and tryptase Tissue destruction, inflammation, and pain. β-hexoaminidase Extracellular matrix (ECM) remodeling. Generation of arachidonic acid.

Phospholipid Cys-LTs, LTB4, PGD2 Inflammation, pain, bronchoconstriction and mediators vascular permeability. Cytokines (ILs) and Inflammation, leukocytes activation, and TNFα proliferation. Chemokines MIP-1α, MIP-1β, and Chemoattraction of leukocytes MCP-1 Growth Vascular Endothelial Growth of different cell types Factors Growth Factor (VEGF), TGF-β, basic fibroblast growth factor (bFGF) and Colony Stimulating Factor (CSF)

MC number is increased in the bronchial smooth muscle of asthmatic patients and its proliferation is mainly regulated by the growth factor; SCF (Boyce, 2003).

Stem cell factor and its receptor, c-Kit

SCF or steel factor is a key growth factor for MCs that mediates MC differentiation, proliferation and activation through binding to its receptor; c-Kit (CD117) (Lewis et al.,

2013; Oliveira et al., 2002). SCF is secreted mainly by bronchial epithelial cells in vivo

(Al-Muhsen et al., 2004) and by different kinds of cultured human airway cells such as bronchial subepithelial myofibroblasts and pulmonary fibroblasts in vitro (Kassel et al.,

1998; S. Zhang et al., 1996). In humans, the gene encoding SCF is located on

12q22-q24; while in mice, the gene is located on steel (Sl) locus on chromosome 10 (Da

Silva et al., 2006; Furitsu et al., 1993)

5

C-kit is a tyrosine kinase receptor that is encoded by the proto-oncogene c-kit and belongs to the type III colony stimulating factor/-derived growth factor receptor families (Babaei et al., 2016; Da Silva et al., 2006; Furitsu et al., 1993; Lotinun et al.,

2016). Generally; tyrosine kinase receptors have three domains; an extracellular - binding domain, a transmembrane hydrophobic domain, and a cytoplasmic domain with tyrosine kinase activity (Babaei et al., 2016; Da Silva et al., 2006). As a result of alternate mRNA splicing, there are four isoforms of c-Kit receptors in humans and two in mice. The mouse c-kit gene is allelic with the Dominant White Spotting (W) locus located on chromosome 5, (Da Silva et al., 2006; Furitsu et al., 1993). A mutation in either the W or

Sl locus will affect hematopoiesis leading to MC depletion, anemia, and a change in the coat color observed as white spotting (Ashman, 1999; Furitsu et al., 1993). KitW-sh mice are on a C57BL/6 background with an inversion mutation upstream of the c-kit promoter region on the W locus which affects regulatory elements, resulting in altered gene expression and MC deficiency (Da Silva et al., 2006; Lotinun et al., 2016; Nigrovic et al.,

2008). In the experiments listed in this dissertation, the KitW-sh mice were used to determine the role of MCs in PGE2 and LTD4-enhanced peripheral inflammation.

C-Kit is expressed in SCF responding cells which include hematopoietic progenitor cells, melanocytes, germinal cells, peripheral eosinophils and MCs (Ashman,

1999; Da Silva et al., 2006). Upon binding of SCF to its Kit receptor, the receptor homodimerizes and transphosphorylation of the receptor occurs in the cytoplasmic domain.

This initiates downstream signaling interactions that involve adaptor proteins such as Grb-

2 (growth factor receptor- bound protein 2). This leads to subsequent activation of MAP kinases, including the extracellular regulated kinase (Erk1/2), p38, and c-jun N-terminal

6

kinase (JNK) subgroups (Da Silva et al., 2006). As a result, SCF induces proliferation, differentiation, chemotaxis and activation of MCs (Da Silva et al., 2006).

A mutation that leads to the loss of c-Kit function is associated with some tumors such as melanoma (Montone et al., 1997). However, other activating mutations in the c-kit gene are also detected in . Furisto et al. showed that in MC leukemia, a point mutation in the c-kit proto-oncogene can lead to continuous activation of the receptor, regardless of the ligand availability, which might be involved in carcinogenesis of MCs

(Da Silva et al., 2006). Other studies linked c-kit activating mutations to mastocytosis, gastrointestinal and uveal carcinogenesis (Ashman et al., 2000; Babaei et al., 2016; Buttner et al., 1998).

Alterations in this growth factor receptor are also associated with inflammation.

SCF and c-kit gene expression are induced in the airway epithelium and subepithelium of asthmatic patients compared to healthy individuals (Al-Muhsen et al., 2004) and SCF intratracheal installation can enhance airway hyperreactivity through the synthesis of cysteinyl leukotrienes (cys-LTs) (Oliveira et al., 2001). Based on all this literature, SCF and c-Kit play a vital role in asthma and other diseases that are associated with a local increase of MC number and activity. This lead us to study the role of SCF in MC proliferation and the regulation of eicosanoid-mediated inflammatory responses as detailed in Chapter IV.

Eicosanoids

Eicosanoids are biologically active lipid derivatives that include leukotrienes (LTs), prostaglandins (PGs), and epoxyeicosatrienoic acids (EETs). Eicosanoids have various pathological roles in and inflammation through alterations in the interactions

7

between different cell types (D. Wang et al., 2010). Eicosanoids are derived from arachidonic acid that is generated from cellular membrane phospholipids by the A2 (PLA2) cytosolic enzyme (Paruchuri et al., 2009; Theron et al., 2014).

Three enzymatic pathways further metabolize arachidonic acid; cyclooxygenase (COX), lipooxygenase (LOX) and P450 epoxygenase, resulting in the formation of PGs, LTs,

(EETs), hydroxyeicosatetraenoic acids (HETEs), and hydroperoxyeicosatetraenoic acids

(HPETEs) (D. Wang et al., 2010). This dissertation will mainly focus on cys-LTs and PGs;

In particular, LTD4 and PGE2.

Leukotrienes

LTs are inflammatory mediators that are synthesized in many cell types including

MC, eosinophils, and endothelial cells. Leukotrienes are derived from arachidonic via the LOX enzymatic pathway and they play important roles in asthma and other inflammatory diseases (Hay et al., 1995; D. Wang et al., 2010).

Biosynthesis of Leukotrienes

LTs are synthesized from arachidonic acid metabolism by the 5-LOX pathway (Fig.

1.1). As a first step, 5-LOX activating protein (FLAP) binds to the arachidonic acid and presents it to the 5-LOX enzyme which acts to produce both hydroperoxyeicosatetraenoeic acid (HPETE) and (LTA4). Next, LTA4 is either metabolized to LTB4 via

LTA4 hydrolase or to cys-LT C4 (LTC4) by its conjugation to a reduced glutathione via

LTC4 synthase (LTC4S). LTC4 then leaves the cell via multidrug resistance transporters and is metabolized to LTD4 via γ-glutamyl peptidase. LTD4 is further metabolized to the terminal stable cys-LT, LTE4, via the action of the dipeptidase enzyme (Kanaoka et al.,

2014; M. Liu et al., 2015; Paruchuri et al., 2008; D. Wang et al., 2010). LTB4 and LTD4 8

have the highest bioactivity and they mediate their effects via G-protein coupled receptors

(GPCR) (D. Wang et al., 2010).

Figure 1.1. Schematic diagram of leukotriene biosynthesis.

Cys-LTs are synthesized by the intracellular mechanism mentioned above in MCs, basophils, eosinophils, and myeloid dendritic cells upon activation. Other cells such as endothelial cells and use the transcellular mechanism to produce their cys-LTs due to the expression of LTC4S and the lack of 5-LOX expression. In this mechanism, the active 5-LOX expressing cells such as release LTA4 to the

LTC4S-expressing cells leading to an extra source of cys-LTs in the inflammatory environment (Laidlaw et al., 2012).

9

Biological and pathological effects of cysteinyl leukotrienes

Cys-LTs are strong inflammatory mediators that are involved in many allergic responses which include both vascular leakage and airway smooth muscle contraction

(Kanaoka et al., 2014). Cys-LTs are also implicated in the pathogenesis of different diseases, such as brain and colorectal cancers (Savari et al., 2014), cardiovascular diseases

(Duah et al., 2013), asthma (Paruchuri et al., 2009) and (Figueroa et al., 2003).

LTE4 levels were shown to be increased in the urine of Aspirin Exacerbated Respiratory

Disease (AERD) patients and in asthmatic patients after allergen challenge (Christie et al.,

1991; Smith et al., 1991).

−/− Kanaoka et al. showed that LTC4S mice have a smaller increase in ear thickness compared to wild-type (WT) mice after an intradermal injection of a mouse monoclonal anti-dinitrophenyl (DNP) IgE followed by intravenous injection of DNP-human serum albumin after 20 h. In this model, the increase in ear thickness corresponded to the extravasation of plasma proteins attributable to changed vascular permeability induced by local MC activation (Kanaoka et al., 2001). Increased vascular permeability is a key feature of anaphylaxis and MCs are one of the main sources of cys-LTs in anaphylaxis (M. Liu et al., 2015).

Cys-LT levels are highly elevated in asthma which is associated with an increase in the activity of MCs and eosinophils in the diseased tissues (Kanaoka et al., 2014; A. P.

Sampson et al., 1992). In asthma, cys-LTs induce vascular leakage (Dahlen et al., 1981), enhance goblet cell mucus secretion (Marom et al., 1982), and decrease mucociliary clearance (Hay et al., 1995). LTC4 and LTD4 inhalation causes more bronchoconstriction than histamine and LTE4 secretion in urine is increased during asthmatic exacerbations

10

(Kanaoka et al., 2014; Laidlaw et al., 2012). In conclusion, LTs are important inflammatory mediators and cys-LTs play an important role in asthma and peripheral vascular inflammation. Therefore, we thought to investigate the role of PGE2 and LTD4 synergism in inducing peripheral vascular inflammation and their roles in Der. f-induced airway inflammation in vivo in Chapter III.

Prostaglandins

The metabolism of arachidonic acid produces another class of bioactive lipids, prostaglandins. Arachidonic acid is converted by cyclooxygenase enzyme (COX) to prostanoids which include prostaglandins and A2 (Fig. 1.2).

Biosynthesis of Prostaglandins

Prostaglandins are synthesized by the COX pathway. The COX1 gene is basally expressed in cells, while COX2 is usually quiescent in normal conditions in most cells, but highly upregulated in inflammatory and carcinogenic conditions (Stables et al., 2011; D.

Wang et al., 2010). COX is a bifunctional enzyme with bis-dioxygenase and peroxidase activities. The bis-dioxygenase component converts the arachidonic acid to the hydroperoxy arachidonate metabolite PGG2. Following that, the peroxidase function of

COX will metabolize PGG2 to PGH2 by reducing the hydroperoxide on carbon 15 position to the corresponding alcohol (Stables et al., 2011).

11

Figure 1.2. Schematic diagram of prostaglandin synthesis pathway.

PGH2 is further metabolized to the biologically functional prostanoids via certain synthase that are differentially expressed in cells and tissues (Stables et al., 2011;

Sugimoto et al., 2007). Each species binds to specific cell surface receptors to exert their function (Table 1.2). 12

Table 1.2. Prostanoid synthases and receptors (Stables et al., 2011) (Sugimoto et al., 2007).

Prostanoid Prostanoid synthase Receptor

PGD2 prostaglandin D synthase DP1 and DP2

PGE2 synthase EP1, EP2, EP3 and EP4

PGF2 prostaglandin F synthase FP

PGI2 prostaglandin I IP

TXA2 thromboxane A synthase TP

Biological and pathological effects of prostaglandins

The synthesis profile of prostanoids differs between the cells according to the expression of these synthase enzymes and the cell activity. For example, MCs produce mainly PGD2, while macrophages produce mainly TXA2 in resting states and PGE2 when activated (Stables et al., 2011). Tissues respond differently to prostaglandins due to two main reasons; the different levels of prostaglandin production and different cellular expression levels of their receptors (D. Wang et al., 2010). Upon synthesis and binding to their corresponding GPCRs, PGs exert their effects in a paracrine or autocrine manners

(Stables et al., 2011). Among the prostanoids in the human body, PGE2 is the most broadly synthesized and it has the most versatile effects. PGE2, a proinflammatory lipid mediator, plays a role in pathological conditions including asthma and pain (D. Wang et al., 2010).

Although PGE2 is a proinflammatory mediator, it has a protective role in lung fibrosis through its action on the c-AMP-dependent protein kinase A (PKA) leading to reduced expression in fibroblasts (Okunishi et al., 2014). PGE2 is shown to have 13

pleiotropic effects; some studies have shown that inhalation of PGE2 leads to inhibition of

MC mediators (Hartert et al., 2000; Kay et al., 2006; Safholm et al., 2015). However, other studies showed that PGE2 enhances MC mediator release (Feng et al., 2006; Safholm et al.,

2015; X. S. Wang et al., 2006). PGE2 has different biological effects on human airways. It has a protective role in mediating bronchodilation; however, it has undesirable effects in triggering the airway sensory nerves. This phenomenon is explained by different receptor expression in the cells resulting in divergent cellular responses to PGE2 (Birrell et al.,

2015). In addition, PGE2 has a beneficiary role in airway remodeling through its inhibition for TGF-β1-mediated fibroblast to myofibroblast differentiation (Bozyk et al., 2011).

However, the exact mechanism of this protective role is not known yet and this question is explored in Chapter V.

Most eicosanoids, including PGs and LTs, mediate their biological effects through binding and activation of G-protein coupled receptors (GPCRs) (D. Wang et al., 2010)

G-protein coupled receptors

GPCRs encompass the main family of cell surface receptors in the human , have many physiological roles, and serve as potential therapeutic targets. Receptors in this family have a structure consisting of seven transmembrane helices connected by three loops each on the intracellular and extracellular sides (Fig. 1.3) (Syrovatkina et al., 2016).

Upon ligand binding, GPCRs are converted to active conformations, causing a cellular response through a multifaceted signaling cascade (Bar-Shavit et al., 2016; Syrovatkina et al., 2016). Binding of different ligands leads to different conformational changes in the receptor and induces specific signaling pathways with different cellular responses (Bar-

Shavit et al., 2016). 14

Figure 1.3. Schematic model of G-protein signaling cycle. When the GPCR is not activated, the binds to guanosine diphosphate (GDP) and upon stimulation by ligand binding to the ligand-binding site, a conformational change of the receptor occurs. This allows the receptor to interact with the Gα subunit, which then exchanges its bound GDP with (GTP), leading to dissociation of the Gβγ dimer, thus enabling it to relay the signal by regulating the activity of additional intracellular signaling molecules. Adapted from (J. Li et al., 2002)

Signals from GPCRs are directly transmitted by heterotrimeric guanine- nucleotide- binding regulatory proteins named G-proteins. G-proteins consist of α, β and γ subunits. β and γ subunits are usually bound together and they are considered as one functional unit, referred as the βγ dimer. When agonists bind to the receptor, the guanosine diphosphate (GDP) bound to the Gα subunit is replaced by guanosine triphosphate (GTP) leading to the dissociation of the Gα subunit from the Gβγ dimer, which initiates downstream signaling pathways (Fig. 1.3). After the GPCR signal exerts its effects, the receptor activity is highly regulated to control the amount and duration of the signal. The 15

Gα subunit has an intrinsic weak GTPase activity that cleaves GTP back to GDP.

Additionally, regulators of G-protein signaling (RGS) is a large GTPase-activating protein family that controls the hydrolysis of GTP to GDP and re-association of Gα to the Gβγ dimer resulting in deactivation of GPCR (Syrovatkina et al., 2016; Watson et al., 1996).

Desensitization also occurs through a number of different cellular processes, including internalization of cell surface receptors, and conformational alteration (Pavlos et al., 2016)

G-proteins are classified into four groups according to their Gα subunit sequence and function. These groups consist of Gα stimulatory (Gαs), Gα inhibitory (Gαi), Gαq and

Gα12. There are different subtypes for each Gα group with Gαi being the largest and most varied one. On the other hand; there are 5 genes for Gβ and 12 genes for Gγ in the mouse and human . Gαs activates adenylyl cyclase, which converts ATP to cAMP. cAMP, in turn, activates its regulated proteins such as PKA. Conversely, Gαi inhibits adenylyl cyclase and reduces cAMP production. Gαq activates phospholipase C-β isoform, which cleaves phosphoinositol 4,5 bisphosphate into inositol triphosphate (IP3) and a membrane-bound diacylglycerol (DAG). Consequently, IP3 opens calcium channels located on the endoplasmic reticulum membrane, and DAG activates protein kinase C

(PKC) to initiate a downstream signaling cascade. Gα12 activates certain types of Rho

Guanine -nucleotide- exchange factor (GEF) proteins (Syrovatkina et al., 2016).

Cysteinyl leukotriene receptors

Cys-LTs act mainly through two main GPCRs, CysLT1R and CysLT2R (Fig. 1.4).

Human CysLT1R gene is located on chromosome Xq12.31 and encodes the receptor which consists of 339 amino acids and has a higher affinity for LTD4 followed by LTC4. Human

CysLT1R is 87% similar to the receptor in mice. CysLT1R is the target for pharmacologic 16

antagonists (Montelukast, , Pobilukast, ) used for asthma treatment

(Laidlaw et al., 2012; M. Liu et al., 2015; Roy et al., 2009). On the other hand, CysLT2R has 38% identity with CysLT1R. The human CysLT2R gene is located on chromosome 13q14, encoding a receptor that consists of 347 amino acids and has an equal affinity for LTC4 and LTD4. Both CysLT1R and CysLT2R have minimal affinity for LTE4.

The human and mouse genes for CysLT2R are 65% similar (Laidlaw et al., 2012; M. Liu et al., 2015; Paruchuri et al., 2009). CysLT2R is the target for the antagonist BAYu9773, which partially inhibits the receptor (Wunder et al., 2010), while the compound,

BayCysLT2, is a more selective for CysLT2R (Ni et al., 2011).

Recently a new CysLT3R was discovered, GPR99, which was previously found as a GPCR for oxoglutarate. GPR99 favors LTE4 binding; LTE4 is the most abundant cys-LT at sites of inflammation due to its stability (Kanaoka et al., 2013; M. Liu et al., 2015)

Figure 1.4: Cysteinyl leukotrienes receptors and their antagonists.

CysLTRs are expressed in many hematopoietic and non- hematopoietic cell types.

Cells can express higher levels of one receptor over the other. For example, lung smooth muscle cells express higher levels of CysLT1R, whereas human umbilical vein endothelial cells express higher levels of CysLT2R. MCs express both CysLT1R and CysLT2R

17

(Laidlaw et al., 2012; M. Liu et al., 2015). Furthermore, CysLT2R downregulates the effect of LTD4 binding to CysLT1R in MCs. Knocking down CysLT2R led to the enhanced

CysLT1R localization on the MC surface without changing the total protein level of

CysLT1R (Jiang et al., 2007).

Intracellular signaling studies performed on CysLT1R cDNA injected Xenopus laevis oocytes and CysLT1R transfected HEK-293 cells showed that CysLT1R is a GPCR that is coupled to Gαq subunit of G-proteins (M. Liu et al., 2015). However, CysLT1R in other cells like human THP-1 cells are coupled to Gαq and Gαi subunits. Therefore, the

CysLT1R signaling can be dependent on the available G-protein subunit and the cell type

(M. Liu et al., 2015).

Prostaglandin E2 receptors (E-Prostanoids)

PGE2 acts through four GPCR subtypes; EP1, EP2, EP3 and EP4, with EP3 having many splice variants. Although PGE2 can bind to all four subtypes, the amino acid similarity between EP1 and EP2 is 30%, EP1 and EP3 is 33% and EP1 and EP4 is 28%.

(Sugimoto et al., 2007). EPs differentially signal upon PGE2 binding; EP1 increases the

2+ Ca flux through coupling to the Gαq subunit (Tabata et al., 2002), EP2 and EP4 signals increase cAMP through coupling to the Gαs subunit, and EP3 decreases cAMP through coupling to the Gαi subunit. Although these are the common pathways for signaling, other potential signaling pathways may also occur (Sugimoto et al., 2007; Woodward et al.,

2011).

EPs have a distinct tissue distribution. EP3 and EP4 are commonly expressed in all tissues in mice, while EP2 is the least abundant and is restricted to some organs, such as the kidney (Sugimoto et al., 2007). PGE2 mediates different processes through EPs,

18

including pyrogenic through EP3 (Ushikubi et al., 1998), ovulation via EP2 (Hizaki et al., 1999), and anti-inflammatory responses in the lung via EP4 (Birrell et al., 2015).

A variety of agonists and antagonists were synthesized for the different EP subtypes. ONO-D1-004 is an agonist for the EP1 receptor and iloprost, a 6α-Carba analog of prostacyclin, has shown to have EP1 agonism in some systems (Lawrence et al., 1992) and possible EP3 agonist activity in others (Whittle et al., 2012). Ono pharmaceuticals have also designed EP1 antagonists like ONO8711 (Kawamori et al., 2001) and ONO8713

(Watanabe et al., 2000). EP1 inhibition has a beneficiary and therapeutic effects for several diseases, including colon cancer, hypertension, and inflammation (Woodward et al., 2011).

In regard to EP2, butaprost is an agonist that has EP2 selectivity ; however, there is no selective and potent antagonist of this receptor available (Sugimoto et al., 2007; Woodward et al., 2011). AH-6809 is the most widely-used EP2 antagonist, despite its EP1 cross- reactivity (Woodward et al., 1995). For EP3, sulprostone is an agonist that has acidic methylsulfonamide in place of C1 carboxylate and has a modest activity for EP3 versus

EP1. L-798106 and L-826266 are highly lipophilic acryloylsulfonamides with EP3 antagonistic effects. EP3 is a therapeutic target for numerous diseases and its agonists are efficient in treating ischemic myocardial infarction, while its antagonists help in the management of pain, inflammation, cough, and lung infection (Woodward et al., 2011).

Finally, a selective agonist for EP4 is ONO-AE1-329, which has a 16-phenyl group (Cao et al., 2002). In addition, the first discovered EP4 antagonist was AH 23848 (Coleman et al., 1994); this was replaced by more selective and effective compounds, such as L-161982 that has a diaryl-acylsulfonamide (Jones et al., 2009). The EP4 receptor has diverse effects, it is protective for the heart in ischemic reperfusion, but aggravates cardiac hypertrophy in

19

adaptation to cardiovascular diseases (Woodward et al., 2011). We used these inhibitors/agonists in our experiments in Chapter III to determine which EP receptor is playing a role in PGE2 and LTD4 synergism in MCs.

Receptor activation is relayed through different intracellular signaling cascades and changes in gene expression to ultimately initiate the proper cellular responses. Of the signaling pathways, kinases including PKG and PKA and the mitogen-activated protein kinase (MAPK) such as Erk are important mediators in cellular responses to prostaglandins. To relay the extracellular signal into a transcriptional response, the signaling molecules will turn on/off the expression of the genes by modulating the activity of the transcriptional factors. c-fos is one of the downstream transcription factors and its phosphorylation (activation) can be regulated through Erk (Schenk et al., 1999; Whitmarsh,

2007).

PKG

Cyclic nucleotides act as cellular secondary messengers and play a significant role in to relay the environmental and extracellular stimuli into intracellular effects (Lorenz et al., 2016). The classical signaling of cGMP starts with the production of nitric oxide (NO) by nitric oxide synthases (NOS) and its and activation of soluble enzyme that converts GTP to cGMP. cGMP binds to and activates its dependent protein kinase-G (PKG), which in turn phosphorylates protein substrates (Lorenz et al., 2016; Tunctan et al., 2013). cGMP is commonly considered as a positive regulator of leukocyte’s lysosomal enzyme release while cAMP is regarded as a negative modulator. (Goldstein et al., 1973; Nanamori et al., 2007; Zurier et al., 1974).

20

cGMP increases MC degranulation through the FcɛRI receptor cross-linking (Nanamori et al., 2007).

cAMP and cGMP-dependent protein kinases (PKA and PKG, respectively) are similar enzymes that have binding domains but with different amino acid compositions and binding affinities for cAMP and cGMP (Lorenz et al., 2016). and degranulation is regulated by PKG (Nanamori et al., 2007). In addition, atrial natriuretic hormone induces histamine release through MC degranulation in both cGMP-dependent and calcium uptake-dependent manners (Chai et al., 2000).

Proliferative and inflammatory genes

Erk

The mitogen-activated protein kinase (MAPK) signaling pathway is a common pathway activated to promote intracellular signaling (Samatar et al., 2014; Shaul et al.,

2007). Four discrete MAPK cascades (Erk, JNK, p38 and BMK) collaborate in transmitting the signals from different ligands resulting in specific cell response depending on which ligand is bound (Abe et al., 2002; Shaul et al., 2007). The two key Erk proteins are ERK1

(44 kDa) and ERK2 (42 kDa) that are expressed by the genes erk1 (Mapk3) and erk2

(Mapk1). Erk is a -threonine kinase that is phosphorylated at the threonine and tyrosine residues in a Thr-Glu-Tyr motif upon activation (Orton et al., 2005; Shaul et al.,

2007). This double phosphorylation is an indirect way to measure Erk activity. Activated

Erk phosphorylates its targeted substrates in the nucleus and the cytoplasm and can affect gene expression (Orton et al., 2005; Yung et al., 1997). Erk is deactivated by dephosphorylating either one or both of the phosphorylated tyrosine and threonine

21

residues. This deactivation is usually achieved by serine/threonine phosphatases (Shaul et al., 2007).

Erk signaling is mostly activated through the RAS–RAF–MEK–Erk pathway. Erk is the key part in the RAS/RAF/MEK/ Erk pathway. Erk is the only kinase that can activate a lot of downstream substrates, affecting over 160 molecules downstream (L. Li et al.,

2016). Erk activity is regulated by feedback loops at multiple levels to guarantee homeostasis and control cell growth (Samatar et al., 2014). c-fos

C-fos is a transcription factor that is regulated by immediate early regulatory genes

(Lee et al., 2016). Like other immediate early genes, the c-fos expression is low or unnoticeable in inactive cells, but its expression is induced within minutes of extracellular stimulation. This transcriptional induction is momentary and the mRNAs transcribed from these genes are usually short-lived (Sheng et al., 1990). C-fos belongs to the fos family that consists of c-fos, fosB, fosL1, and fosL2. Members of the fos family form heterodimer complexes with Jun and ATF/CREB proteins to form the activator protein-1 (AP-1) transcription factor. AP-1 proteins are usually the final target in the signal transduction pathways. They bind to specific sites on the DNA, named AP-1 sites, which are located in the regulatory regions of many cellular genes including IL-4 (E. Hu et al., 1994; Nishad et al., 2016). AP-1 sites are involved cell proliferation and DNA repair (Nishad et al., 2016).

C-fos and c-jun can also repress different gene expressions; c-fos can repress its own promoter in addition to other early response genes promoters. C-fos has different domains for activating gene expression rather than the ones for repressing it (E. Hu et al., 1994).

The expression of fos genes is induced by many factors including several cytokines and 22

growth factors. C-fos is the fastest mRNA and protein to be expressed; the mRNA of this gene can be induced within 15 min to 2 h (E. Hu et al., 1994).

MCs degranulated enhance the c-fos proto-oncogene expression in the (Sequier et al., 1990) and PKCɛ links the MC FcɛRI receptor activation to the expression of c-fos (Razin et al., 1994). Furthermore, CysLT1R activation by LTD4 and

LTE4 enhances c-fos expression through PKCɛ in MCs (Kondeti et al., 2013). c-fos also plays an important role in regulating inflammatory gene expression in asthma. Minimal levels of c-fos are expressed in most human cells in normal conditions, however, its expression level is rapidly and temporarily increased by many inflammatory mediators including histamine, eicosanoids, and cytokines (Barnes, 1996; H. Liu et al., 2014). Based on that, c-fos is a rapidly and transiently-induced transcription factor that plays a role in cells proliferation and inflammation through regulating many growth factors and gene expressions.

MIP-1β

Chemokines (chemotactic cytokines) are small molecular weight proteins, around

8-14 kDa in size, that recruit immune cells to the sites of inflammation and infection (W.

G. Liang et al., 2015; Ren et al., 2010). In humans, there are around 50 different chemokines acting through 20 GPCRs. Macrophage inflammatory protein 1 (MIP-1) is a that has proinflammatory effects with two subtypes; MIP-1α, also named as CC motif chemokine ligand 3 (CCL3), and MIP-1β, also named as CC motif chemokine ligand

4 (CCL4) (Ren et al., 2010). The CCL3 and CCL4 genes are located on chromosome 17q12 within 40 kb of each other (Modi et al., 2006). MIP-1 is induced by many proinflammatory stimuli in most immune cells, including B cells and MCs, and it attracts macrophages to 23

the inflammatory sites (Menten et al., 2002). Upon induction, they can bind to different chemokine receptors (CCR1 and CCR5) that initiate different responses that control both acute and chronic inflammation (Kondeti et al., 2013; Menten et al., 2002; Ren et al., 2010).

Deregulation of MIP-1 proteins is enhanced in many inflammatory conditions and they were studied as a therapeutic target in many diseases such as HIV and cancer (Ren et al.,

2010).

MIP-1α and MIP-1β are extremely acidic proteins with similar tertiary structures, oligomerization of these proteins results in quaternary structures that guard chemokines against degradation by proteases and control their activities (W. G. Liang et al., 2015; Ren et al., 2010). Oligomerization into high molecular weight (> 600 kDa) is a reversible process which allows a control of the monomer levels (W. G. Liang et al., 2015).

The MIP-1 activity is enhanced by interacting with glycosaminoglycans and certain proteases, such as CD26/DDP (Proost et al., 2000)or attenuated by extracellular proteases like cathepsin D (Wolf et al., 2003). Extracellular proteases degrade chemokines by selectively cutting them at the N- and C-termini. Nevertheless, oligomerization buries the receptor-binding sites, particularly at the N-terminus. Thus, chemokine oligomers prolong the half-life of the chemokines by protecting them from proteases but decreases the activity at the monomer level (W. G. Liang et al., 2015). In conclusion, MIP-1 proteins are inflammatory chemokines that recruit macrophages to the inflammatory sites and the equilibrium between monomers, dimers, oligomers, and polymers results in more active chemo-attractants over a longer time.

24

Tumor necrosis factor (TNF)

Tumor necrosis factors, consisting of TNF-α and TNF-β (Lymphotoxin), are cytotoxic proteins released from peripheral blood leukocytes and cell lines of hematopoietic origin upon mitogenic activation (Locksley et al., 2001; Nedwin et al.,

1985). These proteins are encoded by distinct genes located on chromosome 6. Each gene has three introns, two of which are located at different positions and the third has the same location in both genes (Nedwin et al., 1985). In addition, these factors are secreted from different cell types with different kinetics. TNF-α is produced by , mainly macrophages, in addition to other cells, such as MCs and neural cells after 2-24 h of stimulation. However, TNF-β is produced by T- after 24-48 h of stimulation

(Nedwin et al., 1985; Wajant et al., 2003).

Both TNF-α and TNF-β have similar biological activities; both have a role in necrosis and inhibiting proliferation in certain cell types, especially tumor cell lines

(Locksley et al., 2001; Nedwin et al., 1985). In addition, TNF-α has a role in autoimmunity, inflammation and host defense (Locksley et al., 2001).

IL-8

IL-8 (or CXCL8) is a cytokine that is transcribed and translated from the IL-8 gene located on chromosome 4 and consists of 4 exons and 3 introns. IL-8 mRNA expression is a rapid process. The transcripts are noticed after1 h of cell stimulation, peaks after 6 h, and lasts for 6 hs. IL-8 gene expression is regulated depending on the stimulus, the cell type, and the receptor expression (Akdis et al., 2011). IL-8 was experimentally induced by monocytes after exposure to lipopolysaccharide and was shown to enhance migration of neutrophils. Other studies have demonstrated that IL-8 can be secreted by other cells like

25

fibroblasts and endothelial cells, and can affect other cells beside neutrophils (Russo et al.,

2014).

IL-8 is upregulated in and enhances carcinogenesis by promoting angiogenesis, proliferation of cancer cells and endothelial cells, and migration of cancer cells (Russo et al., 2014). In addition, a study showed that human MCs can produce IL-8 upon activation (Moller et al., 1993). MCs can express and release higher concentrations of IL-8 upon incubation with activated T-cells membranes versus incubation with resting

T-cells. These activated MCs also induced neutrophil chemotaxis that was normalized by neutralizing antibodies to IL-8 (Salamon et al., 2005). In line with these studies, the IL-8 concentration was found to be high in the bronchoalveolar lavage (BAL) fluid of patients with acute respiratory distress syndrome and the synovial fluid of patients with rheumatoid (Akdis et al., 2011). Accordingly, many studies have suggested the use of anti–IL-

8 mAbs as a therapeutic intervention for distinct inflammatory diseases (Akdis et al., 2011;

Fudala et al., 2007; Peichl et al., 1999; Taguchi et al., 2005). In conclusion, IL-8 is a cytokine that is induced in inflammation and leads to the activation of neutrophils and other immunological cells.

IL-5

An increased number of eosinophils in the bronchoalveolar tissue is a characteristic pathology of asthma. Eosinophils are derived from myeloid precursor cells in the bone marrow and differentiate mainly under the action of the cytokine, IL-5, before being released into the blood. Eosinophils and basophils are the only cells that express IL-5 receptors (Greenfeder et al., 2001).

26

IL-5 is secreted by MCs, Th2 cells, and eosinophils (Dubucquoi et al., 1994). IL-5 is known to have a role in differentiation and growth of eosinophils in the bone marrow, migration of eosinophils to their designated inflammatory sites, and inhibition of . In addition, IL-5 plays a role in basophil growth, metabolism, and function; there is, however, no direct effect on the degranulation (Corren, 2012). Anti-IL-

5 antibodies have an inhibitory effect on the guinea pig asthmatic models (Mauser et al.,

1993). Also, isolated monocytes from asthmatic patient BAL fluid have higher transcript levels of IL-5 in addition to IL-4 and IL-2 (Krishnaswamy et al., 1993). Based on that, IL-

5 is the cytokine that mainly regulates eosinophil cell maturation and chemotaxis to the inflammatory sites.

IL-13

In asthma, the immune system responds to the allergen in many ways which include

MC hyperplasia, chemotaxis of Th2 cells, eosinophils and basophils, IgE secretion, and changes in the smooth muscle and epithelial layers in the airways. IL-13 and IL-4 are the driving forces for these responses (H. E. Liang et al., 2011). IL-13 is a cytokine that is synthesized and secreted by both T-cells and dendritic cells. IL-13 and IL-4 have many similar biologic functions due to their receptor complexes sharing the IL-4–receptor α- chain, a structural part that relays both receptor signals. Although T-cells secrete 1L-13 cytokines, they do not express their functional receptors. IL-13 is crucial for IgE synthesis and IgE-mediated allergic reactions (de Vries, 1998).

In asthma and other pulmonary diseases, IL-13 enhances goblet cell metaplasia, fibrosis and mucus secretion (Wills-Karp et al., 1998; Zhu et al., 1999). IL-13 enhances

MUC5AC gene expression, which thereby enhances mucus secretion. Exposing normal

27

human bronchial epithelial cells to IL-13; led to goblet cell differentiation, and hyperplasia in addition to increased MUC5AC gene and protein expression (Yasuo et al., 2006).

Table 1.2. Summary of cytokine source and function

Cytokine Source Function

MIP-1 B cells, MCs Macrophage chemotaxis

TNF-α Peripheral blood leukocytes and Necrosis, inhibiting tumor cell cells with hematopoietic origin proliferation, inflammation and host defense IL-8 Monocytes, MCs, fibroblasts Chemotaxis of neutrophils and endothelial cells IL-5 MCs, Th2 cells, and eosinophils Eosinophils growth and differentiation

IL-13 T-cells and dendritic cells IgE-mediated allergic reactions and and mucus secretion

MUC5AC

MUC5AC is a main mucin that is secreted by the epithelium of the small airway. It is highly regulated through gene expression levels and highly enhanced in inflammatory conditions (Davis et al., 2008). Five similar genes exist in humans and mice that are responsible for the production of the following mucins: MUC2, MUC5AB, MUC5AC, and

MUC6. These genes are located on the same chromosome 11p15 in humans and on chromosome 7 F5 in mice. The fifth MUC19 gene is located on chromosome 12q12 in humans and on chromosome 15 E3 in mice (Young et al., 2007).

MUC5-AC and MUC5-AB are produced in different locations. The MUC5AC mucin is mainly produced by the goblet cells located in the surface epithelium while the

MUC5-AB mucin is mainly produced by mucous cells located in the submucosal gland

(Kirkham et al., 2002). The MUC5AC expression is enhanced in allergen-induced airway 28

goblet cell metaplasia and in smoker airways compared to non-smokers (G. Wang et al.,

2012).

Gob-5

The goblet cell-derived protein, CLCA, is a calcium-activated chloride channel that is linked to cancer and many respiratory inflammatory conditions (Patel et al., 2009).

CLCA has different isoforms in both human and mice; Gob-5 is a mouse CLCA3 isoform that is an ortholog to the human CLCA1 isoform. The activity of the Gob-5 protein is related to fluid exudation in the trachea and in the small intestine (Loewen et al., 2005).

Gob-5 gene expression and protein levels are highly elevated in several respiratory diseases including asthma, cystic fibrosis, chronic obstructive pulmonary disease (Erickson et al.,

2015), and airway hyperresponsiveness (Nakanishi et al., 2001). In asthma, increased Th2 cells and their related cytokines (IL-9, IL-4, and IL-13) are related to increased CLCA expression in the asthmatic patient and mouse models of asthma. Furthermore, the expression of mGob-5/hCLCA1 isoforms specifically leads to mucus hypersecretion via enhancing mucin gene expression, especially the MUC5AC gene (Loewen et al., 2005)

Asthma is a disease that results in recurrent and reversible bronchoconstrictions

(Martinez et al., 2013). However, upon repeated exposures to the allergen and continuing inflammation, irreversible structural changes to the respiratory system occur. This includes increased airway smooth muscle thickness, goblet cell metaplasia, and fibrosis (Barnes,

1996).

Fibroblast to myofibroblast differentiation

Fibroblasts are spindle-shaped long cells that are usually located in the extracellular matrix of most body tissues. Fibroblasts can be active, as in the case of cardiac tissues, or 29

inactive, such as in the eye cornea (Watsky et al., 2010). Myofibroblasts are typically active fibroblasts that are distinguished by their expression of alpha-smooth muscle actin (α-

SMA). Also, myofibroblasts have higher collagen and cytokine gene expression, higher contractility, and lower proliferation rate (Desmouliere et al., 1993; Phan, 2002; Watsky et al., 2010). These cells have condensed aggregates of and display specific contacts with adjacent cells and the ECM components (Fig. 1.5) (Phan, 2002).

Figure 1.5. Differentiation of fibroblasts to myofibroblasts. Upon TGF-β1 stimulation, fibroblasts transform to myofibroblasts. Myofibroblasts are characterized by secreting the ECM proteins, expressing focal adhesions to communicate with the ECM and adjacent cells, and their cellular actin is bundled into contractile stress fibers. Adapted from (Falke et al., 2015).

Myofibroblasts can also be derived from other cell types such as endothelial, epithelial and mononuclear cells (Desmouliere et al., 1993). Myofibroblasts originate interstitial fibroblasts in the lung, stellate cells in the liver and pancreas, and glomerular mesangial and interstitial fibroblasts in the kidney (Takeji et al., 2006). These cells produce large amounts of the ECM components including fibronectin (FN) and collagen-I.

Morphologically, they have a large elliptical euchromatic nucleus with one or two nucleoli, rough endoplasmatic reticulum, and a protruding Golgi apparatus. However, in inactive adult fibroblasts, the nucleus is more flat and heterochromatic, and the endoplasmic

30

reticulum is diminished (Kalluri et al., 2006). Myofibroblasts normally function in . When an injury occurs, fibroblasts differentiate to myofibroblasts and migrate to the wound site to lay down the ECM proteins and also help in wound closure by its α-SMA related contractility. In the end, myofibroblasts undergo apoptosis. However, when the apoptotic process fails to degrade the myofibroblast, extra ECM proteins are secreted, resulting in tissue fibrosis (Desmouliere et al., 1993). Zhang et al. showed that IL-1β induces myofibroblast apoptosis through inducible nitric oxide synthase (iNOS) induction and subsequent BCL-2 reduction. In addition, TGF-β1 induces fibrosis by counteracting that effect via inhibiting both the iNOS induction and B-cell lymphoma 2 (BCL-2) regulator protein reduction (H. Y. Zhang et al., 1999). Similarly, fibroblasts isolated from patients with idiopathic pulmonary fibrosis showed less expression of antifibrotic genes involving COX-2, Thy-1, or caveolin-1, which are all known as antifibrotic factors (Phan,

2008)

The most common trigger for the differentiation is TGF-β1, which usually signals through SMAD proteins. Mechanical factors can also play a role in fibroblast differentiation. Increased stiffness and strain of the ECM in the heart can enhance this differentiation through a possible increase in TGF-β1 secretion and its mediated effect

(Yong et al., 2015). Complement factor C1, altered ECM composition, or reactive oxygen species can also activate fibroblast differentiation (Kalluri et al., 2006).

TGF-β1

Transforming growth factor β superfamily consists of the TGF-β, activins, bone morphogenic protein and other associated protein subfamilies (Kubiczkova et al., 2012).

TGF-β subfamily has three isoforms: TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being

31

the predominant one (Derynck et al., 1985). TGF-β is secreted by many structural and immunological cells, including macrophages, MCs, fibroblasts, and platelets (Assoian et al., 1987; Makinde et al., 2007). TGF-β1 is present as a dimer of two monomers that are stabilized by hydrophobic interactions and disulfide bonds (Dubois et al., 1995). TGF-β1 is involved in many cellular functions including cellular differentiation, migration, proliferation, and matrix synthesis (Kubiczkova et al., 2012).

TGF-β mediates its effects through binding to two receptor families, TβRI and

TβRII. TβRI and TβRII are similar glycoproteins with a single transmembrane segment and a serine-threonine protein kinase domain located in the C-terminus. TβRI is distinguished by its GS domain, which becomes phosphorylated upon TGF-β binding and plays a critical role in both receptors’ activation. In addition, TβRI contains a binding region for the negative controller of receptor signaling, FKBP12-binding protein.

However, TβRII is distinguished by its constitutive activity (Watsky et al., 2010). TGF-β1 typically binds to TβRII, causing a conformational change that results in phosphorylation of TβRI at the glycine-serine regulatory region, and subsequent disengagement of the inhibitory FKBP12-binding protein and dimerization of the two receptors (Kubiczkova et al., 2012; Watsky et al., 2010). SMAD2 and/or SMAD3 proteins are attached to TβRI along with another protein called SARA. After TβRI activation, SMAD2/3 are phosphorylated followed by their dissociation from the TβRI-SARA complex, their localization to the , and their binding to SMAD4. The SMAD4 binding results in nucleus translocation and binding to the transcription factors of TGF-β1 related genes (Derynck et al., 2003;

Kubiczkova et al., 2012; Watsky et al., 2010). TGF-β1 can also induce signaling through non-SMAD pathways such as MAPK (Bakin et al., 2002) and NF-κB (Gingery et al.,

32

2008). TGF-β1 mediated SMAD activation is controlled through receptor interacting proteins such as SARA that facilitate TGF-β1 mediated receptor internalization and stabilizes the interaction of SMAD2/3 with TβRI to initiate the intracellular signal

(Penheiter et al., 2002). SMAD6 and 7 negatively regulate TGF-β signaling through binding to TβRI, resulting in the halt of their activation of SMAD2/3 (Massague, 2000).

SMAD is deactivated through dephosphorylation, ubiquitination, and nuclear export (Lonn et al., 2010).

TGF-β1 signaling through SMAD induces differentiation, but additional factors are required. For example, cell adhesion is an essential factor for TGF-β1 to enable its differentiation effect. Fibroblasts respond to TGF-β1 when they are plated on integrin binding proteins, however, cells that are seeded on non-integrin binding proteins do not show any response (Thannickal et al., 2003). Also, a tumor suppressor Lkb 1 gene deletion in mouse fibroblasts results in diminished TGF-β1 induced SMAD activation and differentiation to myofibroblast (Vaahtomeri et al., 2008). Blocking transient receptor potential vanilloid 4 (TRPV4) channels also reduces fibroblast differentiation dramatically

(Gombedza et al., 2017; Rahaman et al., 2014). Another biochemical factor inducing fibroblast differentiation includes angiotensin II, which showed an effect on cardiac fibroblasts (Yong et al., 2015).

TGF-β is linked to the pathogenesis of many diseases (Vignola et al., 1997).

Genetic mutations that result in enhanced TGF-β signaling can promote tumorigenesis

(Levy et al., 2006; Teicher, 2001). Also, TGF-β mediates airway remodeling in patients with asthma and chronic bronchitis (Vignola et al., 1997). TGF-β plays multiple roles in the progression of airway remodeling in asthma; it enhances mucus secretion, increases

33 goblet cell proliferation, and damages the epithelial cell layer. More importantly, it induces sub-epithelial fibrosis through mediating fibroblast propagation, differentiation, and secretion of the ECM proteins (Makinde et al., 2007). In addition, increased stiffness of the

ECM in the heart can increase TGF-β1 levels and its intermediated effects (Yong et al.,

2015).

Fibrotic genes or proteins

α-SMA

α-SMA is expressed by the Acta2 gene located on Chromosome 10 in humans.

Actin is available in six different isoforms in eukaryotic cells. Two striated muscular

(α cardiac and α skeletal), two smooth muscle actins (α in the aorta mainly and γ in the enteric), and two cytoplasmic non-muscular actins that are located in all cells (β and γ)

(Rockey et al., 2013; Ronnov-Jessen et al., 1996). The muscular isoforms are very similar and differ in just few residues of amino acid sequence, whereas the muscular and cytoplasmic actins differ by 10% of their amino acid sequences (Rockey et al., 2013). α-

SMA is a typical isoform of vascular smooth muscle actin and is present in smooth muscle, pericytes, and myofibroblasts (Rockey et al., 2013). In myofibroblasts, α-SMA allows the cells to contract which aids in wound closure (Hinz et al., 2001); however, it concurrently reduces cell (Ronnov-Jessen et al., 1996).

α-SMA incorporates into the myofiboblasts’ stress fibers (bundles of micro- filaments), and it is a critical factor for myofibroblast focal adhesion maturation (Hinz et al., 2003). Upon incorporation into the cytoskeleton fibers, actin assists the cells in contraction and migration. Actin filaments are polymerized against the cellular membranes, resulting in providing the required force for the cell contraction. The structure 34

and movement in the filaments are precisely controlled by a large number of actin-binding proteins which regulate the nucleation, extension, and breaking up of the filaments

(Tojkander et al., 2012).

SM22

SM22 is an actin-associated protein family (Lawson et al., 1997) and it consists of three isoforms: SM22α, SM22β, and SM22γ. SM22α, also called transgelin, SM22β, and

SM22γ are products of the genes TAGLN1, TAGLN2 gene, and TAGLN3 respectively.

Deletion of SM22 reduces ROS production and cellular oxidative stress. SM22α is an F- actin binding protein that helps in actin bundling and play a role in cell differentiation

(Thompson et al., 2012). SM22α is a 22 kDa protein that is expressed in the earliest phases of smooth muscle cell differentiation (Solway et al., 1995). Kawai-Kowase et al. have shown that TGFβ-1 induces SM22α related gene expression in mouse fibroblast via increasing SRF expression. However, bFGF impedes TGFβ-1-mediated SM22 gene expression by attenuating only SRF function but not the expression (Kawai-Kowase et al.,

2004). bFGF also inhibits human airway fibroblast induced differentiation and SM22 expression (Schuliga et al., 2013). TGF-β1 induces SM22α through MRTF-A in mouse and human fibroblasts, and inhibiting MRTF-A led to the attenuation of SM22 expression

(Velasquez et al., 2013).

Rho GTPases and MRTF-A

Rho GTPases are GTPase enzymes that have a role in many cellular functions including migration, differentiation, and adhesion. When Rho is activated by extracellular signals through Rho-GeF, it enhances polymerization of the monomeric globular actin protein (G-actin) into the filamentous actin (F-actin) through Rho-associated kinase 35

(RoCK) and Formins. This results in low levels of G-actin in the cytoplasm (Bian et al.,

2016; Olson et al., 2010).

At low actin polymerization levels, the myocardin-related transcription factor

(MRTF) is reversibly linked to G-actin. Upon Rho activation, MRTF is released from its association with the G-actin, followed by its nuclear translocation to enhance the serum responsive factors (SRF). SRF strongly induces the expression of SMA and actin binding proteins that affect the actin dynamics, cell differentiation, morphology and growth (Bian et al., 2016; McDonald et al., 2015)

The MRTF protein family consists of MRTF-A (also known as MKL1, MAL, and

BSAC), MRTF-B (also known as MLK2 and MAL16), and myocardin. The MRTF family plays an important role in different tissues such as cardiovascular system development, adipogenesis, skeletal homeostasis (Bian et al., 2016; Olson et al., 2010) and asthmatic airway remodeling (Gombedza et al., 2017). Myocardin is specifically expressed in the cardiovascular system; however, MRTF-A and MRTF-B are expressed in a wide range of tissues (Olson et al., 2010).

MRTF-A plays a role in SRF mediated smooth muscle gene expression including

αSMA and SM22 but did not affect the SRF proliferative response. (M. Zhang et al., 2007).

MRTF-A is implicated in the mechanical sensations of the ECM and determining the cellular fate to be differentiated into myofibroblasts. In addition, MRTF-A KO mice are resistant to cardiac, lung and skin fibrosis (Shiwen et al., 2015). Being an important transcription factor in airway remodeling, MRTF-A regulation by NADPH oxidase 4

(NOX4) was explored in Chapter V of this dissertation.

36 Fibronectin

FN is a multifunctional protein that is expressed in fibroblasts, chondrocytes, , and synovial cells (Pankov et al., 2002; To et al., 2011). FN exists as 20 different variants in human, and all these variants are expressed from the same gene and arise due to differently spliced mRNA (Kosmehl et al., 1996). FN protein is secreted as a dimer of two similar monomers that are linked by two disulfide bonds located at the C- terminus (Rocco et al., 1983). Each monomer consists of three different types of FN repeats: type I, II and III. FN has 12 type I repeats, two type II repeats, 15 type III repeats, in addition to a nonhomologous type III connecting segment region (To et al., 2011). FN is a glycosylated protein; glycosylation helps in protecting it from hydrolysis and increases its affinity for specific substrates (Pankov et al., 2002). FN helps in , adhesion, differentiation, and interaction with the ECM. It binds to many cellular integrins, which are heterodimeric receptors located on the cell surface and that aid in the ransmission of contractile binding of the cellular cytoskeleton to the ECM. The main receptor for FN is α5β1 integrin (Plow et al., 2000).

FN is present as a soluble plasma molecule that is usually produced by hepatocytes and has simple splice variants. FN also exists in a less soluble cellular form that has more complex splice variants that differ between cell and tissue type and is usually present in the ECM (Pankov et al., 2002). In the early phase of tissue injury, plasma FN assists in the fibrin clot as well as platelet adhesion, spreading and aggregation. During the late phase, cellular FN is assembled in a 3-D fibrillar network that aids in cell adhesion, migration, and apoptosis (To et al., 2011). In addition, it contributes to the regulation of the deposition of collagen-I and III in the ECM and fibroblast to myofibroblast differentiation.

37 Myofibroblast actin incorporates in adhesion complexes that transduce mechanical forces and allow transmission of contractile forces generated by the intracellular actin and the extracellular tension (Tomasek et al., 2002).

Fibrosis results from both increased ECM protein secretion as well as decreased

ECM degradation. The main player in inhibiting matrix degradation is the plasminogen activator inhibitor-1 (PAI-1) (Cesari et al., 2010).

Plasminogen activator inhibitor-1 (PAI-1)

Plasminogen activator (PA) is an enzyme that converts plasminogen to plasmin which in turn degrades the fibrin in a process called “fibrinolysis”. Plasmin is a serine protease that is an essential component in fibrinolysis due to its role in the final degradation of fibrin and ECM proteins (Cesari et al., 2010). Plasmin is produced from its precursor plasminogen by two types of plasminogen activators: urokinase (uPA) and tissue (tPA) types, which are both inhibited by PA inhibitor (PAI) (Loskutoff et al., 1983).

The most common PAIs are as follows: 1) PAI-1, which was initially found in endothelial cells (Loskutoff et al., 1983), 2) PAI-2, also called placental type (Kruithof et al., 1986), and 3) PAI-3, which is undistinguishable from inhibitor (Kruithof et al., 1986). PAI-1 is produced by many cells, including endothelial cells, smooth muscle cells, fibroblasts and monocytes/macrophages (Cesari et al., 2010). PAI-1 is a protease from the serpin superfamily, which forms 1:1 enzyme-inhibitor complexes that are inactive. PAI-1 is a fast-acting inhibitor for both PAs (Fay et al., 1997). Increased expression of PAI-1 is shown to reduce fibrinolysis, which means more fibrin accumulation

38

and more fibrosis (Aso, 2007). Furthermore, PAI-1-/- mice have shown enhanced vascular fibrinolysis and resistance to lung fibrosis mediated by lung injury (Fay et al., 1997)

Oxidative stress and antioxidants and their role in lung remodeling

Oxidative stress is a state in which there are higher levels of oxidants versus antioxidants in a biological system, which results in an alteration of the biological system redox state in favor of oxidizing. Under oxidative stress, reactive oxygen species (ROS)

· · are produced. ROS includes O2 , OH , and nonradicals such as H2O2 (Drummond et al.,

2011; Kerksick et al., 2005). ROS can be produced by many sources which include the following: 1) mitochondria, where ROS either develop because of a mistake in oxidative pathways or they escape the scavenging enzymes, 2) capillary endothelium, where hypoxia-related cardiovascular diseases and exercise occur , and 3) inflammatory cells, which are usually chemotaxed due to muscle or tissue damage (Kerksick et al., 2005). A disturbed redox state is linked to several pathological and physiological conditions, including diabetes, fibrosis, cancer, senescence and exercise (Drummond et al., 2011;

Kerksick et al., 2005; Riganti et al., 2004).

Superoxides are the most common free radical of ROS. It can be converted to H2O2 by different dismutases in the human body (McCord et al., 1969). Although the chemical structure of H2O2 is not a free radical, it is considered as a ROS due to its reactivity that quickly results in formation of strong hydroxyl radicals, which can lead to DNA damage

(Kerksick et al., 2005).

Since ROS has many related pathologies, antioxidants have been introduced in researches to study ROS related diseases. Diphenyleneiodonium (DPI) is one of the most common antioxidants being used (Fig. 1.6). It is a flavoprotein inhibitor that extracts

39

electrons from FAD, thereby stopping electron transport. DPI inhibits NADPH Oxidase

(NOX), nitric oxide synthase (NOS), xanthine oxidase, and NADPH-cytochrome P450 oxidoreductase (Doussiere et al., 1992).

Glutathione is a common antioxidant, and it is present in almost every cell. It is produced endogenously and has many roles in the body. In addition to its role as an antioxidant, it modulates cells proliferation, cell signaling related to redox state, cysteine storage and passage, immune response, and metabolism of leukotriene and prostaglandins

(Kerksick et al., 2005).

N-Acetyl Cysteine (NAC) is a pharmaceutical compound that is composed of acetylated cysteine residues (Fig. 1.6). It acts as a cysteine source to glutathione, so the latter can be regenerated in the reduced form and act as an antioxidant again to scavenge more free radicals in the cells (Reid et al., 1994). Although NAC is used as a supplement in the market to prevent cancer, it accelerates lung cancer cell proliferation in mice (Sayin et al., 2014). Pharmaceutical-grade NAC is mainly used as a mucolytic agent in respiratory conditions, to counteract acetaminophen overdoses, and to avoid radio-contrast-induced renal side effects (Sansone et al., 2011).

Figure 1.6. The chemical structure of antioxidants used in this dissertation.

40

NOX4 is a major player in oxidative stress induced in carcinogenesis, lung airway remodeling, vascular integrity and heart failure (Amara et al., 2010; Kuroda et al., 2010;

Touyz et al., 2011; C. Zhang et al., 2014). However, the exact mechanism of its mediated effects is not fully understood.

NOX4

NOX is a protein family that includes seven members: NOX1, NOX2, NOX3,

NOX4, NOX5, Duoxes 1, and Duoxes 2. NOX enzymes are transcribed by seven NOX genes in humans and 6 genes in mice, because mice lack NOX5 (Bernard et al., 2014).

NOX4 is located as transcellular protein, and it transports electrons across cellular membranes, resulting in reduction of oxygen into superoxide (Panday et al., 2015). NOX proteins have a conserved structure consisting of a cytosolic C-terminus with NADPH and

Flavin adenine dinucleotide (FAD) binding sites, six transmembrane domains with four histidines that provide two heme-binding sites, and a cytoplasmic N-terminus (Touyz et al., 2011). NADPH molecules in the cytosol provide the electrons to the NOX isoforms, and the oxygen molecules are the terminal receptors of these electrons which results in superoxide formation (Fig. 1.7).

41

Figure 1.7. NOX4 structure and function. Adapted from (Chen et al., 2012).

NOX1, NOX2, and NOX5 release superoxides as a final product while NOX4,

Duoxes1 and 2 release H2O2 due to a possible dismutase activity in the extra loop (E loop) of their structures (Drummond et al., 2011). Different NOX isoforms have different expression levels and roles in several physiological and pathological conditions (Panday et al., 2015). For example, endothelial cells express NOX1, 2, 4 and 5, while fibroblasts express NOX2 and 4 (Panday et al., 2015). NOX2 is located in the endothelial cells lining the blood vessels and inhibits microbe migration from the bloodstream into the surrounding tissues (Drummond et al., 2011). NOX4 enhances endothelial cell survival, propagation, and migration in addition to mediating fibroblast differentiation (Hecker et al., 2009; Touyz et al., 2011).

NOX4 is characterized by its constitutive activity, production of H2O2 as the main reactive oxygen species, and its regulation through transcription levels (Chen et al., 2012). 42

NOX4 is found in the nucleus, mitochondria, stress fibers, and vascular focal adhesions

(Lyle et al., 2009). TGF-β highly increases the expression level of NOX4 in fibroblasts, resulting in their differentiation and enhancing the ECM protein secretion (Amara et al.,

2010).

Physiologically, the NOX-mediated production of ROS is an inflammatory response that is involved in killing the invading microorganisms in macrophages and neutrophils. However, increased NOX4 expression is linked to the pathogenesis of many diseases, including diseased prostatic stroma (N. Sampson et al., 2011), idiopathic pulmonary fibrosis (Amara et al., 2010; Panday et al., 2015) and asthmatic smooth muscle contractility (Sutcliffe et al., 2012). In addition, NOX4 expression is increased in airway smooth muscle of asthmatic patients compared to non-asthmatic people (Sutcliffe et al.,

2012). TGF-β1 enhances NOX4 expression in lung mesenchymal cells through the

SMAD3 pathway, and NOX4-mediated release of H2O2 is essential for TGF-β1–mediated myofibroblast differentiation, ECM protein synthesis, and cell contractility (Hecker et al.,

2009). On the other hand, lung fibrosis is attenuated by the bioactive eicosanoid PGE2 through its inhibition of fibroblast to myofibroblast differentiation. However, the exact role of NOX4 in asthmatic airway remodeling and the mechanism by which PGE2 inhibits fibroblast to myofibroblast differentiation are not known. Chapter V of this dissertation will demonstrate how PGE2 attenuates NOX4-mediated airway remodeling in asthma.

This dissertation will discuss the role of eicosanoids in acute inflammatory responses in MC-dependent pathways in Chapters III and IV and airway remodeling in

Chapter V, both of which contribute to asthma pathogenesis. 43

CHAPTER II

MATERIALS AND METHODS

Reagents

Penicillin-streptomycin and L-glutamine were purchased from Thermo Fisher

Scientific. LTD4, PGE2, MK571 (Cat. # 70720), BaycysLT2 (Cat. # 10532), iloprost, butaprost, sulprostone, L-798, ONO-8711, L-161, and PGD2 Enzyme linked immunosorbent assay (ELISA) kits were purchased from Cayman chemicals (Ann Arbor,

MI). KT5823, (PTX), H7, GF109203X, Rp-cAMPS, H89, and Y27632 dihydrochloride antagonists were from Tocris Bioscience (Minneapolis, Minn); PD98059 was purchased from Calbiochem (San Diego, CA). Fura-2 AM was from Molecular Probes

(Eugene, Ore), c-fos, COX-2, MRTF-A, PAI-1 and all phospho-specific antibodies were obtained from Cell Signaling Technology (Danvers, Mass); α-SMA was from

Sigma-Aldrich (St. Louis, MO), FN antibody was from Abcam (Cambridge, MA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Fitzgerald

(Acton, Mass). All secondary antibodies were purchased from Jackson ImmunoResearch

(West Grove, Pa). Nonspecific small interfering RNA (siRNA) and isoform-specific siRNAs for CysLT1R, EP3, PKG and NOX4 were obtained from Dharmacon (Lafayette,

Colo), and MIP-1β ELISA kit, TGF-β1, and recombinant human SCF were purchased from

R&D Systems (Minneapolis, Minn). IL-5 and IL-10 for human cord blood–derived mast cell (hMC) cultures were purchased from PeproTech (Rocky Hill, NJ). Dermatophagoides farina allergen (Der. f) protein extract was purchased from Greer Laboratories (Lenoir,

44

NC). Sterile phosphate buffered saline (PBS) was purchased from Corning (Corning, NY).

Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA)

Table 2.1. Antagonists/inhibitors

Antagonists/Inhibitors Target Working concentration

MK571 (MK) CysLT1R 1 µM

BaycysLT2 (Bay) CysLT2R 1 µM

L-798 EP3 100 nM

ONO-8711 EP1 2 nM

L-161 EP4 100 nM

PD98059 (PD) MAPK 50 µM

Pertussis toxin (PTX) Gαi 100 ng/ml

H7 PK (general) 10 μM

Rp-cAMPS PKA 50 μM

H-89 PKA 1 μM

KT5823 (KT) PKG 5 μM

GF109203X (GFX) PKC 2 µM

Y27632 dihydrochloride Rho kinase 10 µM

45

H 89 dihydrochloride

KT5823 GF109203X

PD98059 Y27632 dihydrochloride H-7 dihydrochloride ONO-8711

Rp-cAMPS, L-161 triethylammonium salt L-798

Figure 2.1. Structure of the most used inhibitors.

Animals

Six- to eight weeks old BALB/c mice, C57BL/6 mice and KitW-sh mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were maintained at constant humidity (60%) and (20°C) on a 12- h /dark cycle at the Comparative

Medicine Unit, Northeast Ohio Medical University. KitW-sh mice were housed in a barrier facility under specific pathogen free conditions. All animal experiments were performed according to the standard guidelines, as approved by the Animal Care and Committee of

Northeast Ohio Medical University.

46

Intradermal injection of agonists and assessment of ear edema

Six- to eight weeks old WT BALB/c female mice, WT C57BL/6 female mice and

KitW-sh female mice were anesthetized by intraperitoneal injection of 100 μl of ketamine

/xylazine, then intradermal injections of 0.5 µM LTD4, PGE2, or LTD4 plus PGE2 (in a 10-

µL volume) were performed on the right ear and 10 µL of saline was injected in the left ear. In experiments with antagonists, MK571, L-798, or both were injected 30 minutes before the agonists. Afterward, ear thickness was measured with a caliper at 0, 30, 60, 120,

240, and 300 minutes, as a measure of inflammatory edema. Mice were sacrificed after 1 hour (maximum response) by intraperitoneal injection of 100 μL Fatal-Plus. The ears were harvested and fixed in 4% paraformaldehyde for at least 24 h.

Der. f induced airway remodeling

Six to eight weeks old WT C57BL/6 female mice were anesthetized using isofluorane vaporizer. Then, 25 µg/animal in 12.5 µL volume or 12.5 µL of saline were administered to the mice nostrils (intranasally) three times a week for 5 weeks (in studying airway remodeling, Chapter V experiments), or 5 µg/ animal of Der. f with/without

1nanomole LTD4 plus 1nanomole PGE2 in 15 µL total volume or 15 µL of saline were administered to the mice nostrils (intranasally) twicw a week for 5 weeks (Chapter III experiments). After the 5th week, mice were euthanized by intraperitoneal injection of 100

µL Fatal-Plus solution. Bronchoalveolar lavage (BAL) fluid was collected and lungs were dissected and stored in -80ºC for later RNA extraction and quantitative polymerase chain reaction (qPCR) to measure gene expression.

47

BAL Fluid Collection

BAL fluid was collected and cellular content was analyzed. After animal was euthanized, skin was cut by scissors to expose the thoracic cage and neck, and fatty tissue layers were removed to expose the trachea. A one-inch long thread was slid underneath the trachea by forceps, a small cut was made in the upper part of the trachea, and a 20-gauge lavage tube was inserted and tied by the thread to prevent dislodging. Then 1 mL of PBS was slowly injected using an “input” 1 mL syringe via 3-way stopcocks to flush the lung and dislodge all the cells, then BAL fluid was collected using an “output” 1 mL syringe into a 1.5 mL Eppendorf tube on ice. Following BAL fluid collection, total cell number was determined by trypan blue staining, then BAL fluid was cytospun (Cytospin; Thermo

Fisher Scientific) at 550 x g for 5 min at room temperature onto glass slides where they were stained with a Diff Quik Stain Set according to manufacturer protocol. Briefly, slides were dipped consequently in the methanolic fixative 5 times for 1 second each time, buffered solution of Eosin Y 5 times for 1 second each time, buffered solution of thiazine dyes that has methylene blue and azure 5 times for 1 second each time, and finally in water

3-4times for 1 second each time. Slides were allowed to dry and then examined under a microscope and cells were counted. A minimum of 300 cells were counted and differentiated according to their morphology as macrophages, lymphocytes, or polymorphonuclear leukocytes (PMNs), which include eosinophils and neutrophils. The number of differential cells in the BAL fluid was determined from total cell counts and expressed per milliliter of BAL fluid.

48

Tissue processing

The fixed ear tissues were dehydrated through consecutive dehydrating conditions manually to enable paraffin infiltration. Tissues were respectively washed 3 times in PBS for 5 minutes with shaking, 50% ethanol for 20 minutes, 70% ethanol 20 minutes, 80% ethanol 20 minutes, 90% ethanol for 20 minutes, 100% ethanol for 30 minutes three times.

Tissues were then washed with Xylene for 20 minutes for three times. Tissues were then treated with Xylene and molten paraffin wax mix for 1 h at 60-65°C (Incubator) and molten paraffin wax overnight at 60-65°C.

Tissue embedding

After tissue processing, tissues were placed in metal molds and embedded in molten paraffin wax (leica Biosystems, Richmond, IL) at 60-65°C. Next, embedded tissues were cooled for 1 h at room temperature, then at 4°C for several hours. When the blocks were set, they were stored at room temperature until sectioning.

Tissue sectioning

Prior to sectioning, the paraffin blocks were removed from the metal molds and placed in ice cold water to harden the wax and equilibrate the densities across the surface of the block. Tissues were cut into 5 µm sections and floated in a 40°C water bath to smoothen any wrinkles in the sections. Afterward, the sections were lifted out onto

Superfrost plus slides (Thermo Scientific) and placed on slide holder until the sections were completely dry. The slides were saved in slide boxes at room temperature until stained for hematoxylin and eosin (H&E) and toluidine blue (to detect MCs).

49

Tissue staining

The H&E staining was performed to examine the tissue morphology. Hematoxylin is a basic purple dye that stains the basophilic/negatively charged components like nuclear chromatin of cells and tissue. Eosin is an acidic red dye that preferentially stains the acidophilic/positively charged cytoplasmic components (Fischer et al., 2008). Toluidine blue staining was performed to check the recruitment of MCs to the ear of the mice after the experiments involving intradermal injection of PGE2 or LTD4.

Slides deparaffinization and rehydration

Slides of paraffin sections were placed in a solvent safe plastic slide holder and then were placed in a 60°C incubator for 2 h. Tissue sections were deparaffinized and rehydrated by dipping consecutively 3 times in Xylene for 3 minutes, 3 times in 100% ethanol for 3 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, 50% ethanol for 3 minutes then deionized H2O for 5 minutes. Excess water was blotted on kimwipes and slides were then stained.

H&E staining

Deparaffinized and rehydrated slides were stained with Hematoxylin (Poly

Scientific R&D Corp, Bay Shore, NY) for 3 minutes. Slides were then washed with deionized H2O for 1 minute and with tap water for 5 minutes to allow the stain to develop.

Slides were then dipped quickly in 1% acid-ethanol for 8‐12 times to de-stain, followed by tap water rinsing for 1 minute twice, then deionized water for 2 minutes. Excess water was blotted on kimwipes. Slides were stained with eosin (Poly Scientific R&D Corp, Bay

Shore, NY) for 30 seconds then dipped 3 times in 95% ethanol for 5 minutes, 3 times in

50

100% ethanol for 5 minutes, followed by 3 times in Xylene for 15 minutes. Finally, slides were covered with Permount (Xylene based), and coverslips were placed carefully to avoid forming any bubbles. Slides were dried in a hood overnight. Slides were then visualized, and images were taken under a bright field inverted microscope at Χ 20 objective.

Toluidine blue staining

Toluidine blue staining was used to determine hMC maturity and ear sections of mice that were deparaffinized and rehydrated. To determine hMC maturity, 200 µL of PBS was added to the cytospin chamber and cytocentrifuged (Cytospin, ThermoFisher

Scientific, Waltham, MA) at 550 rpm for 5 min at room temperature to moisten the filter papers in the cytospin. Then, 2-5 x 10 4 cells in 200 µL media were cytospun at 550 rpm,

5 min at room temperature.

Cytospun cells and the slides of deparaffinized and rehydrated tissues were air dried then fixed by 4% paraformaldehyde for 15 minutes. After fixing, slides were washed 3 times by gently dipping them in fresh distilled water. Slides were air dried and stained with toluidine blue (Amresco, Solon, OH) for 1 h. Following that, any extra stain was washed from the slides by immersing them in fresh distilled water for 5 times and then allowing them to air dry. Cover slips then were mounted using Permount.

For tissue sections, toluidine blue stained total (all toluidine blue–positive cells) and degranulated MCs (toluidine blue-positive cells with disrupted and diffused staining) sections were visualized at Χ 60 magnification; counted by a blinded participant; and expressed as the MC number per mm.

51

Cell culture and activation

The LAD2 MC line

The LAD2 MC line was a gracious gift from Dr. Arnold Kirshenbaum (National

Institutes of Health). These cells were cultured in StemPro-34 (Invitrogen) complemented with Pen-strep (100 IU/mL), 2mM L-Glutamine and 100 ng/mL SCF (R&D systems). Cell culture medium was hemi-depleted weekly by adding new medium and SCF. Cells were counted and kept in culture at 1x106/mL density. Cells were seeded and SCF-starved overnight before stimulation in all the experiments. For each well/tube to be treated, cells were used at the following densities: 250,000 cells/250µL for western blotting, 1x106/mL for qPCR, 100,000 cells/100µL/well of 96 well plate for ELISA, and 5000/100µL/well of

96 well plate for XTT.

Human cord blood–derived mast cells

Cord blood was obtained from Cleveland Cord Blood Center. Primary hMCs were isolated from cord blood mononuclear cells. 60 cc syringes with 1cc heparin and 19G- butterfly needle were prepared. 40 mL blood was aspirated in each syringe. Then 10 cc of

4.5% dextran (9.0 g of dextran (Fisher) in 200 mL water) was added to each syringe, butterfly needles were removed, and syringes were capped and incubated at 37°C upright for 1 h. Following that, 20 mL of filtered Ficoll-Hypaque (Pharmacia) was added into 50 mL conical tubes (20 mL is needed for each 40-mL blood). A serum/buffy coat containing a mononuclear cell layer at the interface was gently layered on the Ficoll using fresh 19-G butterfly needles. Tubes were centrifuged at 1500 RPM for 30 minutes at room temperature, then serum was aspirated carefully to maintain the interface of mononuclear cells. The interface was pipetted using 10 mL pipettes and pooled into one 50 mL conical 52

tube. PBS was added to a final volume of 50 mL, then the tubes were centrifuged at 1000

RPM, 10 minutes at room temperature. PBS was aspirated and the pellet was lysed hypotonically using 1 mL of 0.2 % filtered NaCl and gently swirled for 1 minute, followed by 1 mL of 1.6 % filtered NaCl with gently swirling for 30 seconds. After that, 48 mL of

PBS was added and the cells were spun at 1000 RPM for 10 minutes at room temperature.

Cells were washed once with 5 mM EDTA/PBS (5 mL of 0.5 M EDTA (Amresco®, Solon,

OH) in 500mL PBS) and spun at 1000 RPM for 10 minutes at room temperature. Finally, cells were counted and re-suspended at 1x106 cells/mL density in RPMI 1640 media supplemented with 10% FBS, 2mM L-Glutamine, Pen-Strep (100UI/mL), MEM Non-

Essential Amino Acids (5 mL of 100 X; Stem Cell Technologies, Vancouver, Canada,),

Gentamicin (2 µg/ml, MP Biomedicals, Solon, OH) and β-Mercaptoethanol (50 μM,

Sigma, St. Louis, MO). SCF (100 ng/mL), IL-6 (50 ng/mL) and IL-10 (10 ng/mL) were added freshly every week. Cells were incubated in a humidified 37°C, 5% CO2 incubator.

Cells were passaged weekly into fresh media containing fresh cytokines and SCF

Human Lung Fibroblasts

Human lung fibroblasts (hLF; CC-2512) were purchased from Lonza

(Walkersville, MD). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)

(Corning) supplemented with 10% FBS, 100 U/mL penicillin-streptomycin, and 2 mM L- glutamine. Cells were kept at 37°C in a humidified 5% CO2 incubator. Media was changed every other day and when cells became confluent, they were passaged at a 1:5 ratio. All cells that were used were below passage 8 for all the experiments. Cells were seeded at

75,000 cells/2 mL/well of a 6 well plate for western blotting and qPCR. After 24h, cells were serum starved in DMEM media supplemented with 0.4% FBS, 2 mM L-glutamine, 53

and 100 U/mL penicillin-streptomycin overnight and then treated in DMEM supplemented with 1% BSA, 2 mM L-glutamine, and 100 U/mL penicillin- streptomycin. siRNA treatment

For LAD2 cells, cells (1*106/well) were seeded overnight and transfected on the next day with the non-specific (NS) or specific siRNA that targets EP3, CysLT1R or PKG in 1 mL of OptiMEM media for 6 h. Then, 1 mL/well of StemPro complete media was added to the OptiMEM media. 24h later, cells were centrifuged and kept in complete Stem-

Pro media for 24 h followed by SCF starvation overnight, treatment for the mentioned time points and lysis.

For hLF cells, cells were seeded (75,000/well) and when cells were 70% confluent, they were treated with NOX4 - specific siRNA (100 nM, Dharmacon, Cat # M-010194-

00) in 1 mL of OptiMEM media / well. After 6 h, 1 mL of DMEM supplemented with 10

% FBS, L-Glutamine, and penicillin - streptomycin was added to each well. On the following day, media was changed to DMED with 10% FBS, L-Glutamine, and penicillin

– streptomycin. On the next day, cells were treated with TGF-β1 for 48 h and were lysed.

Cell lysates and Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE)

MCs were centrifuged after treatment, and the media was aspirated. Then, 150 µL of lysis buffer (1X Rippa lysis buffer plus 1 X of protease and phosphatase inhibitors) was added in addition to 50 µL of 4 X Laemmli sample buffer (Boston Bio Products, Ashland,

MA). hLF were washed with PBS after treatment, and then lysed while scraping the plate

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in ice using 150 µL lysis buffer for each well. Then, 50 µL of 4 X Laemmli sample buffer was added.

Samples were vortexed, centrifuged and heated for 6 minutes at 95°C. 15 µL of the lysates mix were loaded along with 5µL of molecular weight marker (BIO-RAD, Hercules,

CA) into 4-15% SDS-PAGE gels (BIO-RAD, Hercules, CA) with 1 X running buffer.

SDS-PAGE gels were run at 100V until the dye front ran off the gel bottom.

Western blotting

After running the SDS-PAGE, proteins were transferred to Polyvinylidene

Difluoride (PVDF) membranes (Millipore, Billerica, Mass.). A transfer sandwich was created in cold transfer buffer (Table 2.2) as follows: a sponge, three blotting papers, the gel, the charged PVDF membrane, another three blotting papers and finally another sponge.

The cassette was closed and moved into a transfer apparatus that had a magnetic stirrer, ice pack and filled with pre-chilled transfer buffer. The transfer apparatus was placed on ice and on a magnetic stirring plate and then connected to the electrodes and ran at 100 V for

1.5 h.

Table 2.2. Buffers used for western blotting

Buffer Reagents 20% SDS 20 g SDS (Sigma Aldrich, L3771) Volume made up to 100 mL with water Running buffer 14.42 g Glycine (Fisher) 3.03 g Tris-base (Fisher) Volume made up to 1 L with water Transfer Buffer 14.42 g Glycine (Fisher)

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3.03 g Tris-base (Fisher) 200 mL methanol (Fisher, Cat# A412-4) Volume made up to 1 L with water TBS 2.442 g Tris-Base 8.766 g NaCl pH adjusted to 7.6 with HCl Volume made up to 1 L with water

Membranes were washed with PBS once for 5 minutes, then blocked with 5% w/v dry milk in 1X TBS-0.1% Tween -20 (TBS-T) for 1 h. Each primary antibody (Table 2.3) was added over a separate PVDF membrane and left at 4ºC on a shaker overnight. On the next day, membranes were washed in (TBS-T) for 15 minutes, followed by two 5 minute washes. PVDF membranes were then incubated with the proper secondary antibody diluted to 1:5000 in 5% w/v dried non-fat milk and left at room temperature for 1 h on a shaker.

The membranes were washed again in TBS-T, sealed in plastic wrap, and incubated in 1 mL of enhanced chemiluminescence (ECL) solution (Millipore, Billerica, MA) for 1 minute while covered with foil to protect from light. Bands were visualized using imager

(Protein Simple) and quantified using alpha view software. Exposure times varied from 10 seconds to 1 h. Membranes were stripped and reblotted for GAPDH to confirm equal loading.

Table 2.3. Primary antibodies for western blotting

Primary antibody Dilution Host c-fos 1:1000 Rabbit

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P-c-fos 1:1000 Rabbit p-c-kit 1:1000 Rabbit

P-Erk 1:1000 Rabbit

GAPDH 1:25,000 Mouse

COX-2 1:1000 Rabbit

αSMA 1:35,000 Mouse

FN 1:2000 Rabbit

MRTFA 1:1000 Rabbit

PAI-1 1:1000 Rabbit

ELISA

Concentrations of PGD2 and MIP-1β in the supernatants of treated LAD2 cells were assayed by using the PGD2-MOX and MIP-1β ELISA kits according to the manufacturer’s protocols.

For MIP-1β measurement, 96 well plates were pre-coated with mouse anti- human

MIP-1β capture antibody diluted in PBS (1.0 μg/mL) overnight on a shaker at room temperature. On the next day, plates were washed three times with PBS-0.05 % Tween®

20 and blocked with the reagent diluent which consists of 1% BSA in PBS-0.05 % Tween®

20 for 1 h at room temperature. 100,000 cells in 100 µL for each treatment were seeded and treated for 6 h. Supernatants of these cells were applied to previously coated, blocked and washed ELISA plates in 1:20 dilution in reagent diluent along with the standards at

100 μL volume and incubated for 2 h at room temperature. The plates were washed 3 times

57

and incubated with biotinylated goat anti-human MIP-1β detection antibody diluted in reagent diluent (50 ng/mL) for 2 h at room temperature on shaker. Then, the plates were washed and incubated with streptavidin conjugated horseradish-peroxidase secondary antibody for 1 h at room temperature on shaker. Plates were washed again and TMB substrate was added and once the blue color developed, a stop solution of 2 N H2SO4 was added and the plates were read on a Biotek Epoch spectrophotometer plate reader at 450 nm along with 540 nm correction wavelength.

For PGD2 measurement, PGD2 secreted in the medium was converted into the more stable PGD2-MOX form to prevent further degradation. Then, PGD2 was assessed using the PGD2-MOX ELISA assay that depends on the competition between PGD2-MOX and

PGD2-MOX-acetylcholinesterase (AChE) conjugate (PGD2-MOX tracer) to bind to a restricted number of PGD2-MOX-specific rabbit antiserum binding sites. When the substrate for AChE is added, a yellow color develops adversely to the amount of PGD2-

MOX in the samples.

Table 2.4 lists all the blanks and reagents used. First, reagents (1) in Table 2.4. were added to the commercially coated plates, then plates were covered and incubated overnight at room temperature. Next day, plates were washed and reagents (2) shown in Table 2.4. were added. Plates were covered and incubated for 90-120 minutes at room temperature before being read at 412 nm on plate reader.

Table 2.4. PGD2-ELISA reagents.

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Total Non- Maximum Reagent Blank activity specific binding Standard/sample binding EIA Buffer - - 100 µL 50 µL - Standard/Sample - - - - 50 µL 1 Tracer - - 50 µL 50 µL 50 µL AntiSerum - - - 50 µL 50 µL

Tracer - 5 µL - - - 2 Ellman’s reagent 200 µL 200 µL 200 µL 200 µL 200 µL

qPCR

RNA extraction from stimulated cells

After stimulation, cells were lysed using 350 µL of TRK lysis buffer plus 0.2 % β- mercaptoethanol, followed by either storing them at -80ºC or extracting RNA on the same day. Total RNA kit I (Omega bio-tek, Cat. No. R6834-02) was used to extract RNA from the lysates according to the manufacturer instructions. Simply, lysates were homogenized using homogenizer columns followed by adding equal volume of 70% ethanol to the eluent.

The mixture of the lysate and the ethanol was transferred into the HiBind columns and then washed with wash buffer I. The RNA was treated with RNase free DNase I set (Omega bio-tek, Cat. No. E1091-02) for 15 minutes to remove any DNA contamination. Another washing step with wash buffer I was done followed by two washes of wash buffer II and drying the tubes. Finally, the RNA was eluted using 40 µL DEPC water.

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RNA extraction from lung tissues

100 mg of frozen lung tissue (Der. f experiments) was homogenized in 1 mL Trizol.

Then, 0.2 mL of chloroform was added followed by a centrifugation step that resulted in the formation of 3 phases with the upper clear aqueous phase containing the RNA. RNA was transferred to a fresh tube and incubated with 0.5 mL isopropyl alcohol for 10 minutes followed by centrifugation which led to RNA precipitation. Then, the RNA pellet was washed with 1 mL of 75% ethanol and centrifuged again to precipitate the RNA. The pellet was dried at room temperature for 20-30 min., then dissolved in 100 µL DEPC water.

Following RNA isolation, the samples were treated with DNase using Ambion

DNA-free™ DNA Removal Kit (ThermoFisher). Simply, 0.1 volume of 10 X DNase I

Buffer and 1 µL of rDNase I were added and mixed with the RNA, and incubated at 37ºC for 25 minutes. Then, 0.1 volume of DNase Inactivation Reagent was added and mixed.

The mixture was incubated for 2 min. at room temperature and then centrifuged at 10,000 x g for 1.5 minutes. The RNA was passed through RNeasy MinElute spin columns

(Qiagen) and was quantified using the Take-3 program- Gen5 Software on Bio-Tek Epoch

Microplate Spectrophotometer at absorbance of A260 and RNA purity is assessed using the

A260/A280 with the ratio of 1.8-2.1 is indicative of highly purified RNA.

Reverse transcription

1 µg of total RNA was used for reverse transcription using cDNA synthesis kit from

Quanta Biosciences. RNA was reverse-transcribed to cDNA using Transcriptor First

Strand cDNA Synthesis Kit (Roche, Cat. No. 04 379 012 001). The template-Primer and

RT mix for each reaction is shown in table 2.5.

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Table 2.5. Reverse transcription reagents.

Reagent Volume Final Concentration Total RNA Variable 1 µg total RNA Anchored-oligo(dT)18 1 µL 2.5 µM primer, 50 pmol/ µl Random hexamer primer, 2 µL 60 µM 600 pmol/ µl

Transcriptor Reverse 4 µL 1× (8 mM MgCl2) Transcriptase Reaction Buffer, 5× concentration. Protector RNase Inhibitor, 0.5 µL 20 U 40 U/ µl Deoxynucleotide Mix, 10 2 µl 1 mM each mM each. Transcriptor Reverse 0.5 µL 10 U Transcriptase, 20 U/µl Water, PCR-grade Variable To make total volume = 20 µL

The contents of each reaction were added in 0.2 mL PCR tubes and mixed well.

The tubes were placed in a thermal block cycler with a heated lid, and the PCR reaction was programmed as 10 min at 25°C, then 30 min at 55°C followed by 5 min at 85°C to inactivate the transcriptor reverse transcriptase. The reaction is stopped by placing the tube on ice or storing the samples at -20°C for later use.

For qPCR, a 96 well PCR plate was used and each well/reaction was loaded with

7.5 µL SYBR green mix (2X), 4.5 µL water (PCR grade), 1 µL cDNA and 2 µL primer

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mix consisting of 10 µM of the forward primer, and 10 µM of the reverse primer (Table

2.6).

Table 2.6. Primer sequences for target genes.

Target Primer sequence hMIP-1β F: 5’-CCA GCC AGC TGT GGT ATT-3’

R: 5’-CAG TTC AGT TCC AGG TCA TAC A-3’ hTNFα F: 5’-CCA GGG ACC TCT CTC TAA TCA -3’

R: 5’-TCA GCT TGA GGG TTT GCT AC -3’ hCOX-2 F: 5’-CAA CTC TAT ATT GCT GGA ACA TGG A-3’

R: 5’-TGG AAG CCT GTG ATA CTT TCT GTA CT-3’ hCysLT1R F: 5’-TCA ACG TAC CAT TCA CCT TCA T-3’

R: 5’-GCA GCC AGA GAC AAG GTT AT-3’ hCysLT2R F: 5’-CGA CAT GGA AAG TGG GTT TAT G-3’

R: 5’-GCA AAG TAA TAG AGC AGA GGA TTG-3’ hIL-8 F: 5’-TTT GCC AAG GAG TGC TAA AGA-3’

R: 5’-CCA CTC TCA ATC ACT CTC AGT TC-3’ hEP1 F: 5’-TTG TCG GTA TCA TGG TGG TGT CGT-3’

R: 5’-ATG TAC ACC CAA GGG TCC AGG AT-3’ hEP2 F: 5’-ACC CTT GGG TCT TTG CCA TCC TTA-3’

R: 5’-AGG TCA GCC TGT TTA CTG GCA TCT-3’ hEP3 F: 5’-TGG ATC CTT GGG TTT ACC TGC TGT -3’

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R: 5’-AGG TGG AGC TGG ATG CAT AGT TGT -3’ hEP4 F: 5’-TGG TGC GAG TAT TCG TCA ACC AGT -3’

R: 5’-CAA TGC GGC AGA AGA GGC ATT TGA -3’ hGAPDH F: 5’-TGC ACC ACC AAC TGC TTA GC-3’

R: 5’-GGC ATG GAC TGT GGT CAT GAG-3’ hSM22 F: 5’-TGGAGATCCCAACTGGTTTAT-3’

R: 5’-CCCATCTGAAGGCCAATG-3’ mNOX4 F: 5’-CGG GAT TTG CTA CTG CCT CCAT-3’

R: 5’-GTG ACT CCT CAA ATG GGC TTC C-3’ mGAPDH F: 5’-CTCCCACTCTTCCACCTTCG-3’

R: 5’-CCACCACCCTGTTGCTGTAG-3’

Primers for hCOX-1, hPKG-1 and hNOX4 were obtained from QIAGEN (Cat #

PPH01306E, PPH00491C, and PPH06078A respectively). SYBR green PCR master mix was from Quanta Biosciences.

PCR was run using the Roche light cycler® 480 II machine and preincubated at

95°C for one cycle, followed by 45 amplification cycles with each cycle consisting of incubation at 95°C, 60°C, and 72°C respectively for 10 seconds each. No template control and no reverse transcriptase control were included with each qPCR run to ensure that the solutions used for PCR were not contaminated.

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Calcium flux measurement

LAD-2 cells (0.5-1 x106 per sample) were washed twice with CM buffer (500 mL

HBSS, 1 mL of 0.5 M CaCl2, 0.5 mL 1M MgCl2 (Aldrich Chemical Company Inc.), 0.5 g

BSA (Fisher)) and then loaded with a Ca2+ sensitive ratiometric dye, FURA-2AM

(Molecular Probes, cat#F-1221), as 2.5μL of stock per 1 mL of cells. Cells were wrapped with foil and incubated in a 37°C water bath to allow the dye to penetrate the cells. Cells were washed with excess cold CM buffer then were suspended in 0.5 mL/sample in CM buffer, wrapped with foil and kept on ice. Cell samples were loaded in the cuvette with a magnetic stirrer. Cells were pretreated with inhibitors for 10 min. before agonists. The cuvette was placed in the machine and the baseline fluorescence was measured. After 30 seconds of baseline fluorescence, agonists were added and fluorescence was read.

Changes in intracellular calcium were measured by assessing the emitted fluorescence at 510 nm with alternating the excitation between 340 and 380 nm. The intracellular calcium levels were calculated by taking the 340/380 fluorescence ratio.

XTT Proliferation Assay

TACS® XTT cell proliferation assay (Treviagen®, Gaithersburg, MD) was used to measure cell proliferation rate. Briefly; cells were seeded overnight in SCF free medium in 96 well plates (5000 cell in 100µL per well) along with 3 control wells having 100 μL of growth medium only as blank absorbance readings. On the next day, cells were treated as four replicates for each treatment for 72 h. Activated-XTT solution was prepared by mixing 0.1 mL of the activation reagent to 5.0 mL of the XTT reagent, then 50 μL of the activated-XTT solution was added to each well. Cells were incubated at 37°C for 2 h and

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then the plates were read on the Biotek Epoch spectrophotometer plate reader at 475 nm along with 660 nm correction wavelength.

Statistical Analysis

The data was analyzed using GraphPad Prism 7 software (GraphPad, San Diego,

USA) and is represented as mean ± SEM of at least three different experiments. Data was analyzed using two-tailed student’s t-test to compare the means of two sample, one-way

ANOVA followed by post-hoc Tukey analysis to compare the means of more than two samples. Statistical significance was set at * P<0.05; ** P<0.01; *** P<0.001.

In the next chapters of this dissertation, we will demonstrate the results of the following projects:

1. LTD4 and PGE2 synergism in enhancing mast cell inflammatory responses in vitro and vascular edema and der. f induced inflammation in vivo which is published by Elsevier Science Incorporation in the journal of and clinical immunology and data are used with agreement.

2. Modulation of mast cell proliferative and inflammatory responses by leukotriene D4 and stem cell factor signaling interactions which is published by John Wiley and Sons in the journal of cellular physiology and data are used with permission.

3. PGE2 role in NAD(P)H oxidase 4 (NOX4)- regulated asthmatic airway remodeling.

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CHAPTER III

LTD4 AND PGE2 SYNERGIZE TO ENHANCE MAST CELL INFLAMMATORY

RESPONSES IN VITRO AND VASCULAR EDEMA AND DER. F INDUCED

INFLAMMATION IN VIVO

Introduction:

MCs are key inflammatory cells of allergic and inflammatory reactions, derived from myeloid progenitor cells and differentiate upon the effect of growth factors such as

SCF and IL-4 (Bischoff, 2007; Lewis et al., 2013). MCs reside at the body barriers such as skin, vascular and mucosal sites. They bind to the IgE via FcɛRI receptors and they secrete many inflammatory mediators including histamine, cys-LTs, and PGs (Bischoff, 2007;

Gilfillan et al., 2006).

Cys-LTs are inflammatory mediators composed of LTC4, LTD4 and LTE4, with

LTD4 being the most potent cys-LT (D. Wang et al., 2010). Cys-LTs are derived from arachidonic acid via the 5-LOX pathway (Paruchuri et al., 2008) and they are produced in many immune cells such as MCs, macrophages, basophils and eosinophils (Kanaoka et al.,

2014). Cys-LTs mediate their effect through two main GPCRs: CysLT1R,which binds

LTD4 with higher affinity than LTC4 and has low affinity to LTE4, and CysLT2R, which has equal affinity for LTC4 and LTD4 and low affinity to LTE4 (Paruchuri et al., 2008). A newly discovered CysLTR is GPR99, which binds preferentially to LTE4 (Kanaoka et al.,

2013).

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Another group of arachidonic acid derivatives are PGs, which are produced under the COX pathway. PGs consist of PGD2, PGE2, PGF2 and PGI2 (D. Wang et al., 2010).

PGE2 is the most predominant PG in acute inflammatory sites (Stables et al., 2011). PGE2 acts through four GPCRs, EP1, EP2, EP3 and EP4 (D. Wang et al., 2010). The common

2+ signaling pathways for EPs are as follows: EP1 enhances the Ca flux through coupling to the Gαq subunit, EP2 and EP4 increase cAMP through coupling to the Gαs subunit, and EP3 decreases cAMP through coupling to the Gαi subunit (Sugimoto et al., 2007). The specific action of each prostaglandin in each particular tissue is dependent upon the specific EP receptor expressed by that tissue (D. Wang et al., 2010). PGE2 has both protective and potentiating inflammatory effects in asthma. It contribute to asthma pathogenesis by enhancing IgE production in B cells (Gao et al., 2016) and histamine release by MCs (X.

S. Wang et al., 2006). However, inhalation of PGE2 attenuated allergen-(Gauvreau et al.,

1999) and exercise- (Melillo et al., 1994) induced bronchoconstriction in asthmatic patients. Therefore, further elucidation of PGE2’s role in asthma is required for a better understanding of the disease.

The number and activity of MCs are increased in the airways of asthmatic patients

(Bradding et al., 2006). Cys-LTs augment asthma inflammation via enhancing vascular permeability, increasing goblet cell mucus secretion, and decreasing mucociliary clearance

(M. Liu et al., 2015). In addition, PGE2 has multiple roles in peripheral vascular leakage in mouse ear skin, depending on which EP receptor is activated (Omori et al., 2014). The interactions between different mediators in the inflammatory microenvironment are complex and hard to define. This chapter describes how LTD4 and PGE2 synergize to enhance both peripheral inflammation and Der. f induced lung inflammation in vivo. Also,

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a description of MC activation in vitro through CysLT1R, EP3, Gi, PKG, and Erk- dependent pathways will be demonstrated. Our results suggest that dual blocking of the

EP3 receptor and CysLT1R might be a better therapeutic option for asthma.

Results

Combined treatment with PGE2 and LTD4 synergistically potentiates peripheral vascular inflammation in mice

Increased vascular permeability is a feature of acute inflammatory responses. To check possible cross-talk between LTD4 and PGE2 in mediating peripheral vascular edema, the ear of BALB/c mice were injected with saline, PGE2, LTD4, or LTD4 plus PGE2. Then, the tissue edema was measured using calipers which is an indirect way to measure the vascular permeability in vivo. All treatments of LTD4, PGE2, and LTD4 plus PGE2 had the same patterns of increased ear thickness; peaking at 30 minutes and back to the normal levels at 5 h. However, the maximum peaking magnitude was observed with LTD4 plus

PGE2 compared to either LTD4 or PGE2 alone (Fig 3.1 A). The induced ear thickening with

LTD4 plus PGE2 was distinguished by being quick, transient, and about 6-fold higher compared to the control values in the first 30 min. Histological analysis of ear tissues showed a widening of the extracellular space matching with ear thickness (Fig 3.1 B). In addition, the number of degranulating MCs was increased with LTD4 plus PGE2 treatment

(Fig 3.1 B, middle and bottom right panel, arrows point to degranulating MCs, and Fig 3.1

C) compared with other groups, suggesting that LTD4 plus PGE2 can synergize in inducing vascular edema and inflammation through their action on MCs. To further clarify the role of MCs in PGE2 and LTD4 synergism in vivo, the above experiment was repeated in

C57BL/6 and KitW-sh mice (on C57BL/6 background). C57BL/6 WT mice showed the same 68

response of ear edema as BALB/c mice with all the agonists, but the response was less and more transient (Fig 3.1 D). Fascinatingly, LTD4 plus PGE2 together enhanced the inflammation in C57BL/6 WT mice, but the inflammation is significantly reduced in KitW- sh mice (Fig 3.1 D), suggesting that MCs are the effector cells mediating LTD4-PGE2 cross- talk.

Figure 3.1. Ear thickness in mice treated with LTD4 and PGE2. WT BALB/c mice (A-C), WT C57BL/6 and KitW-sh mice (D) were treated with Saline (Sal), 0.5 μM LTD4, 0.5 μM PGE2 or LTD4+PGE2. Ear thickness was measured using a caliper (A and D). H&E staining and toluidine blue staining for ear sections were imaged (B). Quantification (blind analysis) of MC number/mm (C). Results are mean ± SEM from 4-6 mice/group/experiment and three experiments were performed. Data was analyzed using 69

one-way ANOVA followed by post-hoc Tukey analysis.

2+ LTD4 primes PGE2-dependent Ca flux, and enhances c-fos phosphorylation and expression and MIP-1β secretion in MCs

Next, we wanted to investigate the mechanisms by which PGE2 and LTD4 activate

2+ MCs and potentiate inflammation. LTD4 induces Ca flux, c-fos phosphorylation and expression and MIP-1β secretion through CysLT1R in LAD2 cells (Kondeti et al., 2013).

To investigate if PGE2 can modulate LTD4 signal or vice versa, MCs were treated with

PGE2 and/or LTD4 and the cells inflammatory responses were checked. PGE2 induced a weak calcium signal, however, when cells were primed with LTD4, PGE2 signal was significantly amplified (Fig.3.2 A and B). Furthermore, LTD4 with PGE2 stimulation enhanced c-fos phosphorylation and expression (Fig.3.1 C and D) and increased MIP-1β secretion (Fig.3.2 E) compared to LTD4 or PGE2 alone. Of importance, hMCs also displayed increased phosphorylation and expression of c-fos upon treatment with

LTD4+PGE2 (Fig. 3.2 F and G). In addition, stimulating LAD2 cells with the same dose of LTD4 (0.5 µM) and different doses of PGE2 (0.001, 0.01, 0.1, and 0.5 µM), also enhanced c-fos phosphorylation and expression and MIP-1β secretion in a dose-dependent manner (Fig. 3.3 A and B).

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Figure 3.2. PGE2 and LTD4 synergistic effects in MCs. LAD2 cells were loaded with Fura-2AM, stimulated (at the indicated arrows) with.5 µM LTD4 followed by 0.5 µM PGE2 or vise versa, and changes in calcium concentration were measured (A and B). LAD2 cells (C-E) and hMCs (F and G) were stimulated with 0.5 µM LTD4, 0.5 µM PGE2, or both, lysed, and c-fos phosphorylation and expression were measured by western blotting, bands were quantified using alpha view software, data were represented as % control (D and G). LAD2 cells were stimulated and MIP-1β concentration was measured by ELISA (E). The data shown represents mean ± SEM of three separate experiments. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis

CysLTRs and EPs expression in LAD2 cells

To determine which receptors are mediating the crosstalk, first we checked the

LAD2 expression of CysLTRs and EPs using qPCR with specific primers for these

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receptors. Results presented that CysLT1R is expressed at a higher level than CysLT2R and all EPs are expressed, with the highest expression level for EP3 followed by EP2, EP4, and

EP1 respectively (Fig. 3.3 C).

Figure 3.3. PGE2 synergism with LTD4 in dose-dependent manner and LAD2 expression levels of CysLTR and EP receptors. LAD2 cells were treated with 0.5 µM LTD4 and increasing concentrations of PGE2 (A and B). c-fos (A) and MIP-1β (B) levels were analyzed. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis (B). The data shown represents mean ± SEM of three separate experiments. Expression levels of CysLTR and EP receptors were evaluated in LAD2 cells by qPCR (C).

Blocking CysLT1R partially attenuate the LTD4 and PGE2 synergism

To check which CysLTR mediates the synergism, LAD2 cells were pretreated with

MK571 (CysLT1R antagonist) or BayCysLT2 (CysLT2R antagonist) before stimulation

2+ with LTD4 and/or PGE2. MK571 completely blocked LTD4 mediated Ca flux, however, it partially blocked PGE2 mediated signal. However, BayCysLT2 could not block LTD4 or

2+ PGE2 mediated Ca signals (Fig. 3.4 A). In addition, the same response was found for

LTD4 and PGE2 synergistic effects in c-fos expression and phosphorylation (Fig. 3.4 B and

C) and MIP-1β secretion (Fig. 3.4 D); MK571 almost completely blocked LTD4 signal but partially reduced PGE2 and LTD4 potentiation while BayCysLT2 had no effect.

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Figure 3.4. LTD4 and PGE2 synergistic effects are partly sensitive to CysLT1R antagonism. LAD2 cells were loaded with Fura-2 AM then pretreated with MK571 (1 µM) or BayCysLT2 for 10 min. and calcium flux was measured upon stimulation with 0.5 µM LTD4 and 0.5 µM PGE2 (A). For western blotting and ELISA, LAD2 cells were stimulated with 0.5 µM LTD4, 0.5 µM PGE2, or both in the presence or absence of MK571 (1 µM) or BayCysLT2 (1 µM). Cells were lysed and c-fos phosphorylation and expression were measured by western blotting (B) and quantified by alpha-view software (C). MIP-1β secretion was measured by ELISA (D). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

EP3 receptor partially mediates PGE2 and LTD4 enhanced effects

The next aim was to determine which EP receptor is mediating the cross-talk between LTD4 and PGE2. LAD2 cells were stimulated with different EP agonists plus

LTD4. Results showed that Iloprost (Ep1/EP3 agonist) and sulprostone (EP3 agonist) but not butaprost (EP2 agonist) have effects that mimic the PGE2 synergism with LTD4 in

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increasing c-fos phosphorylation and expression (Fig. 3.5 A and B), and MIP-1β secretion

(Fig. 3.5 C). Which indicates that EP3 has a role in this synergism with a possible involvement of EP1 as well.

Figure 3.5. EP3 plays a partial role in PGE2 and LTD4 synergistic responses. LAD2 cells were treated with 0.5 µM LTD4 and/or 0.5 µM PGE2, 10 µM iloprost, 5 µM butaprost, or 100 nM sulprostone. c-fos was analyzed by western blotting (A, B) and MIP- 1β was analyzed by ELISA (C). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

Since EP1 is coupled to Gαq and EP3 is coupled to Gαi, cells were pretreated with the general Gαi inhibitor PTX. PTX showed complete blocking for PGE2, LTD4 and

PGE2+LTD4 mediated c-fos phosphorylation and expression (Fig. 3.6 A and B) and MIP-

1β secretion (Fig. 3.6 C), which increases the possibility of EP3 involvement. To further identify the exact EP receptor, cells were pretreated with different EP receptor antagonists before stimulation. Pretreatment with either ONO-8711 (EP1 antagonist) or L-161 (EP4 antagonist) had no effects on either c-fos (Fig 3.6 D) or MIP-1β (Fig. 3.6 E) increased levels. However, blocking EP3 receptor by L-798 partially attenuated PGE2 plus LTD4 synergized effects without completely blocking them (Fig. 3.7 A-C).

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Figure 3.6. Effect of Gαi, EP1 and EP4 inhibition on PGE2 and LTD4 synergism in MCs. LAD2 cells were pre-treated with PTX (100 ng/ml, 18 h; A-C), ONO-8711 (2 nM), or L- 161 (100 nM) (D, E), then stimulated with 0.5 µM LTD4 and/or 0.5 µM PGE2. c-fos (A, B, D) and MIP-1β (C, E) levels were analyzed. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

Figure 3.7. PGE2 and LTD4 synergistic responses were partially blocked by the EP3 antagonist. 75

LAD2 cells were treated with 0.5 µM LTD4, 0.5 µM PGE2 or both with/without 100 nM L-798 pre-treatment and c-fos (A, B) and MIP-1β (C) were examined. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

LTD4 and PGE2 synergistic responses were completely blocked by antagonizing both

CysLT1R and EP3 at once

Since MK571 and L-798 partially attenuated LTD4 and PGE2 synergism, we thought to check cell responses after using both of the antagonists simultaneously.

Blocking both CysLT1R and EP3 receptors led to a complete attenuation of c-fos phosphorylation and expression (Fig. 3.8 A and B), and MIP-1β secretion (Fig. 3.8 C).

Importantly, blocking both receptors also blocked the LTD4 and PGE2 synergistic c-fos phosphorylation and expression in hMCs (Fig. 3.8 D and E). These data indicate that LTD4 and PGE2 are cross-talking through both CysLT1R and EP3 receptor.

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Figure 3.8. LTD4 and PGE2 synergistic responses were completely attenuated by the combined effect of CysLT1R and EP3 antagonists. LAD2 cells (A-C) and hMCs (D and E) were pretreated with MK571 (1 μM) or/and L- 798 (100 nM) and stimulated with LTD4 (0.5 μM) ± PGE2 (0.5 μM). Western blotting was performed to measure c-fos protein (A, B, D, and E) and ELISA was used to analyze MIP- 1β secretion levels (C). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

LTD4 and PGE2 enhanced inflammatory gene expression

Cytokine and inflammatory gene expressions were checked after cell stimulation with LTD4 and/or PGE2. LTD4 enhanced MIP-1β, TNFα, IL-8 and COX-2 gene transcripts

(Fig. 3.9 A-D). However, PGE2 enhanced only COX-2 gene expression (Fig. 3.9 D). More importantly, stimulation with LTD4 plus PGE2 led to upregulation of all mentioned gene expressions (Fig. 3.9 A-D). COX-1 gene expression wasn’t changed with any of the treatments (Fig. 3.9 E). Also, the protein level of COX-2 was induced by LTD4 and highly upregulated with concurrent stimulation by LTD4 + PGE2 (Fig. 3.9 F).

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Figure 3.9. Inflammatory gene transcripts and COX-2 protein were induced by LTD4 and PGE2. LAD2 cells were stimulated with 0.5 μM LTD4, PGE2, or both. RNA was isolated followed by cDNA synthesis and qPCR analysis using the specific primers for each gene to measure the transcript level of MIP-1β, TNF-α, IL-8, COX-2, and COX-1 (A-E). COX-2 protein level was determined (F) in cell lysates. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

LTD4 and PGE2 enhanced PGD2 secretion and Erk phosphorylation.

LTD4 induces PGD2 secretion and Erk phosphorylation in LAD2 cells (Paruchuri et al., 2009). Therefore, PGE2 and LTD4 synergism was checked on PGD2 secretion and

Erk phosphorylation levels. LTD4 induced PGD2 secretion and this induction was blocked by MK571 (Fig. 3.10 A). PGE2 did not induce any PGD2 secretion. However, when cells were stimulated by both PGE2 and LTD4, the PGD2 secreted levels were highly increased and this increase is partially attenuated by blocking CysLT1R or EP3 but completely blocked by inhibiting both receptors (Fig. 3.10 A). In addition, LTD4 and PGE2 enhanced

Erk phosphorylation by themselves, however, together they highly upregulated Erk phosphorylation and this response was blocked by MEK inhibitor PD989059 (Fig. 3.10 B and C)

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Figure 3.10. LTD4 and PGE2 synergize in PGD2 secretion and Erk phosphorylation. LAD2 cells were stimulated with 0.5 μM LTD4, 0.5 μM PGE2, or both with/without MK571 (1 μM) and/or L-798 (100 nM), or PD989059 pre-treatment. PGD2 secretion levels were measured by ELISA (A), and Erk phosphorylation was measured by western blotting (B) and quantified by alpha view software (C). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

LTD4 plus PGE2 synergistic effects were mediated through PKG and Erk-dependent pathways

Next, we wanted to explore the signaling cascade involved in LTD4 and PGE2 upregulated responses after receptor stimulation. Since PKs were shown to be triggered by

CysLT1R activation (Paruchuri et al., 2008), LAD2 cells were pretreated with different PK inhibitors and stimulated with LTD4 or/and PGE2. Pretreatment with the general PKs

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inhibitor H7 completely blocked LTD4 signals, and LTD4 plus PGE2–enhanced effects (Fig

3.11 A, E, and F). However, pretreatment with a general PKC inhibitor GF109203X completely reduced LTD4 signals, but partially attenuated the increased c-fos and MIP-1β levels by LTD4 plus PGE2 stimulation (Fig. 3.11 B, E, and F). In addition, blocking PKA by using Rp-cAMPS and H-89 had no effect on synergistic effects of LTD4 plus PGE2 (Fig.

3.11 C-F).

Figure 3.11. Effect of PKC and PKA inhibition on LTD4 and PGE2 synergistic effects. LAD2 cells were pre-treated for 30 min with H7 (A, E, and F; 10 μM), GFX (B, E, and F; 2 μM), Rp- cAMPS (C, E, F; 50 μM) and H89 (D, E and F; 1μM), and then stimulated with 0.5 μM LTD4 and/or 0.5 μM PGE2. c-fos (A-E) and MIP-1β (F) levels were analyzed. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

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More importantly, pretreatment of LAD2 with the PKG inhibitor; KT5823, or the

MEK inhibitor; PD98059, significantly attenuated LTD4 plus PGE2 synergistic increase of c-fos phosphorylation and expression (Fig. 3.12 A and B), MIP-1β secretion (Fig. 3.12 C) and PGD2 secretion (Fig 3.12 D).

Figure 3.12. LTD4 and PGE2 enhanced effects are mediated through PKG and Erk. LAD2 cells were stimulated with 0.5 μM LTD4, 0.5 μM PGE2, or both with/without KT5823 (KT; 5 μM), or PD98059 (PD; 50 μM) pre-treatment. c-fos expression and phosphorylation were measured by western blotting (A) and quantified by alpha-view software (B), MIP-1β generation (C) and PGD2 (D) secretions were measured by ELISA. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

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CysLT1R plus EP3 or PKG knockdown resulted in complete attenuation of LTD4 and

PGE2 synergistic effects

To confirm the effects of the used pharmacological inhibitors, CysLT1R, EP3, both

CysLT1R and EP3, and PKG genes were knocked down using specific siRNAs. In addition, scrambled siRNA was used to serve as a control. First, the transfection efficiency was confirmed using qPCR. Transfection with CysLT1R siRNA downregulated the gene expression by 55%, EP3 siRNA downregulated the gene expression by 60%, and PKG siRNA downregulated the gene expression by 64% (Fig. 3.13 A).

Then, cellular responses to PGE2 and LTD4 stimulation after siRNA knockdown were checked. Downregulation of CysLT1R or EP3 partially inhibited both LTD4 and LTD4 plus PGE2–induced responses, however, downregulation of both CysLT1R and EP3 completely inhibited LTD4 plus PGE2–induced effects (Fig 3.13 B-D). In addition, PKG downregulation significantly reduced LTD4 plus PGE2–enhanced effects (Fig 3.13 B-D) compared to the control siRNA treatment. This indicates that LTD4 and PGE2 cross-talk is mediated through both CysLT1R and EP3 receptors and dependent on PKG signaling.

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Figure 3.13. Inhibition of LTD4 and PGE2 synergistic effects by knocking down CysLT1R and/or EP3 and PKG. CysLT1R, EP3, or both and PKG were knocked down in LAD2 cells by corresponding siRNAs (10 nM). qPCR was performed to check the siRNA efficiency (A). siRNA treated samples were stimulated with 0.5µM LTD4 and/or 0.5µM PGE2 (B-D) and c-fos was analyzed by western blotting (B) and quantified by alpha-view software (C). MIP-1β protein level was measured by ELISA (D). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

CysLT1R and EP3 antagonists together attenuates vascular inflammation induced by

LTD4 plus PGE2

To confirm the receptors involved in PGE2 and LTD4 synergism in ear edema, mice were pre-injected with MK571 and L-798 before injecting the agonists. The synergistic ear edema response by PGE2 plus LTD4 was highly attenuated with MK571+L798 treatment

(Fig. 3.14). 83

Figure 3.14. Ear thickness in BALB/c mice treated with LTD4+PGE2 and/or MK571+L- 798. BALB/c mice were treated with Saline (Sal) or LTD4+PGE2 with/without MK571+L798 pretreatment. Ear thickness was measured using caliper. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. Results are mean ± SEM from 4-6 mice/group/experiment and three experiments were performed.

Combined treatment with PGE2 and LTD4 synergistically potentiates Der. f–induced airway remodeling in mice

Asthma is a common disease that is induced by exposure to allergens, mainly house dust mite allergens. Der. f is one the most common dust mite allergen (H. Wang et al.,

2017) and its induced allergy in mice is a widely-used model to study asthma (Cates et al.,

2004; Gombedza et al., 2017; H. Wang et al., 2017). To check the role of LTD4 and PGE2 synergism in asthma, mice responses to Der. f in presence or absence of PGE2 and LTD4 were evaluated. Der. f induced airway thickening, leukocyte infiltration to the airways, and mucus secretion. However, when mice were treated with Der. f plus PGE2 and LTD4, the responses were highly amplified with increased narrowing of the airways, more mucus secretion, and higher numbers of leukocytes in the airway wall (Fig. 3.15 A). The infiltrated 84

cell content was further confirmed by analyzing the BAL fluid (Fig. 3.15 B-D). Differential cell count analysis demonstrated that PGE2 and LTD4 by themselves didn’t have a significant effect on BAL cellular contents. Der. f increased recruitment of lymphocytes and polymorphonuclear leukocytes (PMNs) to the lung, and more importantly, PGE2 and

LTD4 along with Der. f treatment led to potentiated leukocyte (PMNs and lymphocytes) recruitment to the airways compared to Der. f alone (Fig. 3.15 C and D). Macrophage count is increased by Der f, but it is not significantly changed by LTD4 plus PGE2 treatment (Fig.

3.15 B.)

Th2 released cytokines (IL-4, -5, -6, -9, and -13) are important for B cell activation and release of IgE. Secreted IgE binds to its receptor located on the MC surface, resulting in the release of inflammatory mediators from MCs and initiation of an inflammatory response (Busse et al., 2001). IL-5 also plays a role in (Greenfeder et al., 2001) and IL-13 also enhances mucin gene expression including MUC5AC (Yasuo et al., 2006) and Gob 5 (Loewen et al., 2005). Therefore, the inflammatory gene expressions in the lung tissues were analyzed using qPCR. Results revealed that, Der. f alone enhanced Gob 5,

MUC5AC, IL-13 and IL-5 gene transcripts (Fig. 3.15 E-H). In addition, PGE2+LTD4 treatment has none to very minimal effects on these gene transcripts, however when it is introduced with Der. f it highly enhanced Der. f-mediated expression of Gob 5, MUC5AC and IL-3 (Fig. 3.15 E-G) but not IL-5 (Fig. 3.15 H).

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Figure 3.15. PGE2 and LTD4 synergize in potentiating Der. f–induced airway remodeling in mice. C-57BL/6 mice were treated with saline or Der. f (5 µg/animal) intranasally 3 times/week for 3.5 weeks. Mice were euthanized 24 h after the final challenge. Paraformaldehyde-fixed lung sections (5 휇m) were used for the analysis of pulmonary inflammation and remodeling. A) Representative photomicrographs of H&E-stained lung tissue. Original magnification, Χ 20. (B-D) Differential cell count of BAL fluid cytospun cells. (E-H) qPCR was performed to the treated mice lung tissues using the specific primers for (E) Gob 5, (F) MUC5AC, (G) IL-13, and (H) IL-5. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis.

Discussion

MCs are regulatory cells involved in acute inflammatory responses and they are the major player in anaphylactic shock (Liauw et al., 1985). MC activity and number are increased in asthmatic patient airways and in vascular edema (Krystel-Whittemore et al.,

2015; Liauw et al., 1985). MCs release cys-LTs and also express both CysT1R and CysT2R

(Mellor et al., 2003; Mellor et al., 2001). Cys-LTs and PGE2 are arachidonic acid derivatives via two different metabolic pathways (D. Wang et al., 2010).

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Cys-LTs enhance inflammatory responses in asthma and many allergic diseases

(Hay et al., 1995). They induce Ca+2 flux and enhance c-fos phosphorylation and expression, and MIP-1β secretion mainly through their action through CysLT1R in MCs

(Kondeti et al., 2013). PGE2 plays a protective role in inflammation via activation of the

EP2 receptor by inhibiting human lung MC degranulation (Kay et al., 2006) leading to reduced fibrosis (Bozyk et al., 2011). PGE2 also enhances inflammatory responses via EP3 receptor increasing histamine release by human MCs (X. S. Wang et al., 2006) and induction of vascular permeability by MC activation (Morimoto et al., 2014). In this chapter of the dissertation, we explored a) the existence of a potential cross-talk between two groups of eicosanoids, PGE2 and LTD4 in vitro in MCs, b) the receptors and signaling mediators involved, c) the impact of this cross-talk on the inflammatory responses in MCs contributing to peripheral and lung inflammation as seen during asthma. PGE2 and LTD4 together highly potentiated c-fos, MIP-1β, and inflammatory gene expression including

TNF-a, IL-8 and COX-2.

Interestingly; blocking both EP3 and CysLT1R completely blocked the synergism, however, blocking either of the receptors alone partially attenuated the enhanced responses.

This suggests the possibility that the cross talk may operate at the level of intracellular signals like kinases rather than at the receptor level. Our results also revealed that the LTD4-

PGE2 cross talk is relayed through PKG and Erk. PGE2 has been traditionally shown to be coupled to EP2/Gαs/PKA pathway (Kay et al., 2006; Safholm et al., 2015). However, based on our results, we propose that LTD4 induces PGE2 signaling through EP3/Gαi/PKG leading to pathological inflammatory responses (Fig. 3.16).

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Figure 3.16. Schematic model of PGE2 and LTD4 synergism in MCs.

Further, we uncovered another exciting fact that LTD4-PGE2 cross-talk leads to activation of another potent prostaglandin, PGD2. PGD2 levels and its metabolites are markers of MC activation and their levels are highly increased after allergen exposure

(Dahlen et al., 2004). During acute asthmatic incidents, PGD2 is secreted by MCs, resulting in bronchoconstriction and vascular edema (Spik et al., 2005). Further, our in vivo results employing an ear inflammation model also demonstrate that LTD4-PGE2 cross-talk on MC is translated into enhanced permeability and edema as observed during peripheral inflammation.

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One of asthma’s features is enhanced vascular permeability, which is enhanced by cys-LTs (M. Liu et al., 2015). Vascular edema is an inflammatory response that results in an increased vascular permeability and is induced by histamine, which increases both blood flow and endothelial barrier disruption (Ashina et al., 2015). Intradermal injection of PGE2 plus LTD4 in mice ears enhanced ear edema which was blocked by dual antagonism of

W-sh both CysLT1R and EP3 receptors. Also, Kit mice were protected from PGE2 plus LTD4 mediated vascular edema, which suggests that PGE2 and LTD4 synergistic vascular edema is mediated through MC activation.

We further investigated the contribution of this cross-talk in mediating lung inflammation using the dust mite Der. f model of asthma. Again, results demonstrated that

PGE2 and LTD4 enhanced the Der. f-mediated airway thickening, inflammatory cell infiltration, Th2 cytokine and mucin gene expression in the lungs of treated mice.

Therefore, based on the data in this chapter, we propose that asthma can be effectively treated by a combination therapy targeting CysLT1R and EP3 receptors, rather than general steroids or just CysLT1R antagonists like Singulair.

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CHAPTER IV

MODULATION OF MAST CELL PROLIFERATIVE AND INFLAMMATORY

RESPONSES BY LEUKOTRIENE D4 AND STEM CELL FACTOR SIGNALING

INTERACTIONS

Introduction

Human MCs are derived from CD34+ progenitor cells. These progenitor cells leave the bone marrow and travel in the bloodstream as granular mononuclear cells and differentiate once they reach their peripheral destinations. MCs are terminally differentiated cells under the effect of local SCF; they express FcɛRI and c-kit receptors, histamine, tryptase and chymase (Metcalfe et al., 2017). SCF mediates MC differentiation, proliferation and survival through binding to its receptor c-kit. C-Kit is a tyrosine kinase receptor encoded by the c-kit proto-oncogene and expressed by different cells including

MCs and melanocytes (Webster et al., 2006). C-Kit plays a role in hematopoiesis. All hematopoietic cells downregulate their c-kit expression after being terminally differentiated except MCs (Okayama et al., 2006). C-kit over activation mutations were discovered in many cancers such as mastocytosis and gastrointestinal tumors (Webster et al., 2006). “Mastocytosis is a term collectively used for a heterogeneous group of disorders characterized by abnormal proliferation and accumulation of reactive MCs in one or more organ systems” (Jordan et al., 2002). MC hyperplasia is commonly involved in inflammatory conditions such as parasitosis (Metcalfe, 2008) and asthma (Boyce, 2003).

The mechanisms underlying MC hyperplasia are not completely elucidated, and the

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importance of SCF in MC proliferation makes it very important to explore and understand the mechanisms by which SCF control MC function.

During inflammation, different mediators or cells cross-talk with each other to modulate each other’s response (Barnes et al., 1998). In the previous chapter, PGE2 and

LTD4 synergism in potentiating MC inflammatory responses was demonstrated. In addition, we previously found that LTD4 potentiates TNFα- mediated attachment of ThP-

1 cells to endothelial cells via potentiating the vascular cell adhesion molecule 1 (VCAM-

1) expression (Duah et al., 2013). Furthermore, a study in mice found intratracheal installation of SCF can directly produce airway hyperreactivity through cys-LT production.

Additionally, this hyperreactivity is abolished using leukotriene inhibitors (Oliveira et al.,

2001). These studies suggest a potential cross-talk between SCF and LTD4. Further, SCF

(Da Silva et al., 2006) and LTD4 (Paruchuri et al., 2008; Sasagawa et al., 1994) levels are enhanced in asthmatic patients and MCs express both receptors for both mediators and their proliferation is enhanced in many inflammatory conditions (Z. Q. Hu et al., 2007); therefore, we aimed to study if there is a cross-talk between c-kit and CysLTR and how this cross-talk is translated into MC function.

Results

SCF induces c-Kit phosphorylation in a concentration dependent manner

SCF is the main growth factor for MCs and it mediates its effect through the c-Kit receptor (Kondeti et al., 2013). To confirm the c-Kit activation by SCF in LAD2 cells, we treated the cells with different concentrations of SCF and measured the phosphorylation of the receptor. SCF stimulation for 15 minutes resulted in a dose-dependent phosphorylation of c-Kit receptor and the highest phosphorylation was seen with the 100 ng/mL 91

concentration (Fig. 4.1. A and B). This concentration is used in the following experiments since it gave the highest response, and it is also the concentration used to culture LAD2 cells.

Figure 4.1. Dose-dependent phosphorylation of c-Kit receptor by SCF. (A) LAD2 cells were stimulated with the indicated doses of SCF for 15 min and the c-Kit phosphorylation level was determined by western blotting. (B) Densitometric analysis of data shown in A was analyzed using the alpha-view software. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data represents mean ± SEM of three separate experiments.

SCF-mediated c-Kit phosphorylation is enhanced by LTD4

To investigate if there is a cross-talk between c-Kit and CysLTR, the cells were stimulated with cys-LTs with/without SCF and were checked for c-Kit phosphorylation.

Results showed that cys-LTs cannot induce any c-Kit phosphorylation by themselves.

However, only LTD4, of all cys-LTs studied, can potentiate the SCF effect (Fig. 4.2 A and

C). In addition, blocking CysLT1R by MK571 pretreatment impeded this synergistic phosphorylation, suggesting that SCF-LTD4 synergy is mediated via CysLT1R (Fig. 4.2 B and C)

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Figure 4.2. LTD4 and SCF synergistically phosphorylate c-Kit. (A) LAD2 cells were stimulated with 0.5 μM of LTC4, LTD4 and LTE4 with/without 100 ng/mL SCF for 15 min and c-Kit phosphorylation was analyzed by western blotting. (B) c- Kit phosphorylation levels after LAD2 cells stimulation with 0.5 μM LTD4 and/or 100 ng/mL SCF for 15 min in presence or absence of MK571 (1µM) pretreatment. (C) Densitometric analysis of data shown in B using alpha view software. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data represents mean ± SEM of three separate experiments.

LTD4 enhances SCF-mediated proliferation

Because SCF induces MC proliferation (Da Silva et al., 2006), and we showed that

LTD4 enhanced SCF-mediated c-kit phosphorylation, we wanted to check if this synergism is translated into enhanced proliferation as well. Cells were treated with different doses of

SCF with/without LTD4 for 72 h and we measured the cell proliferation using an XTT assay. SCF induced LAD2 cell proliferation in a dose-dependent manner and more importantly; LTD4 enhanced SCF-induced proliferation at higher doses of SCF (100 and

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1000 ng/ml) (Fig. 4.3). Collectively these results indicate that LTD4 synergize with SCF to enhance MC proliferation.

Figure 4.3. LTD4 enhances SCF-induced cell proliferation. LAD2 cells were treated with 0.5 μM LTD4 and/or the indicated doses of SCF for 72 h and XTT assay was used to measure cell proliferation. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data represents mean ± SEM of three separate experiments.

SCF pretreatment augmented LTD4-induced calcium flux

Above mentioned data suggests that LTD4 in presence of SCF enhanced c-Kit- mediated proliferation. Therefore, we asked if this synergism is bidirectional. SCF can

2+ enhance LTD4 mediated inflammatory responses as well employing LTD4-induced Ca flux as a readout. Our lab has shown earlier that LTD4 induces calcium flux via CysLT1R in LAD2 cells (Kondeti et al., 2013; Paruchuri et al., 2009). Therefore, Ca2+ flux was measured upon LTD4 stimulation with/without SCF pretreatment. SCF induced none to minimal calcium flux by itself (Fig. 4.4. B) or by priming with LTD4 (Fig. 4.4. A). LTD4 induced calcium flux by itself (Fig. 4.4 A), but priming cells with SCF significantly amplified LTD4-induced calcium flux (Fig. 4.4 B and C). In addition, this calcium flux was

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blocked by pretreatment with the CysLT1R antagonist, MK571, approving that this signal is mediated by CysLT1R (Fig. 4.4 C).

Figure 4.4. SCF primes LTD4 -induced calcium flux (A, B) LAD2 cells were loaded with Fura-2AM and stimulated with 0.5 μM LTD4 and/or 100 ng/mL SCF (at indicated time arrows) and changes in intracellular calcium concentrations were measured. (C) Quantitative analysis of the three performed experiments. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data represents mean ± SEM of three separate experiments.

SCF enhances LTD4 induced c-fos phosphorylation and expression

We have previously shown that LTD4 induces c-fos phosphorylation and protein expression after 1 h stimulation (Kondeti et al., 2013). Therefore, as a next step, we investigated if SCF can enhance LTD4-mediated c-fos levels. LAD2 cells were stimulated with LTD4 and/or SCF for 1 h and then protein phosphorylation and expression were measured by western blotting. Results indicated that SCF cannot induce any c-fos phosphorylation or expression, however, it can enhance LTD4-mediated effects when cells were treated with LTD4+SCF (Fig. 4.5 A and B). 95

Figure 4.5. SCF and LTD4 synergize to potentiate c-fos phosphorylation and expression. LAD2 cells were stimulated for 1 h with 0.5µM LTD4 and/or 100 ng/mL SCF and western blotting was performed to analyze c-fos phosphorylation and expression levels. (B) Densitometric analysis of c-fos expression shown in (A). Data was analyzed using one- way ANOVA followed by post-hoc Tukey analysis. The data represents mean ± SEM of three separate experiments.

SCF enhances LTD4-induced inflammatory signals

2+ Because SCF enhanced LTD4 mediated Ca flux and c-fos expression and phosphorylation, we explored further if SCF and LTD4 can increase the inflammatory gene expression. As anticipated, inflammatory mediator, LTD4 enhanced COX-2, TNFα, and

MIP-1β gene expression, while SCF did not induce any of these inflammatory genes.

Interestingly, SCF strongly enhanced the LTD4-mediated inflammatory gene expression

(Fig. 4.6 A-C). We further confirmed the synergistic MIP-1β gene expression by LTD4 and

SCF by evaluating it at protein level using an ELISA. We found that LTD4 and SCF synergistically amplified the secretion of MIP-1β protein as well in LAD2 cells.

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Figure 4.6. SCF enhances LTD4-induced inflammatory signals. LAD2 cells were treated with 0.5 μM LTD4 and/or 100 ng/mL of SCF for 2 h, followed by mRNA extraction and cDNA synthesis. Transcript levels of COX-2, TNF-α, and MIP-1β were analyzed using qPCR (A-C). The graph represents fold change in the transcript levels compared to controls from three separate experiments. (D) LAD2 cells were treated with 0.5 μM LTD4 with/without 100 ng/mL SCF for 6 h and MIP-1β protein was analyzed by ELISA. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. The data shown represents mean ± SEM of three separate experiments.

Discussion

Enhanced MC proliferation is a common aspect during inflammation and asthma suggesting that there could be a cross-talk between proliferatory and inflammatory mediators. However, the players involved and the mechanism of action has not been studied in detail. Therefore, to address this, in this chapter, we analyzed the influence of an inflammatory mediator LTD4 and a MC proliferatory cytokine, SCF. We demonstrated a bidirectional crosstalk between CysLT1R and c-Kit receptor. LTD4, through CysLT1R, enhanced SCF-mediated c-Kit phosphorylation and MC proliferation. In addition, SCF 97

2+ enhanced LTD4-mediated Ca flux, c-fos phosphorylation and expression, and COX2,

TNFα and MIP-1β inflammatory gene expression (Fig. 4.7).

Figure 4.7. A schematic model of SCF and LTD4 synergism in MCs.

2+ LTD4 is the most potent cys-LT and it induces Ca flux, c-fos phosphorylation and expression, and enhances inflammatory MIP-1β secretion in MCs (Kondeti et al., 2013).

Cys-LTs are linked to the pathogenesis of asthma and they have the strongest effect in inducing contraction of the airway smooth muscle (Kondeti et al., 2013; Paruchuri et al.,

2009). Because both SCF and LTD4 levels are elevated in asthmatic patients, and MCs express c-Kit (Laidlaw et al., 2011), CysL1TR (Mellor et al., 2001) and CysLT2R (Mellor et al., 2003), and SCF is shown to induce airway hyperreactivity via cys-LT synthesis

(Oliveira et al., 2001), a plan was made to investigate if there is any cross-talk between these receptors and how these receptors modulate each other’s response.

In the current chapter, it was shown that LTD4 alone could not induce any c-Kit phosphorylation or MC proliferation, however, when present with SCF, it potentiated SCF-

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mediated effects and this potentiation is mediated through CysLT1R. More interestingly, this synergism is bidirectional. SCF can’t induce any Ca2+ flux or inflammatory signals,

2+ but when cells were primed with SCF prior to LTD4 stimulation, the Ca flux is greatly increased. Also, when cells were treated with SCF+LTD4, the total c-fos protein and the phosphorylated levels, and the transcripts of the mentioned inflammatory genes were highly enhanced. These results suggest that in MCs, c-Kit and CysLT1R synergize to enhance MC proliferation and inflammatory signals, which results in MC hyperplasia and hyperreactivity in inflammatory conditions such as asthma.

Recently it was shown that SCF potentiated MC activation under acute conditions, however, it inhibited allergen-induced MC degranulation upon prolonged contact. This occur due to altered cytoskeleton reorganization by SCF-mediated down-regulation of the

Src kinase Hck expression (Ito et al., 2012). However other studies have shown that SCF induces airway hyperreactivity via MC activation (Oliveira et al., 2001) and it was also hypothesized that SCF is the reason behind MC hyperplasia and hyperreactivity in some diseases, as observed in the airways of asthmatic patients (Da Silva et al., 2006).

In conclusion, these results propose that the cross-talk between c-Kit and CysLT1R increases MC proliferation and inflammatory signal production. SCF signaling through c-

Kit is crucial for MC physiology. However, locally released cys-LTs at the site of inflammation enhance MC proliferation by enhancing the reactivity of c-Kit receptor.

Likewise, the c-Kit cross talks with CysLT1R to potentiate LTD4 effects by enhancing MC inflammatory responses. Both these events lead to a boosted pathological cycle. Therefore, targeting both CysLT1R and c-Kit receptors might be a novel therapeutic strategy for asthma treatment.

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CHAPTER V

PGE2 ROLE IN NAD(P)H OXIDASE 4 (NOX4)- REGULATED ASTHMATIC

AIRWAY REMODELING

Introduction:

Persistent inflammation in asthma results in irreversible airway structural changes

(Ishmael, 2011), which include increased smooth muscle mass, subepithelial fibrosis with increased ECM proteins, epithelial layer changes such as goblet cell metaplasia and ciliary cell loss, and inflammation (Bergeron et al., 2009).

Subepithelial fibrosis is the most studied feature of the remodeled airways. Studies showed that TGF-β proteins are the driving force for fibrotic changes due to three main reasons: 1) TGF-β proteins are highly existing in asthmatic BAL fluid before and after antigen challenge, 2) TGF-β proteins are secreted by eosinophils, MCs, and fibroblasts from asthmatic and non-asthmatic patients, 3) many studies correlated TGF-β1 expression levels with the thickness of basement membrane and number of fibroblasts and/or severity of the disease (Elias et al., 1999). TGF-β1 mediates fibroblast to myofibroblast differentiation through SMAD-dependent and SMAD-independent pathways (Derynck et al., 2003). Myofibroblasts are distinguished by the enhanced expression of αSMA, bundling of the actin into stress fibers, and secretion of the ECM proteins such as collagen and FN (Desmouliere et al., 1993; Pankov et al., 2002; Thannickal et al., 2003). We have shown previously that TGF-β1 through TRPV4 channel mediates asthmatic airway

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remodeling in vivo and fibroblast differentiation in vitro in two separate but Rho-dependent signaling pathways (Gombedza et al., 2017).

NOX4 is an enzyme that belongs to the NOX family, which produces superoxides

− (O˙2 ) via transferring electrons through the cell membrane from NAD(P)H to the molecular oxygen (Kuroda et al., 2010). NOX family includes seven members; NOX1,

NOX2, NOX3, NOX4, NOX5, Duox1 and Duox2. All NOXs structure has a six transmembrane domains and a C- terminal cytosolic domain (von Lohneysen et al., 2010).

The NOX4 isoform is widely expressed in different organs, including the heart and the lungs (Amara et al., 2010; Kuroda et al., 2010). NOX4 is distinguished by its constitutive

− activity, its robust production of H2O2 instead of O˙2 , and its activity that is not regulated by the C-terminal binding proteins (Chen et al., 2012; Kuroda et al., 2010). NOX4 expression and activity is a driving force for fibroblast differentiation (Amara et al., 2010;

N. Sampson et al., 2011), lung fibrosis (Amara et al., 2010), cardiac failure (Kuroda et al.,

2010), and atherosclerosis (Chen et al., 2012). However, the mechanism by which NOX4 expression is enhanced or its role in asthmatic airway remodeling remains unclear.

TGF-β1-mediated fibroblast differentiation is regulated by NOX4 (Amara et al.,

2010), and counteracted by PGE2, but neither of these mediator mechanisms nor the interaction between them is clearly understood. Here we demonstrate that NOX4 modulates TGF-β1- mediated responses through MRTF-A and PAI-1 expressions and

PGE2 inhibits TGF-β1- enhanced expression of NOX4. Further work is needed to identify the involved EP receptor in this pathway. In addition, NOX4 expression is enhanced in

Der. f- induced airway remodeling, which indicates that NOX4 might be a major player in asthma pathogenesis.

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Results

NOX4 expression and activity is enhanced by TGF-β1 stimulation

To determine the effect of TGF-β1 on NOXs expression in hLF, cells were treated with TGF-β1 for 48 h then the expression levels of all NOXs (NOX1-5) were evaluated.

TGF-β1 stimulation only increased NOX4 levels, around 100 folds, but not other NOXs

(Figure 5.1).

Figure 5.1. NOXs expression levels in hLF. hLF were treated with TGF-β1 for 48h (A and B). NOX1-NOX5 gene expression was measured by qPCR. Data was analyzed using two-tailed student’s t-test. Results are means ± SEM from 3 independent experiments.

NOX4 is crucial for TGF-β1-mediated fibroblast differentiation

The fact that TGF-β1 induced only NOX4 expression prompted further investigation in order to understand the significance of this induction. The role of NOX4 in fibroblast differentiation was analyzed by pretreating the cells with different antioxidants; NAC (Fig. 5.2 A and B) and DPI (Fig. 5.2 C and D) at two different doses (5 and 10 mM for NAC and, 0.5 and 1 µM for DPI). Then, the levels of αSMA and FN proteins were examined. Antioxidant pretreatment reduced TGF-β1-enhanced αSMA and FN

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protein expression significantly. This confirms that oxidative stress is playing a role in cell differentiation mediated by TGF-β1.

To confirm the effect of NOX4 in TGF-β1-mediated hLF differentiation in more specific way, NOX4 gene was knocked down using NOX4 specific siRNA. The transcription efficiency was checked by qPCR (Fig. 5.2 top panel) and αSMA and FN protein levels were examined using western blotting. A huge reduction in TGF-β1 mediated

αSMA and FN protein expression levels was observed via knocking down NOX4 (Fig. 5.2

E and F). This indicates that NOX4 is a crucial player in TGF-β1- mediated differentiation.

Figure 5.2. NOX4 role in fibroblast to myofibroblast differentiation. hLF were treated with TGF-β1 with/without NAC (A and B) or DPI (C and D) for 48 h and αSMA and FN protein levels were analyzed using western blotting (A and C) and quantified using alpha view software (B and D). hLF were transfected with NOX4 siRNA (siNOX4) or scrambled siRNA (siNS), then treated with TGF-β1 for 48h. αSMA and FN protein levels were analyzed using western blotting (E) and NOX4 transcripts were checked by qPCR (F, top panel). αSMA protein and FN protein of the data shown in E were quantified (F, middle and bottom panels). Data was analyzed using one-way ANOVA

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followed by post-hoc Tukey analysis. Results are means ± SEM from 3 independent experiments.

NOX4 mediates TGF-β1-induced MRTF-A and SM22 expression

SMA transcription is regulated by the SRF and co-activators of the myocardin family (Zhao et al., 2007). Cellular stress, mechanical forces, and migration stimulate Rho

GTPases, which participate in actin cytoskeleton polymerization into stress fibers, and translocation of MRTF-A transcription factor into the nucleus (Shiwen et al., 2015; Zhao et al., 2007). It is known from previous work in our lab that Rho and MRTF-A are downstream players of TGF-β1 (Gombedza et al., 2017), so the next step aimed to determine the role of NOX4 in the TGF-β1 signaling pathway. TGF-β1 stimulation enhanced MRTF-A protein expression levels and this expression is significantly attenuated by using antioxidants; NAC (Fig. 5.3 A and B) and DPI (Fig. 5.3 C and D). Also, knocking down NOX4 almost completely blocked TGF-β1 enhanced MRTF-A levels (Fig.5.3 E and

F).

Since Rho activation regulates MRTF-A and actin dynamics (Olson et al., 2010;

Velasquez et al., 2013) and it was previously shown that TGF-β1 enhances Rho activity

(Gombedza et al., 2017), next aimed to check if Rho inhibition can affect NOX4 transcription mediated by TGF-β1. The cells were pretreated with the Rho antagonist Y27, then stimulated with TGF-β1 and analyzed for their expression for NOX4 gene. Y27 pretreatment did not show any reduction effect on TGF-β1 mediated NOX4 transcription

(Fig. 5.3 H). These results suggest that NOX4 is located upstream of Rho/MRTF-A in the

TGF-β1 mediated signaling pathway, and it regulates its downstream effects.

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MRTF-A mediates TGF-β1 induced SM22 gene expression, which is a smooth muscle contractile gene, in cardiac fibroblast differentiation (Velasquez et al., 2013). The aforementioned data showed that MRTF-A is downstream of NOX4, therefore, the effect of NOX4 blocking on SM22 gene expression was checked. NOX4 siRNA treatment led to inhibition of TGF-β1 mediated SM22 gene expression (Fig.5.3 G), which further confirms the essential role of NOX4 in fibroblast differentiation.

Figure 5.3. NOX4 inhibition attenuates TGF-β1 mediated MRTF-A and SM22 expression. hLF were pretreated with NAC (A and B) or DPI (C and D) and then stimulated with TGF- β1 for 48 h and MRTF-A protein levels were analyzed using western blotting (A and C) and quantified using alpha view software (B and D). hLF were transfected with NOX4 siRNA (siNOX4) or scrambled siRNA (siNS), then treated with TGF-β1 for 48 h. MRTF- A protein level was analyzed using western blotting and quantified (E and F top panel) and SM22 transcripts were checked by qPCR (F, bottom panel). hLF were pretreated with Rho inhibitor (Y27) and then stimulated with TGF-β1 for 48 h and NOX4 transcripts were analyzed by qPCR. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. Results are means ± SEM from 3 independent experiments.

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TGF-β1-mediated PAI-1 expression is regulated by NOX4

Fibrotic diseases involve both matrix synthesis and inhibition of matrix degradation. PAI-1 is an enzyme that prevents the activity of plasminogen activators. There are two main forms of plasminogen activators; the urokinase (uPA) and the tissue (tPA) types. Both of these activators convert plasminogen to the active enzyme plasmin which is a serine protease that cleaves the fibers (Cesari et al., 2010). TGF-β1 enhances PAI-1 levels in the cells, thereby slowing down the matrix degradation and enhancing fibrosis

(Gombedza et al., 2017). Since the data showed that TGF-β1 mediates fibrosis through

NOX4, next aim we wanted to examine the role of NOX4 in TGF-β1 mediated PAI-1 expression. Pretreatment with the antioxidants NAC (Fig. 5.4 A and B) and DPI (Fig. 5.4

C and D), and NOX4 blocking via NOX4 specific siRNA (Fig. 5.4 E and F) highly attenuated PAI-1 protein expression, which indicates that NOX4 mediates fibrosis through both enhancing matrix synthesis and repressing matrix degradation. This allows for accumulation of deposited proteins in the ECM which enhances fibrosis.

Figure 5.4. NOX4 mediates TGF-β1 enhanced PAI-1 expression. 106

hLF were pretreated with the antioxidants, NAC (A and B) or DPI (C and D), or transfected with NOX4 siRNA. Then, cells were stimulated with TGF-β1 for 48h and PAI-1 protein levels were analyzed (A, C, and E) and quantified (B, D, and F). Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis. Results are means ± SEM from 3 independent experiments.

PGE2 attenuates TGF-β1 mediated αSMA, FN, and NOX4 expression

PGE2 attenuates TGF-β1-mediated pulmonary fibroblast to myofibroblast differentiation (Bozyk et al., 2011) and NOX4 expression and function is crucial for fibroblast differentiation as shown in the previous figures. Therefore, we explored the role of PGE2 in TGF-β1 mediated hLF differentiation and NOX4 expression. Consistent with literature (Bozyk et al., 2011; Thomas et al., 2007), PGE2 treatment attenuated αSMA and

FN expression induced by TGF-β1 treatment (Fig. 5.5 A and B). More interestingly, TGF-

β1-induced NOX4 expression was also attenuated by PGE2 (Fig. 5.5 C). This suggests that

PGE2 blocks NOX4 expression and thereby inhibits fibroblast to myofibroblast differentiation and it confirms that PGE2 has different effects in different environments.

Chapter III of this dissertation discussed an inflammatory role for PGE2 in the airway and

MC inflammation, however, in this chapter an antifibrotic role for PGE2 is demonstrated.

Further research is needed to examine the exact EP receptor that attenuates the airway remodeling.

Figure 5.5. PGE2 attenuates TGF-β1-induced αSMA, FN and NOX4 expression. 107

hLF were pretreated with PGE2 then stimulated with TGF-β1 for 48h. Afterward, αSMA and FN proteins (A and B) and NOX4 gene (C) expressions were measured by western blotting and qPCR respectively. Results are means ± SEM from 3 independent experiments.

NOX4 is upregulated in Der. f–induced airway remodeling in vivo

Previously we have shown that intranasal installation of Der. f protein (25 µg) three times a week for 5 weeks induced inflammation and airway remodeling in the treated

C57BL/6 WT mice (Gombedza et al., 2017). To determine if NOX4 is upregulated in response to Der. f challenge, NOX4 transcripts in the lungs of the same challenged mice were analyzed. Results expressed that NOX4 levels are upregulated in the lungs of Der. f - treated mice compared to saline treated control mice (Fig. 5.6), which indicates that NOX4 is a predisposing factor for airway remodeling is asthma. However, how PGE2 affects

NOX4 mediated airway remodeling in this asthma model is not explored yet and it will be planned for future work. Also, further work is needed to explore the exact EP receptor that mediates the PGE2‘s protective role in fibroblast differentiation.

Figure 5.6. NOX4 expression is enhanced in the lungs of mice that were challenged with Der. f allergen. WT C57BL/6 mice were treated with saline or Der. f (25µg), three times a week for five weeks. 24 h after the last dose, mice were dissected and lungs were harvested and stored at -80ºC. Mice lungs were homogenized and RNA was extracted using chloroform and trizole method. RNA was reversely transcribed and used for qPCR using specific primers for 108

NOX4 and GAPDH. Relative expression to GAPDH is shown in the figure. Results are mean ± SEM from 4-6 mice/group/experiment and three experiments were performed. Data was analyzed using one-way ANOVA followed by post-hoc Tukey analysis.

Discussion

Airway remodeling is a major contributor to lung function loss in asthma. Airway remodeling includes airway wall thickening, subepithelial fibrosis, and increased submucosal tissue, adventitia, and smooth muscle mass (Busse et al., 2001). Fibrosis is a dynamic process that occurs due to an imbalance between the fibrotic and anti-fibrotic cellular pathways. TGF-β1 is a soluble protein that has a major role in fibrosis and its level increases in BAL fluid of asthmatic patients and correlates with the severity of the disease

(Elias et al., 1999). Other than soluble factors, oxidative stress mediated by NOX4 enzyme plays a role in mediating fibroblast differentiation and NOX4 levels were found to be enhanced in lungs of IPF patients (Amara et al., 2010). However, the mechanism by which

NOX4 mediates fibrosis and its role in asthmatic airway remodeling is not yet known.

Although the bioactive eicosanoid PGE2 is shown to have inflammatory effect in Chapter

III, it also plays a protective role in airway remodeling through limiting TGF-β1-induced differentiation (Birrell et al., 2015; Bozyk et al., 2011). In the present chapter, we explored

1) the role of NOX4 in TGF-β1-mediated fibroblast to myofibroblast differentiation in vitro in hLF, 2) the effect of PGE2 on the enhanced NOX4 transcripts, and 3) the transcription level of NOX4 in airway remodeling using the dust mite Der. f model of asthma. TGF-β1 treatment up-regulated NOX4 gene expression and blocking NOX4 highly attenuated

TGF-β1-induced α-SMA, SM22, FN, MRTF-A and PAI-1 expression in hLF. More importantly, PGE2 treatment led to a reduction in NOX4 expression mediated by TGF-β1;

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confirming the PGE2 protective role in airway remodeling and suggesting further exploration to determine the exact EP receptor mediating this effect (Fig. 5.7).

Figure 5.7. Shematic diagram of PGE2 role in NOX4-regulated fibroblast differentition

We further investigated the involvement of NOX4 in mediating airway remodeling using the dust mite Der. f model of asthma. Results demonstrated that NOX4 gene expression is highly upregulated in Der. f-challenged mice compared to saline challenged controls which proposes the essential role of NOX4 in airway remodeling. Based on the data in this chapter, we propose that NOX4 is a crucial player in airway remodeling and

PGE2 attenuates NOX4 and its mediated effects. Therefore, with more studies to be performed in future, introducing a specific EP agonist in the treatment plan of asthma might be a viable therapeutic option to control airway remodeling and improve asthma disease progression.

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CHAPTER VI

SUMMARY

In Chapter III of this dissertation, a novel cross-talk between LTD4 and PGE2 through CysLT1R and EP3 receptor was demonstrated. This cross-talk enhances peripheral edema represented by increased thickness of mouse ear after intradermal injection of LTD4 plus PGE2 compared to each mediator alone. Blocking CysLT1R and EP3 receptor simultaneously completely abolished the induced inflammation. Furthermore, the number of degranulated MCs in the swollen ears is enhanced, which imply that MCs are the effector cells mediating this synergism between LTD4 and PGE2. To confirm this, we analyzed synergistic peripheral edema in MC-deficient mice; KitW-sh. Interestingly, the enhanced

W- edema in mouse ear that is mediated by the LTD4 and PGE2 cross-talk was absent in Kit sh mice, which confirms the role of MCs in this inflammation. Therefore, we performed further experiments in vitro in MCs to analyze the above-mentioned cross-talk. LTD4 and

PGE2 were shown to cross talk via CysLT1R and EP3, in potentiating MC inflammatory response by enhancing c-fos phosphorylation and expression, MIP-1β secretion, PGD2 excretion, and inflammatory gene expression. This arbitrated synergism is mediated through PKG, Gαi, and Erk signaling pathway. In addition, LTD4 and PGE2 enhanced Der. f induced lung inflammation by increasing airway wall thickness, leukocyte infiltration, and IL-13 gene expression as well as goblet cell mucin gene expression.

Based on this, targeting both CysLT1R and EP3 receptor might be a better therapeutic option for asthma patients than the just commercially available CysLT1R antagonist, Singulair.

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In Chapter IV of this dissertation, it was demonstrated that LTD4 can also cross- talk with the growth factor, SCF in MCs. SCF induces c-kit phosphorylation and subsequent MC proliferation, however when LTD4 is present with SCF in the same inflammatory microenvironment, SCF- mediated effects were amplified resulting in higher number of MCs at the inflammatory sites. Also, this cross-talk is bidirectional, which means that LTD4 induces inflammatory responses in MCs, however, when SCF and LTD4

2+ are present at the same time, the LTD4 mediated effects represented by Ca flux, c-fos phosphorylation and expression, MIP-1β secretion, and inflammatory gene expression were amplified resulting in a more inflammatory microenvironment.

In the last part of this dissertation, it was shown that TGF-β1 induced NOX4 expression and NOX4 inhibition blocked TGF-β1 mediated αSMA, FN, MRTF-A and

PAI-1 expression. Furthermore, PGE2 blocked TGF-β1-induced NOX4 expression and fibroblast differentiation, which confirms the protective role of PGE2 in airway remodeling and proposes further work to be done to investigate the specific EP agonist that can be introduced in the therapeutic approach for asthmatic people suffering from airway remodeling. Also, the NOX4 expression is upregulated in response to Der.f -induced airway remodeling, which indicates that airway remodeling in asthma might occur due to enhanced oxidative stress mediated by NOX4.

In conclusion, asthma is a disease that results from the interplay between different inflammatory molecules. Our results suggest that identification of the cross-talk between these molecules and specific targeting of molecules/receptors involved in this cross-talk might provide better therapeutic options than blocking individual signaling pathways.

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APPENDICIES

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APPENDIX A1

LIST OF PUBLICATIONS

The papers below were included in the basis of this dissertation:

I. Al-Azzam, N., Kondeti, V., Duah, E., Gombedza, F., Thodeti, C. K., & Paruchuri, S. (2015). Modulation of mast cell proliferative and inflammatory responses by leukotriene d4 and stem cell factor signaling interactions. J Cell Physiol, 230(3), 595-602. doi: 10.1002/jcp.24777

II. Kondeti, V., Al-Azzam, N., Duah, E., Thodeti, C. K., Boyce, J. A., & Paruchuri, S. (2016). Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell-dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3. J Allergy Clin Immunol, 137(1), 289-298. doi: 10.1016/j.jaci.2015.06.030

Additional peer-reviewed papers not included in the dissertation:

III. Kondeti, V., Duah, E., Al-Azzam, N., Thodeti, C. K., Boyce, J. A., & Paruchuri, S. (2013). Differential regulation of cysteinyl leukotriene receptor signaling by protein kinase C in human mast cells. PLoS One, 8(8), e71536. doi: 10.1371/journal.pone.0071536

IV. Duah, E., Adapala, R. K., Al-Azzam, N., Kondeti, V., Gombedza, F., Thodeti, C. K., & Paruchuri, S. (2013). Cysteinyl leukotrienes regulate endothelial cell inflammatory and proliferative signals through CysLT(2) and CysLT(1) receptors. Sci Rep, 3, 3274. doi: 10.1038/srep03274

V. Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., . . . Paruchuri, S. (2017). Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation. FASEB J. doi: 10.1096/fj.201601045R

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APPENDIX A2

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Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell–dependent manner through cysteinyl leukotriene receptor 1 and E- prostanoid receptor 3

Vinay Kondeti, PhD,a Nosayba Al-Azzam, MS,a Ernest Duah, MS,a Charles K. Thodeti, PhD,b Joshua A. Boyce, MD,c,d,e and Sailaja Paruchuri, PhDa Akron and Rootstown, Ohio, and Boston, Mass

Background: Although arachidonic acid metabolites, cysteinyl Conclusions: Our results unravel a unique LTD4-PGE2 leukotrienes (cys-LTs; leukotriene [LT] C4,LTD4, and LTE4), interaction affecting mast cells through CysLT1R and EP3 and prostaglandin (PG) E2 are generated at the site of involving Gi, protein kinase G, and Erk and contributing to inflammation, it is not known whether crosstalk exists between vascular inflammation in vivo. Furthermore, current results also these 2 classes of inflammatory mediators. suggest an advantage of targeting both CysLT1R and EP3 in Objective: We sought to determine the role of LTD4-PGE2 attenuating inflammation. (J Allergy Clin Immunol crosstalk in inducing vascular inflammation in vivo, identify 2015;nnn:nnn-nnn.) effector cells, and ascertain specific receptors and pathways Key words: involved in vitro. Mast cells, prostaglandin E2, leukotriene D4, CysLT1R, Methods: Vascular (ear) inflammation was assessed by injecting E-prostanoid receptor 3, prostaglandin D2, c-fos, protein kinase G, agonists into mouse ears, followed by measuring ear thickness extracellular signal-regulated kinase, macrophage inflammatory b and histology, calcium influx with Fura-2, phosphorylation and protein 1 expression of signaling molecules by means of immunoblotting, PGD2 and macrophage inflammatory protein 1b generation by using ELISA, and expression of transcripts by using RT-PCR. Mast cells (MCs) are recognized as critical components of our Candidate receptors and signaling molecules were identified by immune system. They are vital in the initiation and amplification using antagonists and inhibitors and confirmed by using small of acute inflammatory responses and play an important role in interfering RNA. triggering asthma exacerbations through the elaboration of several soluble inflammatory mediators.1,2 MCs reside in connec- Results: LTD4 plus PGE2 potentiated vascular permeability and edema, gearing the system toward proinflammation in wild-type tive tissues and are located in close proximity to the blood vessels. W-sh Activation of MCs stimulate the formation of leukotrienes (LTs) mice but not in Kit mice. Furthermore, LTD4 plus PGE2, through cysteinyl leukotriene receptor 1 (CysLT R) and E- and prostaglandins (PGs), both of which initiate vascular 1 3 W-sh/W-sh sh prostanoid receptor (EP) 3, enhanced extracellular signal- changes. Kit mice have the W-sash (W ) inversion mu- regulated kinase (Erk) and c-fos phosphorylation, inflammatory tation and remarkable deficiency in MCs, providing a great model 4 gene expression, macrophage inflammatory protein 1b system to analyze MC function in vivo. Cysteinyl leukotrienes (cys-LTs; LTC ,LTD, and LTE ) are arachidonic acid deriva- secretion, COX-2 upregulation, and PGD2 generation in mast 4 4 4 cells. Additionally, we uncovered that this synergism is mediated tives generated by MCs, eosinophils, basophils, and macro- phages5 through the action of 5- enzyme. All PGs through Gi, protein kinase G, and Erk signaling. LTD4 plus are derived from PGH and generated through arachidonic acid PGE2–potentiated effects are partially sensitive to CysLT1Ror 2 through the action of PGH synthase (also known as COX). MCs EP3 antagonists but completely abolished by simultaneous treatment both in vitro and in vivo. express both COX-1 and COX-2. COX-2 is upregulated by in- flammatory stimuli driving PGD2 generation under inflammatory conditions. In MCs PGH2 derived from both COX-1 and COX-2 is 6 converted to PGD2 by a terminal hematopoietic PGD2 synthase. Although not a product of MCs, PGE2, a metabolite of PGH2 a b 7 From the Department of Chemistry, University of Akron; the Department of Integrative through the action of PGE2 synthase, is the most ubiquitous Medical Sciences, Northeast Ohio Medical University, Rootstown; the Departments of c d e PG, with prominent and complex functions in inflammation, Medicine and Pediatrics, Harvard Medical School, Boston; and the Division of asthma, and allergic diseases. Remarkably, MCs not only Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston. Supported by National Institutes of Health grant HL098953 and by James Foght Assistant generate cys-LTs but also express corresponding receptors and 8 Professor endowed professorship grant (to S.P.). respond to them. Two known G protein–coupled receptors, Disclosure of potential conflict of interest: V. Kondeti and S. Paruchuri have received termed cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl research support from the National Institutes of Health (HL098953) and the James L. leukotriene receptor 2 (CysLT R), specifically recognize cys-LTs and Martha J. Foght Assistant Professorship. The rest of the authors declare that they 2 have no relevant conflicts of interest. and mediate their biologic functions. CysLT1R binds LTD4 with Received for publication January 4, 2015; revised June 18, 2015; accepted for publication higher affinity than LTC4, whereas CysLT2R has equal affinity 5 June 23, 2015. for LTD4 and LTC4. GPR17, another cys-LT receptor, has been Corresponding author: Sailaja Paruchuri, PhD, Department of Chemistry, KNCL 406, identified and is expressed primarily in the brain,9 and GPR99 185 E Mill street, Akron, OH 44325. E-mail: [email protected]. has been recently identified as a cys-LT receptor with a preference 0091-6749/$36.00 10 Ó 2015 American Academy of Allergy, Asthma & Immunology for LTE4. Mice lacking LTC4 synthase have reduced numbers of http://dx.doi.org/10.1016/j.jaci.2015.06.030 MCs in the airway mucosa after sensitization and challenge by

1 2 KONDETI ET AL J ALLERGY CLIN IMMUNOL nnn 2015

Chemicals (Ann Arbor, Mich). KT5823, PD98059, pertussis toxin (PTX), Abbreviations used H7, GF109203X, Rp-cAMPS, and H89 inhibitors were from Tocris Bioscience cys-LT: Cysteinyl leukotriene (Minneapolis, Minn). Fura-2 AM was from Molecular Probes (Eugene, Ore),

CysLT1R: Cysteinyl leukotriene receptor 1 phospho-specific antibodies were from Cell Signaling Technology (Danvers, CysLT2R: Cysteinyl leukotriene receptor 2 Mass), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody EP: E-prostanoid receptor was from Fitzgerald (Acton, Mass). All secondary antibodies were obtained Erk: Extracellular signal-regulated kinase from Jackson ImmunoResearch (West Grove, Pa). Nonspecific small interfering GAPDH: Glyceraldehyde-3-phosphate dehydrogenase RNA (siRNA) and isoform-specific siRNAs for CysLT1R, EP3, and PKG were hMC: Human cord blood–derived mast cell obtained from Dharmacon (Lafayette, Colo), and the macrophage inflammatory LT: leukotriene protein 1b (MIP-1b) ELISA kit was from R&D Systems (Minneapolis, Minn). MC: Mast cell Cytokines for hMC cultures were obtained from PeproTech (Rocky Hill, NJ). MIP-1b: Macrophage inflammatory protein 1b PG: Prostaglandin PK: Protein kinase Intradermal injection of agonists and assessment of PTX: Pertussis toxin ear edema SCF: Stem cell factor Mice anesthetized with ketamine/xylazine received intradermal injections

siRNA: Small interfering RNA of 0.5 mmol/L LTD4, PGE2, and LTD4 plus PGE2 (in a 10-mL volume) in the right ear and 10 mL of saline in the left ear in the presence or absence of MK571, L-798, or both. At 0, 30, 60, 120, 240, and 300 minutes after the in- tradermal injection, ear thickness was measured with a caliper. Mice were 11 allergen, suggesting the prominence of cys-LTs in MC function. killed 60 minutes after the indicated treatment, ear tissues were fixed in 4% We have previously demonstrated that stimulation of human cord paraformaldehyde and embedded in paraffin, and 4-mm-thick sections were blood–derived mast cells (hMCs), LAD2 cells, or both with LTD4 cut and stained for hematoxylin and eosin and toluidine blue (to detect potently induces calcium flux and cytokine generation8 through MCs). Total (toluidine blue–positive cells that are compact) and degranulated MCs (toluidine-positive cells with no clearly defined cell membrane and CysLT1R. Additionally, MC proliferative and inflammatory re- diffuse) in the toluidine blue–stained sections were visualized at 360 magni- sponses are modulated by LTD4 and stem cell factor (SCF) signaling interactions.12 PGs have also been shown to elicit vaso- fication and presented in Fig 1, B; counted in each section by a blinded dilation and an increase in blood flow. Among PGs, PGE is the observer; and expressed as the number of MCs per millimeter. Representative 2 images of intact and degranulated MCs were shown in Fig 1, B. most abundantly synthesized PG at the inflammation site and is regarded as an important regulator of inflammation.13 The deci- sive effect of PGE2 is the outcome of specific E-prostanoid recep- Cell culture 14 tor (EP) 1 to 4 activation through which the signal is transduced. The LAD2 MC leukemia line15 was a kind gift from Dr Arnold Kirshen- 8 EP1 is coupled to intracellular calcium mobilization through Gq; baum (National Institutes of Health) and cultured as described previously. however, EP2 and EP4 are coupled to stimulation of adenylyl Primary hMCs were derived from cord blood mononuclear cells cultured for 6 to 9 weeks in RPMI supplemented with SCF, IL-6, and IL-10.16 cyclase through Gs, and EP3 is coupled to the inhibition of ad- enylyl cyclase through Gi. Different splice variants are generated by means of alternative splicing of the C-terminal tail of the EP 3 Calcium flux receptor and can couple to different signal transduction pathways. 6 LAD2 cells (0.5 to 1 3 10 /sample) were washed and labeled with Fura 2- Eight human EP3 isoforms are known thus far, which are iden- AM for 30 minutes at 378C. Cells were stimulated with PGE2 (0.5 mmol/L) with tical, except for their carboxyl termini.14 or without LTD4 (0.5 mmol/L) priming, and the changes in intracellular calcium The interactions among various mediator systems that partic- levels measured by using excitation at 340 and 380 nm and emission at 510 nm ipate in inflammatory responses are complex, and it is difficult to were recorded in a fluorescence spectrophotometer (Hitachi F-4500).8 define the unique contribution of any single element. In the current study we show that LTD4 and PGE2 synergistically poten- tiate peripheral inflammation in vivo and MC activation in vitro Cell activation LAD2 cells were stimulated with 0.5 mmol/L of LTD4, PGE2, or both for 15 through CysLT1R-, EP3-, Gi-, protein kinase (PK) G–, and extra- cellular signal-regulated kinase (Erk)–dependent pathways. minutes for the phosphorylation of Erk or 1 hour for expression of c-fos, 2 hours for expression of inflammatory gene transcripts, 3 hours for COX-2 pro- Furthermore, our results indicate that blocking EP3 together tein expression, and 6 hours for measurement of cytokine and PGD levels. with CysLT R could be a better therapeutic target to control 2 1 LTD4 responses were dose dependently inhibited by MK571 (with maximum inflammation. inhibition at 1 mmol/L), and PGE2 responses were attenuated by L-798 (in a dose-dependent manner, with maximum inhibition at 100 nmol/L). Therefore 1 mmol/L MK571 and 100 nmol/L L-798 were used in all the subsequent ex- METHODS periments. Transfection of isoform-specific siRNA smart pool constructs from Animals Dharmacon (10 nmol/L) were carried out with siLentFect transfection reagent Six- to 8-week-old BALB/c mice, C57BL/6 mice, and KitW-sh mice (W-sh) (Bio-Rad Laboratories, Hercules, Calif) for 48 hours, according to the manu- were obtained from Jackson Laboratories and maintained at the Comparative facturer’s protocol. Medicine Unit, Northeast Ohio Medical University. All animal experiments were done in accordance with standard guidelines, as approved by the Animal Care and Use Committee of Northeast Ohio Medical University. Cell lysates and Western blotting After stimulation with the respective agonists, LAD2 cells, hMCs, or both (0.5 3 106) were lysed with lysis buffer (BD Biosciences, San Jose, Calif) sup- Reagents plemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and LTD4,PGE2, MK571, BayCysLT2, iloprost, butaprost, sulprostone, L-798, phosphatase inhibitor cocktail (Pierce, Rockford, Ill). Immunoblotting was 17 ONO-871, L-161, and PGD2 ELISA kits were purchased from Cayman performed, as described previously. Western blots were incubated with J ALLERGY CLIN IMMUNOL KONDETI ET AL 3 VOLUME nnn, NUMBER nn

FIG 1. Effect of LTD4 and PGE2 on ear edema in mice in vivo and MC activation in vitro. Wild-type (WT) BALB/c mice were treated with saline (Sal), 0.5 mmol/L LTD4, 0.5 mmol/L PGE2, or LTD4 plus PGE2. A, Ear thickness. B, Hematoxylin and eosin staining and toluidine blue staining. C, Quantification (blind analysis)

of MCs per millimeter. D, Ear thickness in C57BL/6 and W-sh mice treated with 0.5 mmol/L LTD4, PGE2, and LTD4 plus PGE2. Results are means 6 SEMs from 4 to 6 mice per group per experiment and 3 experiments performed. E-I, LAD2 cells (Fig 1, E-H) and hMCs (Fig 1, I) were stimulated with 0.5 mmol/L LTD4, 0.5 mmol/L PGE2, or both, and calcium flux (Fig 1, E and F), c-fos (Fig 1, G and I), and MIP-1b (Fig 1, H) were analyzed. *P < .05, **P < .01, and ***P < .001.

ECL, and the bands were visualized with an imager (ProteinSimple, San Jose, Chemicals (Ann Arbor, Mich) and R&D Systems, respectively, according to

Calif) and quantified by using Alpha View SA (ProteinSimple). The blots were the manufacturer’s instructions. PGD2 secreted into medium was first converted stripped and reprobed with GAPDH antibody. into PGD2-MOX and then assessed by using the PGD2-MOX ELISA kit.

Real-time quantitative PCR Statistical analysis b a Expressions of CysLT1R, CysLT2R, EP1,EP2,EP3,EP4,MIP-1 ,TNF- , Blots presented are representative of 3 experiments performed, and data are IL-8, COX-1, COX-2, and PKG transcripts were determined with real-time expressed as means 6 SEMs from at least 3 experiments, except where PCR performed on the LightCycler 480 (Roche). Total RNA was isolated otherwise indicated. Significance was determined by using the Student t test, from LAD2 cells after respective treatment with an RNeasy Mini Kit (Qiagen, as well as 1-way ANOVA, followed by Tukey post hoc analysis. Hilden, Germany), and cDNA was synthesized with the cDNA synthesis kit from Quanta BioSciences (Gaithersburg, Md). Real-time PCR was performed by using primers mentioned in Table E1 in this article’s Online Repository at www.jacionline.org, the levels of respective genes relative to GAPDH were RESULTS DD analyzed, and the cycle threshold values relative to control were expressed Combined treatment with PGE2 and LTD4 as the fold change. synergistically potentiates peripheral vascular inflammation in mice ELISA To elucidate the possible interaction between LTD4 and PGE2, Concentrations of PGD2 and MIP-1b in the supernatants were assayed by we first evaluated the increase in vascular permeability and edema using the PGD2-MOX and MIP-1b ELISA kits purchased from Cayman in the mouse ear, a widely used model to assess peripheral 4 KONDETI ET AL J ALLERGY CLIN IMMUNOL nnn 2015

18,19 vascular inflammation, in response to LTD4 plus PGE2 CysLT1R inhibition partially attenuates LTD4-primed compared with agonists alone. LTD4 or PGE2 and LTD4 plus PGE responses m 2 PGE2 (0.5 mol/L, a dose at which both the agonists generated We then sought to identify the receptors involved in mediating 8 maximum response; data not shown) were injected into the ears this synergy by using MK571 (CysLT1R antagonist ) and Bay- 20 of BALB/c mice, and tissue edema was assessed. The patterns CysLT2 (CysLT2R antagonist ). MK571 pretreatment abolished of ear edema induced by LTD4, PGE2, and LTD4 plus PGE2 LTD4-induced calcium influx but attenuated LTD4-primed, were similar, peaking at 30 minutes and returning to baseline at PGE2-generated calcium influx by 30% (Fig 2, A). LTD4 plus 300 minutes. However, we observed a significant enhancement PGE2–stimulated c-fos phosphorylation and expression and in the magnitude of ear edema with LTD4 plus PGE2 compared MIP-1b secretion were reduced with MK571 preincubation with LTD4 or PGE2 alone (Fig 1, A). The increase in ear thickness (Fig 2, B-D), whereas BayCysLT2 had no effect (Fig 2). with LTD4 plus PGE2 was rapid, transient, and approximately 6- fold higher compared with control values (0.32 6 0.05 vs 0.05 6 Effects of LTD plus PGE are partially mediated 0.01) in the first half hour. We also observed an increase in ear 4 2 thickness in response to both LTD (approximately 3-fold, through EP3 4 We next investigated which of the putative EPs were interacting 0.15 6 0.01) and PGE2 (approximately 1.6-fold, 0.08 6 0.01) individually during the first 30 minutes compared with control with CysLT1R and enhancing PGE2 responses. Stimulation of values. Histologic analysis of ear tissues revealed expansion of LAD2 cells with iloprost (EP1/EP3 agonist) and sulprostone (EP3 the extracellular space correlating with ear thickness (Fig 1, B). agonist) in the presence of LTD4 enhanced calcium flux (data b Interestingly, we found a robust increase in the number of degra- not shown), c-fos (Fig 3, A and B), and MIP-1 expression (Fig 3, C), which is similar to what is seen with LTD4 plus PGE2,sug- nulating MCs with LTD4 plus PGE2 treatment (Fig 1, B, middle and bottom right panel, arrows point to degranulating MCs, and gesting the involvement of EP3 and a possible involvement of EP1 in this synergism. The EP2 agonist butaprost had no effect (Fig 3, Fig 1, C) compared with other groups, suggesting that LTD4 A-C). Because EP1 and EP3 act dominantly through Gq- and Gi- plus PGE2 can synergistically potentiate peripheral inflammation 14 through their action on MCs. To elucidate the role of MCs, we mediated signaling pathways, respectively, we used PTX, a a repeated the above experiment in C57BL/6 and W-sh mice (on G i inhibitor, and evaluated LTD4 plus PGE2 synergy. Pretreat- C57BL/6 background). Although we observed a similar pattern ment of LAD2 cells with PTX (100 ng/mL for 18 hours) of ear inflammation in C57BL/6 wild-type mice as in BALB/c completely abolished the enhanced activation of c-fos and MIP- b mice with all the agonists, the response was more transient and 1 by LTD4 and PGE2 (see Fig E2, A-C, in this article’s Online Re- pository at www.jacionline.org), suggesting the involvement of smaller (Fig 1, D). LTD4 plus PGE2 synergistically enhanced the inflammation in C57BL/6 wild-type mice, which is signifi- EP3. Furthermore, we found that the synergistic responses by cantly attenuated in W-sh mice (Fig 1, D). LTD4 plus PGE2 were partially sensitive to the inhibition of EP3 by L-798 (EP3 antagonist, 100 nmol/L; Fig 3, D-F), which is similar to what is seen with CysLT1R inhibition. Neither ONO- LTD4 primes PGE2-dependent calcium flux and 8711 (EP1 antagonist, 2 nmol/L) nor L-161 (EP4 antagonist, 100 potentiates c-fos phosphorylation and MIP-1b nmol/L) had any effect on LTD4 plus PGE2–mediated c-fos expres- b production in MCs sion/phosphorylation or MIP-1 generation (see Fig E2, D and E). Next, we investigated the molecular mechanisms through which LTD4 and PGE2 activate MCs and potentiate inflammation. Synergistic responses to LTD4 and PGE2 were The expression pattern of cys-LT receptor transcript in LAD2 completely attenuated by blocking both CysLT1R cells is similar to that in hMCs, with high levels of CysLT R 1 and EP3 simultaneously compared with CysLT R (see Fig E1, C, in this article’s Online 2 Next, we speculated that blocking both CysLT1R and EP3 Repository at www.jacionline.org). Additionally, LAD2 cells ex- simultaneously might completely block the synergistic responses. press all 4 EPs (EP >EP >EP >EP; see Fig E1, C). Further- 3 2 4 1 We observed that although the effects mediated by LTD4 and more, Western blot analysis of LAD2 cells and hMCs confirmed PGE2 are partially blocked by CysLT1R and EP3 antagonists the expression of CysLT1R, CysLT2R, and EP1-4 (see Fig E1, D). alone (40% and 35% for c-fos and 58% and 57% for MIP-1b, hMCs expressed higher CysLT1R levels compared with LAD2 respectively), combined treatment with MK571 and L-798 cells, and the expression pattern of EP is comparable. 3 completely blocked the effects of LTD4 plus PGE2 (78% for c- First, we analyzed the ability of LAD2 cells to flux calcium in fos phosphorylation and expression and 92% for MIP-1b; response to PGE2 and LTD4. We observed that PGE2 induced Fig 4, A-C). The combination of MK571 and L-798 also attenu- modest calcium flux (Fig 1, E, top panel, and Fig 1, F) and ated augmentation of c-fos and MIP-1b generation by LTD4 LTD4 priming before PGE2 stimulation led to an approximately plus sulprostone (data not shown). Importantly, in hMCs 6 6 2.5-fold increase (0.42 0.05 vs 0.16 0.04; Fig 1, E, bottom MK571 plus L-798 totally inhibited c-fos phosphorylation and panel, and Fig 1, F). Furthermore, LTD4 and PGE2 together expression (Fig 4, D and E), suggesting a functional relevance increased phosphorylation and expression of c-fos (Fig 1, G) for this interaction and associated signaling. and secretion of MIP-1b (Fig 1, H). Importantly, in hMCs also, LTD4 plus PGE2 treatment significantly enhanced c-fos phos- phorylation and expression (Fig 1, I). Stimulating LAD2 cells LTD4 and PGE2 treatment upregulated with a constant dose of LTD4 (0.5 mmol/L) and varying concen- inflammatory genes, PGD2 generation, and Erk trations of PGE2 (0.001, 0.01, 0.1, and 0.5 mmol/L), we observed phosphorylation in LAD2 cells dose-dependent c-fos phosphorylation and expression and MIP- We then investigated whether stimulation of MCs with LTD4 1b secretion (see Fig E1, A and B). plus PGE2 would upregulate any other inflammatory J ALLERGY CLIN IMMUNOL KONDETI ET AL 5 VOLUME nnn, NUMBER nn

FIG 2. LTD4-primed and PGE2-mediated calcium, c-fos, and MIP-1b responses in MCs are partly sensitive to CysLT1R inhibition. LAD2 cells were stimulated with 0.5 mmol/L LTD4, 0.5 mmol/L PGE2, or both in the pres- ence or absence of MK571 (1 mmol/L) and BayCysLT2 (1 mmol/L), and calcium flux (A), c-fos (B and C), and MIP-1b (D) were analyzed. *P < .05 and ***P < .001.

FIG 3. EP3 relays synergistic activation of c-fos and MIP-1b in response to PGE2 and LTD4. LAD2 cells were treated with LTD4 (0.5 mmol/L) 6 PGE2 (0.5 mmol/L) or iloprost (10 mmol/L), butaprost (5 mmol/L), or sulpro- stone (100 nmol/L) 6 L-798 (100 nmol/L), and c-fos (A, B, D, and E) and MIP-1b (C and F) were analyzed. *P < .05, **P < .01, and ***P < .001. 6 KONDETI ET AL J ALLERGY CLIN IMMUNOL nnn 2015

FIG 4. Combined effect of CysLT1R antagonist and EP3 antagonist on synergistic responses to LTD4 and PGE2. LAD2 cells (A-C) and hMCs (D and E) were preincubated with MK571 (1 mmol/L) and L-798 (100 nmol/L) separately or in combination and treated with LTD4 (0.5 mmol/L) 6 PGE2 (0.5 mmol/L), and c-fos (Fig 4, A, B, D, and E) and MIP-1b (Fig 4, C) were analyzed. *P < .05, **P < .01, and ***P < .001.

chemokines and cytokines. LTD4 treatment upregulated MIP- and F). Furthermore, the PKA blockers Rp-cAMPS and H-89 1b,TNF-a,IL-8,andCOX-2,whereasPGE2 upregulated had no effect on synergistic LTD4 plus PGE2 effects (see Fig COX-2 alone. LTD4 plus PGE2 treatment significantly upregu- E3, C-F). Interestingly, pretreatment of LAD2 cells with the lated MIP-1b (145 6 30; Fig 5, A), TNF-a (90 6 11; Fig 5, PKG inhibitor KT5823 (5 mmol/L) or PD98059 attenuated B), IL-8 (72 6 20; Fig 5, C), and COX-2 (60 6 10; Fig 5, D)tran- LTD4 plus PGE2 synergistic responses (Fig 6, C-F). We then scripts, and the expression of COX-1 remained unchanged (Fig knocked down CysLT1R, EP3,bothCysLT1RandEP3,and 5, E). Consistently, we observed a significant 3-fold upregula- PKG in LAD2 cells by using protein-specific siRNAs (10 tion of COX-2 protein with LTD4 plus PGE2 treatment (Fig 5, nmol/L) and nonspecific siRNAs as a control and analyzed F). Furthermore, LTD4 plus PGE2 treatment upregulated PGD2 LTD4 plus PGE2 effects. Transfection of MCs with CysLT1R, secretion compared with control values (1630 6 51 vs 1000 6 EP3, and PKG siRNAs significantly and specifically downregu- 75 pg/mL), and combined treatment with both MK571 and L- lated CysLT1R (55%), EP3 (60%), and PKG (64%) expression 798 inhibited this secretion (Fig 6, A). Then we investigated (see Fig E4 in this article’s Online Repository at www. the ability of LTD4,PGE2, and a combination of both in inducing jacionline.org). Importantly, downregulation of CysLT1Ror Erk phosphorylation. PGE2 and LTD4 stimulation enhanced EP3 partially inhibited both LTD4-andLTD4 plus PGE2– phosphorylation of Erk, and LTD4 plus PGE2 further potentiated induced responses, but knockdown of both CysLT1RandEP3 this effect, which is sensitive to the MEK inhibitor PD989059 completely inhibited LTD4 plus PGE2–induced synergy (Fig 7, (50 mmol/L; Fig 6, B). A-C). Also, knockdown of PKG significantly inhibited LTD4 plus PGE2–induced effects (Fig 7, A-C), whereas the control siRNA did not affect LTD4-orPGE2-induced inflammatory LTD4 plus PGE2 signals operate through PKG and responses. Erk-dependent pathway We next examined the signaling involved in LTD4 plus PGE2 synergism downstream of receptor activation. PKs are known to Combined treatment with CysLT1R and EP3 8,21 be activated downstream of CysLT1R activation. A general antagonists attenuates vascular inflammation PK inhibitor, H7 (10 mmol/L), completely blocked LTD4,as induced by LTD4 plus PGE2 well as LTD4 plus PGE2–induced effects (see Fig E3, A, E, Next, we determined the effect of blocking both CysLT1R and and F, in this article’s Online Repository at www.jacionline. EP3 simultaneously in evoking vascular inflammation. The syner- org). However, the general PKC inhibitor GF109203X (2 gistic ear edema response caused by combined treatment with mmol/L) inhibited LTD4 signals but modestly blocked enhanced PGE2 and LTD4 was substantially attenuated with MK571 plus c-fos and MIP-1b effects by LTD4 plus PGE2 (see Fig E3, B, E, L-798 treatment (Fig 7, D). J ALLERGY CLIN IMMUNOL KONDETI ET AL 7 VOLUME nnn, NUMBER nn

FIG 5. Inflammatory gene induction and COX-2 protein by LTD4 and PGE2 in LAD2 cells. LAD2 cells were stimulated with 0.5 mmol/L LTD4, PGE2, or both; transcript levels of MIP-1b, TNF-a, IL-8, COX-2, and COX- 1 were analyzed by means of real-time PCR at 2 hours of stimulation (A-E); and COX-2 protein levels were determined (F) in cell lysates. *P < .05, **P < .01, and ***P < .001.

DISCUSSION on the receptor subtype, receptor coupling, and the cell type. Edema formation is a prominent feature of the inflammatory For instance, PGE2 blocks FcεRI-mediated exocytosis of rat peri- response and serves an important function in local host defense toneal MCs29 and human lung MCs30 in vitro through an increase and tissue repair. Inflammatory responses result in the movement in cyclic AMP levels (EP2,EP4, or both) but activates murine bone 31 of fluid and plasma proteins into the extracellular space from marrow-derived mast cells through EP3 in vitro. On the contrary, 22 leaky blood vessels in response to various chemical mediators. PGE2 in hMCs did not suppress FcεRI-dependent exocytosis but In the current study, for the first time, we demonstrate that 2 major caused exocytosis on its own when the cells were primed with 16 eicosanoids, LTD4 and PGE2, both derived through alternate path- IL-4. It is likely that the suppressive effects of PGE2 on MC acti- ways from arachidonic acid, synergistically enhanced vascular vation largely reflects EP2 signaling, and in agreement with this, inflammation in vivo (edema formation) and MC activation EP2 protein expression is downregulated in MCs in the nasal in vitro. Although W-sh mice have been well characterized as polyp mucosa of patients with aspirin-exacerbated respiratory dis- 32 MC deficient, they have additional defects beyond just MC defi- ease. PGE2 has been shown to stimulate chemotaxis and adhe- 33,34 ciency, and hence there exists a possibility that the in vivo vascular sion of MCs through EP3. In a recent report Morimoto 19 permeability attenuated in W-sh mice could be mediated through et al demonstrated that EP3 signaling in MCs generated PGE2- other cell types in addition to MCs. A number of mediators have induced vasodilatation and subsequent edema formation and been implicated in the regulation of inflammation in an MC- speculated that EP3-mediated MC activation might be involved mediated mechanism.23 Although MCs are primarily known to in antigen-independent innate immune reactions. Interestingly, be activated in an antigen-dependent manner, evidence also sug- we identified that the LTD4 plus PGE2 synergism is also EP3 19,22,24 gests a role for them in antigen-independent activation. dependent, strengthening the idea that EP3 activation might They are regulatory cells throughout the course of acute inflam- contribute to a proinflammatory role of PGE2. 25 mation, from its initiation to resolution, and they contribute to LTD4 plus PGE2 treatment also upregulated transcripts for in- the development of allergic disease.26 MC activation in patients flammatory mediators, such as TNF-a and IL-8, both of which are with various kinds of inflammatory diseases is significant from chemoattractants for neutrophils. Interestingly, we observed syn- a clinical perspective. Translational implications of LTD4 plus ergistic COX-2 induction by LTD4 plus PGE2 treatment and PGE2 crosstalk provoked us to examine the candidate molecules PGD2 generation, revealing another major finding that LTD4,in and signaling mechanisms involved. We identified that LTD4- concert with PGE2, can generate proinflammatory metabolite PGE2 synergism was mediated through CysLT1R and EP3.Itis PGD2 and amplify associated signaling. MCs express both known that G protein–coupled receptors interact with one another, COX-1 and COX-2, and COX-2 is upregulated by inflammatory modulating each other’s function both positively and nega- stimuli, suggesting a mechanism that can amplify PGD2 genera- 27,28 tively. Also, PGE2 relays differential responses depending tion under inflammatory conditions. Interestingly, PGD2 has been 8 KONDETI ET AL J ALLERGY CLIN IMMUNOL nnn 2015

FIG 6. LTD4 and PGE2 crosstalk causes a synergistic increase in PGD2 secretion and Erk phosphorylation and is mediated through PKG and Erk. LAD2 cells were stimulated with 0.5 mmol/L LTD4, 0.5 mmol/L PGE2,or both in the presence or absence of MK571 (1 mmol/L), L-798 (100 nmol/L), KT5823 (KT;5mmol/L), or

PD98059 (PD;50mmol/L) before treatment, and PGD2 secretion (A and F), Erk phosphorylation (B), c-fos (C and D), and MIP-1b generation (E) were analyzed. *P < .05, **P < .01, and ***P < .001.

recently shown to synergize with LTE4 to stimulate diverse TH2 signaling component from LTD4 and another component from 35,36 functions and TH2 cell and neutrophil crosstalk. It is possible PGE2 signaling, rather than crosstalk at the receptor level. that during inflammation, LTD4 plus PGE2 treatment not only ac- Although we found PKG and Erk are effector molecules in this tivates MCs and initiates inflammation, but also the product of signal, the convergence of these signaling events are still elusive. this signaling, PGD2, can potentially perpetuate inflammation Studies from EP knockout mice suggest that EP3 is mainly through combined action with LTE4. Both LTD4 and PGE2 have responsible for PGE2-induced MC activation and associated 8,16 31 been demonstrated to phosphorylate Erk in MCs. In agree- proinflammatory signaling pathways. High expression of EP3 ment, we noted phosphorylation of Erk by both agonists and is observed in the brain, and recent findings suggest an injurious observed enhanced Erk phosphorylation with LTD4 plus PGE2. role for the PGE2-EP3 signaling axis in modulating brain injury 38 Furthermore, we identified that LTD4-PGE2 synergy is relayed and inflammation after intracerebral hemorrhage. Along through PKG and Erk downstream of CysLT1R and EP3. Surpris- similar lines, enhanced EP3 expression and signaling were found ingly, we found that PKC signals, which are vital in various in patients with diabetes and have been speculated as a new ther- 17,21,37 aspects of CysLT1R, or PKA signals, which mediate EP- apeutic target for b-cell dysfunction in patients with type 2 induced effects,16 are dispensable for this crosstalk, but it is diabetes.39 dependent on PKG and Erk. All the above studies point to the pathologic role of EP3. Cur- We have recently shown that LTD4 can influence SCF re- rent work alludes to the advantage of blocking CysLT1R along 12 sponses, potentiating MC proliferation. We also found that with EP3, and it is tempting to speculate that the combined treat- LTD4 potentiates endothelial cell adhesion mediated by TNF- ment of EP3 and CysLT1R antagonists might contribute effec- a,20 suggesting that cys-LTs can modulate inflammation by acting tively to targeting inflammation compared with the available in concert with other inflammatory mediators. Indeed, findings CysLT1R antagonists alone and could open new avenues in clin- from the present study revealed that LTD4 and PGE2 produced ical approaches for MC-mediated inflammatory diseases. at the inflammation site can synergistically activate MCs and Although PGE2 alone did not induce significant inflammatory further enhance inflammation. Notably, this enhanced inflamma- transcripts and MIP-1b generation, it potentiated all the inflam- tion by LTD4 and PGE2 both in vitro and in vivo is blocked only by matory readouts in concert with LTD4, suggesting that cys-LTs the combined treatment of CysLT1R and EP3 antagonists and not could modulate PGE2 signaling and blocking LTD4 plus PGE2 by either antagonist alone. This argues for the possibility that the could be a potential therapeutic target to combat initiation and synergy is achieved through the contribution of a specific progression of inflammation. J ALLERGY CLIN IMMUNOL KONDETI ET AL 9 VOLUME nnn, NUMBER nn

FIG 7. Simultaneous inhibition of CysLT1R and EP3 on LTD4 plus PGE2–mediated MC activation in vitro and ear edema in vivo. CysLT1R, EP3, or both and PKG were knocked down in LAD2 cells by corresponding siR- NAs (10 nmol/L), and c-fos (A and B) and MIP-1b (C) were analyzed with LTD4 plus PGE2 treatment. D, Ear thickness in BALB/c mice treated with 0.5 mmol/L LTD4 plus PGE2 6 MK571 (1 mmol/L) or L-798 (100 nmol/L). Results in Fig 7, D, are means 6 SEMs from 4 to 6 mice per group per experiment and 3 experiments per- formed. *P < .05, **P < .01, and ***P < .001.

6. Urade Y, Ujihara M, Horiguchi Y, Igarashi M, Nagata A, Ikai K, et al. Mast cells Key messages contain spleen-type prostaglandin D synthetase. J Biol Chem 1990;265:371-5. 7. Woodward DF, Jones RL, Narumiya S. International Union of Basic and Clinical d LTD4 synergizes with PGE2 and activates MCs in vitro Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years and vascular inflammation in vivo through CysLT1R of progress. Pharmacol Rev 2011;63:471-538. 8. Paruchuri S, Jiang Y, Feng C, Francis SA, Plutzky J, Boyce JA. Leukotriene and EP3, which might contribute to MC-mediated allergic inflammation. E4 activates peroxisome proliferator-activated receptor gamma and induces prostaglandin D2 generation by human mast cells. J Biol Chem 2008;283: d LTD4-PGE2 synergism triggers diverse MC inflamma- 16477-87. tory responses through Gi-, PKG-, and Erk- 9. Ciana P, Fumagalli M, Trincavelli ML, Verderio C, Rosa P, Lecca D, et al. The dependent pathways. GPR17 identified as a new dual nucleotides/cysteinyl- leukotrienes receptor. EMBO J 2006;25:4615-27. d LTD4 plus PGE2 crosstalk is efficiently blocked by 10. Kanaoka Y, Maekawa A, Austen KF. Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand. simultaneous inhibition of CysLT1R and EP3 but not by either of the agonists alone, suggesting that vascular J Biol Chem 2013;288:10967-72. 11. Kim DC, Hsu FI, Barrett NA, Friend DS, Grenningloh R, Ho IC, et al. Cysteinyl inflammation can be targeted efficiently by combining leukotrienes regulate Th2 cell-dependent pulmonary inflammation. J Immunol currently available CysLT1R antagonists with EP3 2006;176:4440-8. antagonists. 12. Al-Azzam N, Kondeti V, Duah E, Gombedza F, Thodeti CK, Paruchuri S. Modu- lation of mast cell proliferative and inflammatory responses by leukotriene d4 and stem cell factor signaling interactions. J Cell Physiol 2015;230:595-602. 13. Stables MJ, Gilroy DW. Old and new generation lipid mediators in acute inflam- REFERENCES mation and resolution. Prog Lipid Res 2011;50:35-51. 1. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai 14. Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem 2007;282: M. Mast cells as ‘‘tunable’’ effector and immunoregulatory cells: recent advances. 11613-7. Annu Rev Immunol 2005;23:749-86. 15. Kirshenbaum AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven MA, et al. Charac- 2. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997;77:1033-79. terization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 3. Hart PH. Regulation of the inflammatory response in asthma by mast cell products. established from a patient with mast cell sarcoma/leukemia; activation following Immunol Cell Biol 2001;79:149-53. aggregation of FcepsilonRI or FcgammaRI. Leuk Res 2003;27:677-82. 4. Grimbaldeston MA, Chen CC, Piliponsky AM, Tsai M, Tam SY, Galli SJ. Mast 16. Feng C, Beller EM, Bagga S, Boyce JA. Human mast cells express multiple EPs for cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating prostaglandin E2 that differentially modulate activation responses. Blood 2006; mast cell biology in vivo. Am J Pathol 2005;167:835-48. 107:3243-50. 5. Kanaoka Y, Boyce JA. Cysteinyl leukotrienes and their receptors: cellular distribu- 17. Paruchuri S, Hallberg B, Juhas M, Larsson C, Sjolander A. Leukotriene D(4) acti- tion and function in immune and inflammatory responses. J Immunol 2004;173: vates MAPK through a Ras-independent but PKCepsilon-dependent pathway in in- 1503-10. testinal epithelial cells. J Cell Sci 2002;115:1883-93. 10 KONDETI ET AL J ALLERGY CLIN IMMUNOL nnn 2015

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1 Online Repository

2 Figure legends

3 Figure E1. Dose-dependent increase in PGE2-LTD4 responses and expression of CysLTR

4 and EP receptors in LAD2 cells. LAD2 cells were treated with 0.5 µM LTD4 and the increasing

5 concentrations of PGE2 and (A) c-fos, (B) MIP1β were analyzed. (C) LAD2 cells were analyzed

6 for the expression pattern of CysLTR and EP receptors by real-time PCR (D) Protein profile of

7 CysLTR and EP receptors in LAD2 cells and hMCs.

8 Figure E2. Effect of Gi, EP1 and EP4 inhibition on PGE2-LTD4 responses in LAD2 cells.

9 LAD2 cells were pre-incubated with PTX (100 ng/ml, 18 h; A-C), ONO-8711 (EP1 antagonist; 2

10 nM), and L-161 (EP4 antagonist; 100 nM) in (D, E), stimulated with 0.5 µM LTD4 and/or 0.5 µM

11 PGE2 and (A, B, D) c-fos, (C, E) MIP1β were analyzed.

12 Figure E3. Effect of PKC and PKA on synergistic responses to LTD4 and PGE2. LAD2 cells

13 were pre-incubated for 30 min with (A, E, F- H7; 10 μM), (B, E, F- GFX; 2 µM), (C, E, F- Rp-

14 cAMPS; 50 µM) and (D, E, F- H89; 1 µM), treated with 0.5 µM LTD4+PGE2 and (A-E) c-fos and

15 (F) MIP1β were analyzed.

16 Figure E4. Efficiency of siRNA-mediated knockdown of CysLT1R, EP3, and PKG in LAD2

17 cells. CysLT1R and /or EP3 receptors and PKG were knocked down in LAD2 cells by transfecting

18 corresponding siRNAs or non-specific siRNA pool (10 nM). Extent of knock down and specificity

19 of (A) CysLT1R (B) EP3 and (C) PKG were analyzed by real-time PCR.

20

21 Figure E1 A 0.001 0.001 0.01 0.01 0.01 0.1 0.1 0.1 0.5 0.1 10 10 1 PGE2 (M) - - Ilo (M) - - Buta (M) - - 1 5 Sulp (M) - - 1 p-c-fos c-fos

GAPDH

LTD 4 -++++ + -++++ -++++ ++ -++++ + (0.5M)

B CD sulp (M)

15000 Control LTD₄(0.5M) LAD2 hMC * 0.6 CysLT1R PGE2 (M) Ilo (M) ** 0.3 10000 ** ** CysLT2R * ** * (pg/ml) Buta (M)  * 0 EP1 to GAPDH ₁ ₂ ₃ ₄

* R R ₁ ₂

EP EP EP EP EP MIP1 5000 2 Fold change compared

CysLT CysLT EP3

0 EP4 1 1 1 5 10 10 0.1 - 0.1 0.5 0.1 0.1 GAPDH 0.01 0.01 0.01 0.01 0.001 0.001 Figure E2

A D

p-c-fos

c-fos p-c-fos

GAPDH c-fos B ** GAPDH 500 Control 400 NS 400 NS 400 PTX 300 300 300 * 200 200 200 100 100

c-fos (% control) 100 c-fos (% control) c-fos (% control) 0 0 0 (-)PGE2 LTD4 LTD 4+PGE2 C E 10000 20000 NS Control *** Control 8000 15000 ONO-8711 PTX L-161 (pg/ml) 6000 (pg/ml)  ** 10000 4000 

MIP1 5000 2000 MIP1 0 0 (-)PGE2 LTD4 LTD 4+PGE2 (-) LTD4 PGE2 LTD 4 + PGE2 Figure E3

A BDC

p-c-fos

c-fos

GAPDH

E F ** 600 Control 12000 Control H7 * GFX H7 ** Rp-cAMPS GFX 400 8000 H89 Rp-cAMPS

(pg/ml) H89 *  ** 200 4000 c-fos (% control) MIP1

0 0 LTD (-) PGE2 4 LTD 4 + PGE2 (-) PGE2 LTD 4 LTD 4 + PGE2 BC AB

siRNA Fold change 0.5 1.5 0 1 CysLT *** *** 1 R iN siRNA siRNA Fold change 0.5 1.5 0 1 *** EP 3 ***

Fold change 1.5 0.5 1 0 PKG *** Figure E4 Kondeti Page 1

1 Online Repository

2 Table E1: List of primer and siRNA sequences:

3 h MIP 1β Forward: 5’-CCA GCC AGC TGT GGT ATT-3’

4 Reverse: 5’-CAG TTC AGT TCC AGG TCA TAC A-3’

5 h TNFα Forward: 5’-CCA GGG ACC TCT CTC TAA TCA -3’

6 Reverse: 5’-TCA GCT TGA GGG TTT GCT AC -3’

7 h IL-8 Forward: 5’-TTT GCC AAG GAG TGC TAA AGA-3’

8 Reverse: 5’-CCA CTC TCA ATC ACT CTC AGT TC-3’

9 h COX-2 Forward: 5’-CAA CTC TAT ATT GCT GGA ACA TGG A-3’

10 Reverse: 5’-TGG AAG CCT GTG ATA CTT TCT GTA CT-3’

11 h CysLT1R Forward: 5’-TCA ACG TAC CAT TCA CCT TCA T-3’

12 Reverse: 5’-GCA GCC AGA GAC AAG GTT AT-3’

13 h CysLT2R Forward: 5’-CGA CAT GGA AAG TGG GTT TAT G-3’

14 Reverse: 5’-GCA AAG TAA TAG AGC AGA GGA TTG-3’

15 h EP1 Forward: 5’-TTG TCG GTA TCA TGG TGG TGT CGT-3’

16 Reverse: 5’-ATG TAC ACC CAA GGG TCC AGG AT-3’

17 h EP2 Forward: 5’-ACC CTT GGG TCT TTG CCA TCC TTA-3’

18 Reverse: 5’-AGG TCA GCC TGT TTA CTG GCA TCT-3’

19 h EP3 Forward: 5’-TGG ATC CTT GGG TTT ACC TGC TGT -3’

20 Reverse: 5’-AGG TGG AGC TGG ATG CAT AGT TGT -3’

21 h EP4 Forward: 5’-TGG TGC GAG TAT TCG TCA ACC AGT -3’

22 Reverse: 5’-CAA TGC GGC AGA AGA GGC ATT TGA -3’

23 h GAPDH Forward: 5’-TGC ACC ACC AAC TGC TTA GC-3’

24 Reverse: 5’-GGC ATG GAC TGT GGT CAT GAG-3’

25 Primers for COX-1 and PKG-1 were obtained from QIAGEN (330001 PPH01306E and 330001

26 PPH00491C respectively). SYBR green PCR master mix was from Quanta Biosciences. Kondeti Page 2

1 All siRNA constructs used were purchased from Dharmacon.

2 h CysLT1R- siGENOME SMART pool (Cat# M-005475-02-0005)

3 Target Sequence 1: GAAACUAAACCCUGUGAUU

4 Target Sequence 2: GCAGAAGUCCGUGGUCAUA

5 Target Sequence 3: GGUCUUGCAUUAUGUGUCA

6 Target Sequence 4: CAUUCUUUGUCCAGCGUGA

7 h EP3- siGENOME SMART pool (Cat# M-005713-02-0005)

8 Target Sequence 1: CGGUCGUCAUCGUCGUGUA

9 Target Sequence 2: CUUAAUAGCUGUUCGCCUG

10 Target Sequence 3: CGGGAGAGCAAGCGCAAGA

11 Target Sequence 4: GGACUAGCUCUUCGCAUAA

12 h PKG1- siGENOME SMART pool (Cat# M-004658-04-0005)

13 Target Sequence 1: CCUAUAACAUCAUAUUGAG

14 Target Sequence 2: GGAUUGACAUGAUAGAAUU

15 Target Sequence 3: GGACAGGACUCAUCAAGCA

16 Target Sequence 4: GAACAAAGGCCAUGACAUU

17 Non-Targeting- siGENOME Pool#2 siRNA (Cat# D-001206-14-05)

18

19

20

21

22

23

24

25

26 APPENDIX A3

138

NIH Public Access Author Manuscript J Cell Physiol. Author manuscript; available in PMC 2016 March 01.

NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: J Cell Physiol. 2015 March ; 230(3): 595–602. doi:10.1002/jcp.24777.

Modulation of mast cell proliferative and inflammatory

responses by Leukotriene D4 and Stem Cell Factor signaling interactions

Nosayba Al-Azzam1, Vinay Kondeti1, Ernest Duah1, Farai Gombedza1, Charles K. Thodeti2, and Sailaja Paruchuri1,* 1Department of Chemistry, University of Akron, Akron, OH 2Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH

Abstract Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT1R and CysLT2R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD4-induced calcium, c-fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation.

Keywords

calcium; c-fos; c-Kit; cys-LTs; LTD4; mast cells; MIP1β, proliferation; calcium; stem cell factor

Introduction Mast cells (MCs) are stem cell factor (SCF)-dependent hematopoietic cells that are ubiquitously distributed throughout the body (Gurish and Boyce, 2006; Wedemeyer et al., 2000) and they initiate inflammatory responses to allergens and infectious agents (Okayama and Kawakami, 2006). MCs play an important role in asthma through the secretion of several soluble inflammatory mediators. c-Kit is a member of the type III subclass of receptor tyrosine kinases, comprising of an N-terminal extracellular ligand binding domain, a transmembrane domain and a cytoplasmic kinase domain, which is activated by ligand- mediated receptor dimerization (Mani et al., 2009). C-Kit activation by SCF is crucial for

*Corresponding Author: Sailaja Paruchuri, PhD, Department of Chemistry, KNCL 406, 185 E Mill street, Akron, OH 44325; Phone: 3309722193; [email protected]. The authors have no conflict of interest. Al-Azzam et al. Page 2

the survival and proliferation of human MCs and and the deregulation of the c-kit/SCF axis can lead to uncontrolled proliferation as seen during systemic mastocytosis. Mastocytosis is

NIH-PA Author Manuscript NIH-PA Author Manuscriptthe disturbance NIH-PA Author Manuscript of the homeostatic mechanisms that control the accumulation, proliferation, survival, and turnover rates of MCs, contributing to inflammation, and remodeling. The mechanistic basis of MC hyperplasia in asthma (or in any allergic disease) is not completely understood. The importance of c-Kit signaling in MC proliferation makes it crucial to understand the basic mechanisms by which Kit regulates MC function. Although the role of c-Kit signaling is extensively studied in porcine aortic endothelial cells (Blume-Jensen et al., 1994; Blume-Jensen et al., 1993), its role in MC proliferation is still not well known.

Cysteinyl leukotrienes (cys-LTs), comprising of LTC4, LTD4 and LTE4 are potent bronchoconstrictors and they play an important role in asthma and airway inflammation (Davidson et al., 1987; Drazen and Austen, 1987). They are derivatives of arachidonic acid generated by MCs, eosinophils, basophils, macrophages, and myeloid dendritic cells (Kanaoka and Boyce, 2004), and act through two main receptors, CysLT1R and CysLT2R (Heise et al., 2000; Lynch et al., 1999). MCs not only generate cys-LTs, but also express CysLT1R and CysLT2R (Mellor et al., 2003; Mellor et al., 2001). We and others have previously shown that stimulation of human cord blood-derived MCs hMCs) with LTD4 potently induces calcium flux and cytokine generation through CysLT1R (Mellor et al., 2002; Paruchuri et al., 2008).

During inflammation, various mediators prime each other’s responses, resulting in amplified inflammatory milieu. For example, SCF-induced prolonged activation of mast cells has been shown to play critical role in the progression of allergen-induced airway hyper responsiveness (AHR) and chronic airway hypersensitivity (Hundley et al., 2004). Also, SCF has been implicated in the induction of airway hyper-reactivity during allergy-induced pulmonary responses in mouse models (Campbell et al., 1999). Interestingly, SCF-induced airway hyper-reactivity has been shown to depend on leukotriene production (Oliveira et al., 2001). Enhanced proliferation of MCs is commonly seen at the site of inflammation and this increase in MC number correlates with the severity of AHR. Given that both SCF and leukotrienes are produced at the site of inflammation and induce AHR together with the fact that MCs express receptors for both SCF and leukotrienes and enhanced MC proliferation is seen during inflammation, we asked if c-Kit and CysLT1R can cross-talk and influence MC proliferatory and inflammatory phenotypes. Therefore, in the current study, we tested the MC proliferatory and inflammatory responses in the presence of both SCF and LTD4. We hypothesized that the growth factor SCF can boost LTD4-mediated inflammatory signals and an inflammatory mediator, LTD4, can increase SCF-induced, c-Kit-mediated proliferative responses. Our results suggest that LTD4 and SCF synergistically enhance MC proliferative (c-Kit phosphorylation and proliferation) and inflammatory responses (c-fos phosphorylation, expression, and MIP1β production).

Materials and Methods Reagents

LTC4, LTD4, LTE4, and MK571 were purchased from Cayman Chemicals, Fura-2 AM from Molecular Probes, All phospho-specific antibodies and corresponding controls from Cell

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Signaling Technology, GAPDH antibody from Fitzgerald (Acton, MA), XTT proliferation assay kit was from Trevigen (Gaithersburg, MD), BrdU proliferation assay kit was from

NIH-PA Author Manuscript NIH-PA Author ManuscriptCalbiochem, NIH-PA Author Manuscript MIP1β ELISA kit was from R&D systems. All cytokines were purchased from R&D systems.

Cell culture The LAD2 MC leukemia line (Kirshenbaum et al., 2003) was a generous gift from Dr. Arnold Kirshenbaum, NIH. LAD2 cells were cultured in stemPro-34 (Invitrogen) supplemented with 2mM L-Glutamine (Invitrogen), Pen-strep (100 IU/ml) (Invitrogen), and SCF (R&D systems) (100 ng/ml). Cell culture medium was hemi-depleted every week with fresh medium and 100 ng/ml SCF. Cells were SCF-starved overnight before stimulating with SCF in all the experiments. LAD2 cells are dependent on SCF for their proliferation and retain excellent responses to cys-LTs in inducing calcium flux, secretion of MIP1β and other chemokines, similar to isolated hMCs. (Paruchuri et al., 2008; Paruchuri et al., 2009). The expression pattern of cys-LT receptors in these cells are also similar to hMCs with high levels of CysLT1R compared to CysLT2R (Paruchuri et al., 2008). Bone marrow derived mast cells (BMMCs) were isolated from C57BL/6 mice. Mice were euthanized according to guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) of the Northeast Ohio Medical University (NEOMED). BMMCs were cultured in 80% RPMI 1640 supplemented with 10% fetal bovine serum, 2mM L-Glutamine, Pen-Strep (100UI/ml), Sodium Pyruvate (1mM), Non-Essential Amino acids, HEPES buffer (25mM) and β- Mercaptoethanol (50uM) and 20% WEHI-3 cell conditioned medium for 4–6 weeks. Maturity of BMMCs was examined by Toluidine blue staining and >90% mature BMMCs were used for experiments.

Cord blood was obtained from Cleveland Cord Blood Center and MCs were isolated as described (Mellor et al., 2002). Briefly, heparin-treated cord blood was sedimented with 4.5% dextran solution and the buffy coat was layered onto Ficoll-Hypaque and mononuclear cells (MNC) were obtained after centrifugation at the interphase. Erythrocytes were further removed from MNC by hypotonic lysis and cultured in RPMI-1640 (Gibco), 10%FBS, 2 mM L-glutamine, 0.1 mM non- essential amino acids, Penicillin-Streptomycin, Gentamicin and 0.2µM 2-Mercaptoethanol in the presence of SCF (100ng/ml), IL-6 (50 ng/ml) and IL-10 (10 ng/ml). Non-adherent cells were transferred to fresh medium containing cytokines every week for 6–9 weeks. Maturity of hMCs was examined by Toluidine blue staining and >90% mature hMCs were used for experiments.

Calcium flux Cells were cultured in SCF-free medium overnight. Thereafter, cells (0.5–1 × 106/sample) were washed and labeled with fura 2-AM for 30 minutes at 37°C. Cells were further washed and stimulated with 500 nM of LTD4 and/or 100 ng/ml of SCF, and the changes in intracellular calcium were measured using excitation at 340 and 380nm in a fluorescence spectrophotometer (Hitachi F-4500) as described earlier (Paruchuri et al., 2008). In some experiments, MK571 was added 10 minutes before the addition of indicated agonists. The relative ratios of fluorescence emitted at 510 nm were recorded and displayed as a reflection of intracellular calcium concentration.

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Cell activation

Cells were either stimulated with 500 nM LTD4 and/or 100 ng/ml SCF (or with indicated NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript concentrations) for indicated time points (phosphorylation of c-Kit for 15 min, phosphorylation and expression of c-fos for 1h, measurement of cytokines at transcript level for 2h, and protein level for 6h). The concentration of MIP1β was measured with ELISAs according to the manufacturer’s protocol (Paruchuri et al., 2008).

Cell lysates and western blotting After stimulation with the respective agonists, MCs (0.5×106) were lysed with lysis buffer (BD Bioscience) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Pierce). Immunoblotting was performed as described previously (Paruchuri et al., 2002). Briefly, lysates were subjected to 4–12% SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with respective primary phospho- and total antibodies diluted in 1× TBS, 5% dry milk, 0.1% Tween-20 (1:1000) overnight at 4°C on shaker, and then with secondary antibody (peroxidase-conjugated anti- rabbit or anti-mouse). Western blot was incubated with ECL, and the bands were visualized using imager (Protein Simple) and quantified using Alpha View software (Protein Simple).

Real-time Quantitative PCR The expressions of COX-2, MIP1β and TNFα transcripts were determined with real-time PCR performed on Light cycler 480 (Roche). Cells were cultured and treated as described above, and total RNA was isolated with an RNAeasy minikit (Qiagen) according to manufacturer’s instructions. RNA concentration was determined using a Take 3 module of Epoch micro plate reader (Biotek) at 260/280 nm. 1µg of total RNA was used for reverse transcription using cDNA synthesis kit from Quanta Biosciences, containing MgCl2, dNTPs, recombinant RNAse inhibitor protein, qScript Reverse Transcriptase, random primers, oligo (dT) primers and stabilizers. Gene expression was assayed by quantitative real-time PCR on LightCycler® 480 II (Roche Applied Science) using LightCycler® 480 SYBR Green I Master mix, cDNA prepared as described above and COX-2, MIP1β, TNFα and GAPDH forward and reverse Primers. Following are the forward (F) and reverse (R) primers used

MIP1β– F- CCAGCCAGCTGTGGTATT R-CAGTTCAGTTCCAGGTCATACA TNFα- F- CCAGGGACCTCTCTCTAATCA R-TCAGCTTGAGGGTTTGCTAC COX-2- F-CAACTCTATATTGCTGGAACATGGA R-TGGAAGCCTGTGATACTTTCTGTACT GAPDH- F-TGCACCACCAACTGCTTAGC R-GGCATGGACTGTGGTCATGAG

The ΔΔCt values for COX-2, MIP1β, and TNFα were calculated relative to the GAPDH levels and values were expressed as fold change over the control (Duah et al., 2013).

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Cell proliferation Cells were plated at a density of 5000 cells/well of 96 well plate, cultured in SCF-free NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript medium overnight, and treated with increasing concentration of SCF and/or 500 nM of LTD4. After 72 h, the proliferation was assayed either by XTT assay (Trevigen) or BrdU ELISA (Millipore) according to the manufacturer’s protocol.

Data Analysis Data is expressed as mean ± SEM from at least three experiments except where otherwise indicated. Significance was determined using one way ANOVA and post-hoc analysis.

Results SCF induces concentration-dependent phosphorylation of c-Kit in MCs SCF is the major growth factor for the proliferation of MCs, and it relays its responses through the , c-Kit (Galli et al., 1995; Iemura et al., 1994; Tsai et al., 1991). To confirm if c-Kit is activated in response to SCF stimulation in LAD2 cells, we first determined the effect of SCF on phosphorylation of c-Kit receptor, by treating the cells with different concentrations of SCF. Stimulation with SCF for 15 minutes led to dose- dependent phosphorylation (Fig. 1A, B) of c-Kit receptor. SCF induced phosphorylation of c-Kit receptor at doses as low as 1ng/ml with a maximum response at 100 ng/ml concentration.

C-Kit phosphorylation is synergistically activated by SCF and LTD4 To determine if there is crosstalk between c-Kit and CysLTRs, we stimulated LAD2 cells with cys-LTs with or without SCF and analyzed c-Kit phosphorylation. We could not detect any significant phosphorylation of c-Kit by cys-LTs alone. However, LTD4 significantly strengthened SCF-induced c-Kit phosphorylation (Fig. 2A, B, C). We also found similar synergistic activation of c-Kit by SCF and LTD4 in BMMCs (Suppl. Fig. 1). This synergism with SCF was not observed with LTC4 or LTE4 treatment (Fig. 2A). Further, we found that treatment of cells with CysLT1R antagonist, MK571 prior to stimulation with SCF and LTD4, inhibited this synergistic effect suggesting that it is mediated through CysLT1R (Fig. 2B, C).

LTD4 potentiates SCF-induced MC proliferation

The fact that LTD4 augmented c-Kit phosphorylation by SCF prompted us further to understand the significance of this potentiation. We analyzed if LTD4 treatment together with SCF would amplify LAD2 cell proliferation by using XTT assay. Consistent with earlier results (Laidlaw et al., 2011), we found that SCF promoted proliferation of LAD2 cells in a dose-dependent manner (Fig. 3). Interestingly, a significant increment in cell proliferation was observed with combined stimulation of SCF and LTD4 at high doses of SCF. These findings indicate that LTD4 can enhance c-Kit responses in LAD2 cells and increase their proliferation. We also found that hMCs cultured in the presence of both SCF and LTD4 induced around 2 fold increase in proliferation compared to SCF treatment alone

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(Suppl. Fig. 2A). Taken together, these results suggest that LTD4 in concert with SCF can modulate primary mast cell (hMC) and MC cell line (LAD2) proliferation. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

SCF pre-treatment amplifies CysLT1R function as measured by LTD4-induced calcium flux

Above results clearly depict that combined stimulation of SCF and LTD4 enhances c-Kit- mediated proliferative responses. Next, we asked if this cross-talk is bi-directional i.e. if combined stimulation with SCF can augment LTD4-mediated inflammatory responses, the same way as LTD4 can modulate SCF-induced proliferation. We have previously shown that stimulation of LAD2 cells with LTD4 induces calcium flux, c-fos phosphorylation and expression and MIP1β generation (Kondeti et al., 2013; Paruchuri et al., 2008). First, we asked if SCF pre-treatment influences LTD4-mediated calcium flux. We have previously shown that LTD4 was the most potent agonist among the cys-LTs for eliciting calcium flux and completely desensitized MC to the calcium fluxes induced by the two other cys-LTs (Paruchuri et al., 2008). We observed that SCF (100 ng/ml) induced minimal or no calcium flux by itself (Fig. 4B) or followed by priming with LTD4 (Fig. 4A). However, stimulating cells with SCF prior to LTD4 significantly amplified LTD4-induced calcium flux (Fig. 4B, C). Further, this calcium flux was inhibited by prior treatment with CysLT1R antagonist, MK571, confirming that this signal is mediated by CysLT1R (Fig. 4C).

SCF and LTD4 treatment synergistically amplifies c-fos phosphorylation and expression

Next, we investigated if SCF has the potential to amplify other LTD4-induced MC responses such as c-fos phosphorylation and expression. We have reported earlier that stimulation of LAD2 cells with LTD4 led to phosphorylation and increased expression of c-fos (Kondeti et al., 2013). SCF treatment alone did not induce significant c-fos phosphorylation or expression, however, both LTD4-induced c-fos phosphorylation and expression were synergistically enhanced in the presence of SCF (Fig. 5A, B), further suggesting the ability of SCF/c-Kit to modulate LTD4-induced responses. We also found that treatment of hMCs with a combination of SCF and LTD4 caused potentiation of c-fos phosphorylation and expression compared to treatment with either of the agonists (Suppl. Fig.2B) further confirming the synergistic activation in primary MCs.

Amplification of inflammatory signals by LTD4 and SCF treatment COX-2 activation, MIP1β secretion and up-regulation of other inflammatory cytokine expression are major MC responses downstream of CysLT1R signaling induced by LTD4 (Paruchuri et al., 2008; Paruchuri et al., 2009). These responses further enhance inflammation by recruiting other immune cells. To find out if SCF can increase LTD4- induced MC inflammatory signals, we measured LTD4-induced inflammatory signals in the presence or absence of SCF. LTD4 stimulation alone, but not SCF, induced the expression of COX-2, TNF-α, and MIP1β in LAD2 cells. Interestingly, we found that LTD4-induced expression of these inflammatory genes is significantly boosted by SCF (Fig. 6 A, B, C). Finally, consistent with our transcript data, we found that LTD4 and SCF synergistically increased the secretion of MIP1β protein in LAD2 cells (Fig.6 D). However, either LTD4 or SCF treatment, or the combination of both failed to induce any degranulation (data not shown) as determined by β-hexosaminidase assay using p-NAG (p-Nitrophenyl-N-acetyl-β- D-glucosaminidine) as substrate.

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Discussion

NIH-PA Author Manuscript NIH-PA Author ManuscriptIn the NIH-PA Author Manuscript present study, we demonstrate that an inflammatory mediator, LTD4, and a growth factor, SCF synergistically regulate MC function. We clearly demonstrate that LTD4 can prime SCF-induced c-Kit phosphorylation and proliferation of MCs, and that SCF in turn can strengthen LTD4-induced calcium flux and inflammatory responses such as c-fos phosphorylation and expression, as well as MIP1β production, suggesting a potential cross- talk between the LTD4 and SCF receptors, CysLT1R and c-Kit (Fig. 7).

MCs are mediators of the early phase of allergic inflammation. MCs are released into the blood stream as progenitor cells, and are then recruited to the tissues where they undergo maturation (Gurish and Boyce, 2006; Kirshenbaum et al., 1999; Metcalfe et al., 1997). SCF is an important regulator of MC growth, differentiation, survival and chemotaxis (Grimbaldeston et al., 2006; Prussin and Metcalfe, 2006; Tkaczyk et al., 2006). SCF acts through c-Kit tyrosine kinase receptor which activates downstream signaling molecules such as PI3 Kinase and AKT to exert its effects on MCs (Ali et al., 2004; Moller et al., 2005). SCF was also shown to enhance allergen induced FCεRI mediated degranulation of MCs and release of inflammatory mediators (Gilfillan and Tkaczyk, 2006; Ito et al., 2012; Jensen et al., 2007). These inflammatory mediators may further activate MCs and boost inflammation.

Cys-LTs are produced by MCs and were also shown to activate MCs. We have previously shown that LAD2 cells express both CysLT1R and CysLT2R and that cys-LTs (LTC4, LTD4 and LTE4) induce calcium influx, c-fos expression and phosphorylation (Kondeti et al., 2013; Laidlaw et al., 2011; Paruchuri et al., 2008). Both c-Kit signaling and CysLTR signaling are shown to be mitogenic for MCs. In addition, CysLT1R has been shown to transactivate c-Kit receptor in hMCs (Jiang et al., 2006). Interestingly, patients with systemic mastocytosis showed significantly higher urinary excretion of cys-LTs than controls (Raithel et al., 2011). Further, LTC4 synthase knockout mice were unable to develop MC hyperplasia in the inflamed mucosal surface of the lung in a model of allergen- induced pulmonary inflammation (Kim et al., 2006). However, it is not known if these pro- inflammatory mediators act in concert with SCF. In the present study, though we did not observe any c-Kit phosphorylation or proliferation with LTD4 alone, stimulation of MCs with SCF induced phosphorylation as well as MC proliferation. Interestingly, c-Kit phosphorylation and MC proliferation was augmented in the presence of both SCF and LTD4 suggesting the existence of a cross-talk between their receptors. LTD4, but not LTC4 or LTE4, increased c-Kit phosphorylation by SCF, indicating a specific role for LTD4 in this cross-talk. We also showed that this potentiation of c-Kit is mediated through Cys-LT1R. Notably, we found that stimulating MCs with SCF prior to LTD4 enhanced LTD4-induced calcium flux. In contrast, SCF alone did not induce any calcium flux. Furthermore, we found that c-fos phosphorylation and expression, COX-2, TNF-α and MIP1β transcript expression, as well as MIP1β protein secretion are amplified by combined treatment with SCF and LTD4. Thus, our findings suggest that while LTD4 synergistically activates SCF-induced proliferative signals, SCF in turn potentiates LTD4-induced inflammatory signals in MCs. The concept of this bi-directional cross-talk between LTD4 (CysLT1R) and SCF (c-Kit) signaling is novel and may have important implications in targeting MC-mediated

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inflammatory responses. Importantly, our results suggest that the inflammatory microenvironment, but not a single molecule, dictate the inflammatory phenotype of MCs.

NIH-PA Author Manuscript NIH-PA Author ManuscriptSCF NIH-PA Author Manuscript was previously shown to enhance FCεRI mediated responses in MCs (Gilfillan and Tkaczyk, 2006; Ito et al., 2012; Jensen et al., 2008; Jensen et al., 2007). However, recently it was shown that while acute treatment of SCF potentiated inflammatory phenotype of MCs, prolonged treatment of SCF inhibited PGE2 and allergen-induced MC degranulation (Gilfillan and Tkaczyk, 2006; Ito et al., 2012; Jensen et al., 2007). The underlying molecular mechanism could be alterations in the cytoskeleton by SCF-mediated regulation of src kinase, hck (Ito et al., 2012; Smrz et al., 2013).

In conclusion, our results suggest that the cross-talk between SCF and LTD4 induces MC proliferation, gene regulation, and cytokine production. SCF signaling through c-Kit may regulate baseline maintenance of MCs. However, locally derived cys-LTs produced at the site of inflammation enhance MC numbers by cross talking with c-Kit receptor. Similarly, this c-Kit and CysLT1R crosstalk also potentiates LTD4 effects enhancing MC inflammatory responses leading to enhanced pathological loop (Fig. 7). Although SCF-induced MC activation in allergen mediated AHR was shown to be dependent on leukotrienes (Oliveira et al., 2001), it is not known whether cys-LTs can modulate SCF receptor. Thus, our findings that LTD4 can enhance c-Kit-dependent proliferative response and SCF potentiate LTD4- mediated inflammatory responses are very intriguing and may provide basis for novel therapeutic targets for. asthma and allergic diseases

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work is supported by National Institutes of Health Grants (HL098953) and by James Foght Assistant Professor to S.P.

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Lynch KR, O'Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueroa DJ, Zeng Z, Connolly BM, Bai C, Austin CP, Chateauneuf A, Stocco R, Greig GM, Kargman S, Hooks SB, Hosfield E, Williams DL Jr, Ford-Hutchinson AW, Caskey CT, Evans JF. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature. 1999; 399(6738): 789–793. [PubMed: 10391245] Mani M, Venkatasubrahmanyam S, Sanyal M, Levy S, Butte A, Weinberg K, Jahn T. Wiskott-Aldrich syndrome protein is an effector of Kit signaling. Blood. 2009; 114(14):2900–2908. [PubMed: 19643989] Mellor EA, Austen KF, Boyce JA. Cysteinyl leukotrienes and diphosphate induce cytokine generation by human mast cells through an interleukin 4- regulated pathway that is inhibited by leukotriene receptor antagonists. J Exp Med. 2002; 195(5):583–592. [PubMed: 11877481] Mellor EA, Frank N, Soler D, Hodge MR, Lora JM, Austen KF, Boyce JA. Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional distinction from CysLT1R. Proc Natl Acad Sci U S A. 2003; 100(20):11589–11593. [PubMed: 13679572] Mellor EA, Maekawa A, Austen KF, Boyce JA. Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells. Proc Natl Acad Sci U S A. 2001; 98(14):7964–7969. [PubMed: 11438743] Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev. 1997; 77(4):1033–1079. [PubMed: 9354811] Moller C, Alfredsson J, Engstrom M, Wootz H, Xiang Z, Lennartsson J, Jonsson JI, Nilsson G. Stem cell factor promotes mast cell survival via inactivation of FOXO3a-mediated transcriptional induction and MEK-regulated phosphorylation of the proapoptotic protein Bim. Blood. 2005; 106(4):1330–1336. [PubMed: 15855272] Okayama Y, Kawakami T. Development, migration, and survival of mast cells. Immunol Res. 2006; 34(2):97–115. [PubMed: 16760571] Oliveira SH, Hogaboam CM, Berlin A, Lukacs NW. SCF-induced airway hyperreactivity is dependent on leukotriene production. Am J Physiol Lung Cell Mol Physiol. 2001; 280(6):L1242–L1249. [PubMed: 11350804] Paruchuri S, Hallberg B, Juhas M, Larsson C, Sjolander A. Leukotriene D(4) activates MAPK through a Ras-independent but PKCε-dependent pathway in intestinal epithelial cells. J Cell Sci. 2002; 115(Pt 9):1883–1893. [PubMed: 11956320] Paruchuri S, Jiang Y, Feng C, Francis SA, Plutzky J, Boyce JA. Leukotriene E4 activates peroxisome proliferator-activated receptor γand induces prostaglandin D2 generation by human mast cells. J Biol Chem. 2008; 283(24):16477–16487. [PubMed: 18411276] Paruchuri S, Tashimo H, Feng C, Maekawa A, Xing W, Jiang Y, Kanaoka Y, Conley P, Boyce JA. Leukotriene E4-induced pulmonary inflammation is mediated by the P2Y12 receptor. J Exp Med. 2009; 206(11):2543–2555. [PubMed: 19822647] Prussin C, Metcalfe DD. 5. IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol. 2006; 117(2 Suppl Mini-Primer):S450–S456. [PubMed: 16455345] Raithel M, Zopf Y, Kimpel S, Naegel A, Molderings GJ, Buchwald F, Schultis HW, Kressel J, Hahn EG, Konturek P. The measurement of leukotrienes in urine as diagnostic option in systemic mastocytosis. J Physiol Pharmacol. 2011; 62(4):469–472. [PubMed: 22100848] Smrz D, Bandara G, Beaven MA, Metcalfe DD, Gilfillan AM. Prevention of F-actin assembly switches the response to SCF from chemotaxis to degranulation in human mast cells. Eur J Immunol. 2013; 43(7):1873–1882. [PubMed: 23616175] Tkaczyk C, Jensen BM, Iwaki S, Gilfillan AM. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses. Immunol Allergy Clin North Am. 2006; 26(3):427–450. [PubMed: 16931287] Tsai M, Takeishi T, Thompson H, Langley KE, Zsebo KM, Metcalfe DD, Geissler EN, Galli SJ. Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc Natl Acad Sci U S A. 1991; 88(14):6382–6386. [PubMed: 1712491] Wedemeyer J, Tsai M, Galli SJ. Roles of mast cells and basophils in innate and acquired immunity. Curr Opin Immunol. 2000; 12(6):624–631. [PubMed: 11102764]

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Figure 1. Dose dependent phosphorylation of c-Kit receptor by SCF (A) LAD2 cells were stimulated with the indicated doses of SCF for 15 minutes and the c- Kit phosphorylation was assessed by western blotting using phospho-specific c-Kit antibodies. Blots were stripped and re-blotted for GAPDH to confirm equal loading. The data shown are representative of three separate experiments. (B) Densitometric analysis of data shown in A. The data represents mean ± SEM of three separate experiments. The significance was tested using one way ANOVA and post-hoc analysis *P<0.05, **P<0.001.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 12 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 2. LTD4 and SCF synergistically phosphorylate c-Kit c-Kit phosphorylation was analyzed by western blotting in LAD2 cell lysates (A) stimulated with 500 nM of LTC4, LTD4 and LTE4 in presence or absence of 100ng/ml SCF for 15 minutes. (B) Phospho-c-Kit levels upon stimulation with 500 nM LTD4 and/or of 100 ng/ml SCF for 15 minutes with/without MK571 (1µM) pre-treatment (30 min). (C) Densitometric analysis of phospho-c-Kit levels upon SCF and /or LTD4 stimulation in the presence or absence of MK571 (1µM). The data represents mean ± SEM of three separate experiments. The significance was tested using one way ANOVA and post-hoc analysis *P<0.05.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 13 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 3. LTD4 enhances SCF-mediated cell proliferation Proliferation of LAD2 cells stimulated with 500 nM LTD4 and/or the indicated dose of SCF was measured by XTT assay. Changes in cell proliferation were expressed as percentage of control. The data represents mean ± SEM of three separate experiments. Data was analyzed with one way ANOVA and post-hoc analysis. *P<0.05.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 14 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 4. SCF primes LTD4 -induced calcium flux (A, B) LAD2 cells were loaded with Fura-2AM and stimulated with 500 nM LTD4 and 100ng/ml SCF at indicated times arrows) and changes in intracellular calcium concentration were measured. (C) Quantitative analysis of the three experiments performed. The data represents mean ± SEM of three separate experiments. Data was analyzed with one way ANOVA and post-hoc analysis. *P<0.05, **P<0.001.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 15 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 5. SCF potentiates LTD4-induced c-fos phosphorylation and expression LAD2 cells were treated with 500 nM LTD4 and/or 100 ng/ml of SCF for 1 h and the phospho- and total c-fos levels were evaluated by western blotting. (B) Densitometric analysis of c-fos expression shown in (A). The data represents mean ± SEM of three separate experiments. Data was analyzed with one way ANOVA and post-hoc analysis. P<0.05.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 16 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 6. SCF augments LTD4-induced inflammatory gene repertoire LAD2 cells were treated with 500 nM LTD4 and/or 100ng/ml of SCF for 2 h, followed by mRNA extraction and cDNA synthesis. Transcript levels of COX-2 (A), TNFα (B), and MIP1β (C) were analyzed in these cDNAs using respective real time primers and were analyzed compared to GAPDH. The graph represents fold change in the level of transcripts compared to controls from three separate experiments. (D) LAD2 cells were treated with 500 nM LTD4 in the presence or absence of 100ng/ml SCF for 6h. Culture medium was collected and analyzed for secreted MIP1β protein by ELISA. The data shown represents mean ± SEM of three separate experiments. Data was analyzed with one way ANOVA and post-hoc analysis. *P<0.05, **P<0.001.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Al-Azzam et al. Page 17 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 7. Schematic showing the possible cross-talk between LTD4 and SCF in mast cells LTD4 alone induces calcium influx via CysLT1R leading to activation of c-fos and generation of inflammatory chemokine, MIP1β in MCs, while SCF alone enhances their proliferation through the activation of C-kit. However, simultaneous stimulation of MCs with both LTD4 and SCF synergistically enhance each other’s responses via cross-talk between associated signaling leading to augmented inflammatory and proliferatory responses.

J Cell Physiol. Author manuscript; available in PMC 2016 March 01. Modulation of mast cell proliferative and inflammatory responses by

Leukotriene D4 and Stem Cell Factor interactions

Nosayba Al-Azzam1, Vinay Kondeti1, Ernest Duah1, Farai Gombedza1, Charles K.

Thodeti2 and Sailaja Paruchuri1*

1 Department of Chemistry, University of Akron, Akron, OH, 2 Department of Integrative

Medical Sciences, Northeast Ohio Medical University, Rootstown, OH

*Corresponding Author:

Sailaja Paruchuri, PhD, Department of Chemistry, KNCL 406, 185 E Mill street, Akron,

OH 44325; Phone: 3309722193; E-mail: [email protected]

Contract grant sponsor: NHLBI; Contract grant number: HL098953.

Contract grant sponsor: James Foght Assistant Professorship

Supplementary figure legends

Supplementary figure 1-LTD4 and SCF synergistically phosphorylate c-Kit in

BMMCs- c-Kit phosphorylation in BMMC lysates was analyzed after treatment with 500 nM of LTC4, LTD4 and LTE4 in the presence or absence of 100ng/ml mouse SCF for 15 minutes. The data represents a representative blot of two experiments performed.

Supplementary figure 2- LTD4 enhances SCF-mediated cell proliferation and SCF potentiates LTD4-induced c-fos phosphorylation and expression in hMCs- (A) MCs were treated with 500 nM LTD4 in the presence or absence of 100ng/ml SCF for 1 h and the phosphor, total c-fos and GAPDH levels were evaluated by western blotting. The data represents a representative blot of two experiments performed. (B) hMCs stimulated with 500 nM LTD4 in the presence or absence of 100ng/ml SCF was measured by BrdU ELISA. Changes in cell proliferation were expressed as percentage of control. The data represents mean ± SEM of three separate experiments. Data was analyzed with student's t-test *P<0.05.

Supplementary Figure 1

BMMCs

P-c-Kit

-++- --+ + SCF ---+ --- + LTC4 -+-- +- - - LTD4 --+- -+- - LTE4 Supplementary Figure 2

AB hMCs * SCF SCF + LTD 4 P-c-Fos

c-Fos

(% control) GAPDH cell proliferation

SCF (ng/ml) - 100 APPENDIX A4

139

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For any similar use for a non-accredited course or one offered by a corporation, an institute or corporation must obtain prior written permission from Elsevier and a fee may be required. Anyone interested in such permission should contact the Elsevier Global Rights department at [email protected]

Interlibrary loan [7 above]

The interlibrary loan (ILL) policy for electronic journals is included in each institution‟s ScienceDirect subscription agreement. In short, this allows and provides for the use of electronic journal articles as a source for fulfillment of ILL requests, with some restrictions.

Elsevier grants subscribing institutes the right to use articles from subscribed ScienceDirect content as source material for ILLs subject to the following conditions:

- Each ILL request must come from an academic or other noncommercial, noncorporate research library located in the same country as the subscriber - Each requested article must be printed by the subscriber (not applicable to US customers) and mailed, faxed or transmitted by Ariel (or a similar ILL system) to the requesting library

If a corporate or commercial entity is seeking a ScienceDirect article, instead of requesting ILL, that entity may use ScienceDirect‟s Pay per View service on a guest basis. For details, see www.info.sciverse.com/sd/ppv. Please also refer to http://myelsevier.com

Scholarly sharing of articles [8 above]

Current ScienceDirect subscription agreements permit authorized users to transmit excerpts of subscribed content, such as an article, by e-mail or in print, to known research colleagues for the purpose of scholarly study or research. Recipients of such scholarly sharing do not themselves have to be affiliated with an institute with a ScienceDirect subscription agreement.

Live reference

If they are both affiliated with the same institute, then a librarian can take a user electronically to any Elsevier article to which that institute has subscribed access. The librarian can also e-mail an Elsevier article, included in the library‟s subscription, to the user.

If, however, someone is not affiliated with the same institute as the librarian, then that user is not an authorized user at the librarian‟s institution. The librarian therefore cannot link the user directly to Elsevier articles or e- mail the Elsevier articles to the user. The librarian can, however, explain to the user how to request Elsevier articles through ILL or acquire them through ScienceDirect‟s Pay per View service.

Use by library guests [14 above]

Any institute with a current ScienceDirect subscription may allow members of the general public to use terminals physically located at that institute‟s library to access, search, browse, view and print articles in subscribed Elsevier journals. Some libraries impose their own usage restrictions on such „walk-in users‟. Commercial entities that wish to make copies for commercial purposes should obtain prior authorization from Elsevier or a local reproduction rights organization.

Access Rights

This section provides information on access to Elsevier content that is free at the point of use.

Sponsored articles and open access journals

Elsevier offers authors a variety of Creative Commons licences for its author-pays journals and is piloting a range of options. More information on Creative Commons licences can be found at: http://creativecommons.org/licenses

However, there may be some individual journals (including those owned by third parties on whose behalf the journal is published by Elsevier) for which Elsevier provides a bespoke user licence.

Open archives

This involves making journal articles from a subscription journal freely available on ScienceDirect after a journal specific embargo period. This can be done in two ways:

1. Journal Level

Here a specific journal makes articles within the journal freely available after a specific time period following publication. Elsevier has a number of journals that offer free access after a limited embargo period.

To see a list of Elsevier open archive journals, please follow this link.

2. Institutional Level

Here selected articles associated with a particular institution are made freely available after a specific time period following publication. Elsevier is running two pilots to understand the uptake and use of this delayed access model. We do not make all articles from that institution available and these pilots have been set up using a list of journals from which articles may be selected for inclusion in the pilot.

Under both models, once the embargo period has passed, the content is available for personal, non-commercial use on the basis of the standard terms and conditions of use for www.sciencedirect.com.

Procedia and third party journals hosted by Elsevier Elsevier hosts on ScienceDirect certain content that is free at the point of use- principally Procedia and a number of third party journals for which the peer review and publication process is managed by that third party.

This content is available for personal, non-commercial use on the basis of the standard terms and conditions of use for www.sciencedirect.com.

Free walk-in access via public libraries Any public library or institute with a current ScienceDirect subscription may allow members of the general public to use terminals physically located at that institute‟s library to access, search, browse, view and print articles in subscribed Elsevier journals. Some libraries impose their own usage restrictions on such „walk-in users‟.

Licensed Rights

Purchase of individual articles Anyone may use Pay per View on ScienceDirect and purchase individual full-text journal articles. This service allows users to purchase direct access to articles. HTML and PDF access is instant and available for 24 hours on ScienceDirect; in addition, purchased articles can be downloaded and stored locally for future use. Articles from a number of Elsevier‟s journals are also available as part of DeepDyve (www.deepdyve.com) , the largest online rental service for professional and scholarly research articles. use

Reprints of articles For customers who wish to purchase individual or commercial reprints of an article published by Elsevier, permission can be obtained from Elsevier.

Short quotes and reproduction of material from articles Anyone may in written work quote from an article published by Elsevier, as long as the quote comprises only a short excerpt such as one or two sentences. An appropriate citation, including the journal title, must be provided.

If the intended use is for scholarly content, noncommercial research or educational purposes, an institution or academic may, without seeking permission from Elsevier, use:

- a single text extract of fewer than 100 words or a series of extracts totaling no more than 300 words - a maximum of 2 figures from a journal article or a total of 5 from a journal volume

These guidelines reflect Elsevier‟s endorsement of the International Association of Scientific, Technical & Medical Publishers‟ 2008 guidelines for quotation and other academic uses of excerpts from journal articles. For more information on these guidelines, see www.stm-assoc.org/document-library/ (see Guidelines for Quotations from Journal Articles).

If the intended use or the material needed differs from the categories described above, Elsevier‟s prior written permission must be obtained.

Photocopies of articles National copyright laws generally permit photocopying of an article for personal use.

Elsevier requires permission and a fee for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, copying for resale and copying for all forms of document delivery. Special rates are available for educational institutions (which do not have a subscription with Elsevier) wishing to make photocopies for nonprofit classroom use. (Authors will have the right to use their own material in the classroom as set out in the Author Rights section.)

How to obtain permission from Elsevier Anyone may request permission via Rightslink, the Copyright Clearance Center‟s service available at the top of the HTML version of every journal article on ScienceDirect. Alternatively, e-mail requests to [email protected] or (for individual or commercial reprints) to [email protected] For more information, see:

OBTAINING PERMISSION TO USE ELSEVIER MATERIAL

APPENDIX A5

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License Number 4104221003111 License date May 08, 2017 Licensed Content Publisher John Wiley and Sons Licensed Content Publication Journal of Cellular Physiology Licensed Content Title Modulation of Mast Cell Proliferative and Inflammatory Responses by Leukotriene D4 and Stem Cell Factor Signaling Interactions Licensed Content Author Nosayba Al­Azzam,Vinay Kondeti,Ernest Duah,Farai Gombedza,Charles K. Thodeti,Sailaja Paruchuri

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