Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Hojabrpour, Payman
Master of Science - MSc
University of British Columbia
Idiopathic Pulmonary Fibrosis (IPF) is a progressive and lethal disease characterized by injury and accumulation of fibroblasts, and abnormal remodeling of the lung parenchyma. IPF is a rare disease with no effective treatment. Due to etiology of IPF, several pathogenic mechanisms have been implicated in its development including: aberrant repair of injured epithelium, consistent fibroblast activation and epithelial-to-mesenchymal transition (EMT). The main focus of this research is on the regulation of a bifunctional enzyme called Leukotriene A4 Hydrolase (LTA4H), with pro and anti-inflammatory functions. While LTA4H catalyzes the conversion of leukotriene A4 to the proinflammatory mediator leukotriene B4 (LTB4), it has an aminopeptidase activity that has shown to degrade N-alpha-Proline-Glycine-Proline (N-α-PGP) and non-acetylated PGP, leading to resolution of acute neutrophils driven inflammation in collagen break down during repair process. Chronic neutrophilic inflammation is observed in lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), mediating extensive tissue damage and contributing to organ dysfunction. These studies demonstrated that tissues and fibroblasts cultured from lungs tissues of IPF patients to contain more PGP content and lack extracellular LTA4H and matrix metalloproteinases 8 and 9 (MMP 8/9). In addition, LTA4H expression was much lower in fibrotic lungs when compared to normal patients. Immunohistochemistry (IHC) analysis of the tissue microarray of the IPF and normal patients indicated a reduction in LTA4H in basal, central and lingual part of the lung. In addition to LTA4H studies, it has been also demonstrated that phosphorylation of the c-Jun N-terminal kinases 1/2(JNK), a mitogen-activated protein kinase family which are responsive to stress stimuli, might be linked to remodelling and progression of the IPF. While the expression of JNK was not different across the IPF and normal patients, activated JNK was elevated in the IPF fibroblasts. In addition, the same fibroblast cells were shown to produce more pro-collagen I A; while utilizing the phospho-JNK (P-JNK) inhibitor 1/2, reduced the collagen formation in the IPF fibroblast cells.Further studies are needed to understand the role of LTA4H and PGP breakdown as well as P-JNK pathway in IPF patients, with a hope to control this devastating disease.
REDUCTION OF NEUTROPHILIC INFLAMMATION IN IDIOPATHIC PULMONARY FIBROSIS VIA METABOLISM OF N-ALPHA-PGP BY LEUKOTRIENE A4 HYDROLASE by PAYMAN HOJABRPOUR B.Sc., University of British Columbia, 1998 B.IT, Computer Programming, BCIT 2004 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POST DOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) SEPTEMBER 2018 © Payman Hojabrpour, 2018 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: submitted by Payman Hojabrpour in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Experimental Medicine Examining Committee: Dr. Vincent Duronio Supervisor Dr. Nasreen Khalil Supervisory Committee Member Dr. Michael Cox External Examiner Additional Examiner Additional Supervisory Committee Members: Supervisory Committee Member Supervisory Committee Member iii Abstract Idiopathic Pulmonary Fibrosis (IPF) is a progressive and lethal disease characterized by injury and accumulation of fibroblasts, and abnormal remodeling of the lung parenchyma. IPF is a rare disease with no effective treatment. Due to etiology of IPF, several pathogenic mechanisms have been implicated in its development including: aberrant repair of injured epithelium, consistent fibroblast activation and epithelial-to-mesenchymal transition (EMT). The main focus of this research is on the regulation of a bifunctional enzyme called Leukotriene A4 Hydrolase (LTA4H), with pro and anti-inflammatory functions. While LTA4H catalyzes the conversion of leukotriene A4 to the proinflammatory mediator leukotriene B4 (LTB4), it has an aminopeptidase activity that has shown to degrade N-alpha-Proline-Glycine-Proline (N-α-PGP) and non-acetylated PGP, leading to resolution of acute neutrophils driven inflammation in collagen break down during repair process. Chronic neutrophilic inflammation is observed in lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), mediating extensive tissue damage and contributing to organ dysfunction. These studies demonstrated that tissues and fibroblasts cultured from lungs tissues of IPF patients to contain more PGP content and lack extracellular LTA4H and matrix metalloproteinases 8 and 9 (MMP 8/9). In addition, LTA4H expression was much lower in fibrotic lungs when compared to normal patients. Immunohistochemistry (IHC) analysis of the tissue microarray of the IPF and normal patients indicated a reduction in LTA4H in basal, central and lingual part of the lung. In addition to LTA4H studies, it has been also demonstrated that phosphorylation of the c-Jun N-terminal kinases 1/2(JNK), a mitogen-activated protein kinase family which are responsive iv to stress stimuli, might be linked to remodelling and progression of the IPF. While the expression of JNK was not different across the IPF and normal patients, activated JNK was elevated in the IPF fibroblasts. In addition, the same fibroblast cells were shown to produce more pro-collagen I A; while utilizing the phospho-JNK (P-JNK) inhibitor 1/2, reduced the collagen formation in the IPF fibroblast cells. Further studies are needed to understand the role of LTA4H and PGP breakdown as well as P-JNK pathway in IPF patients, with a hope to control this devastating disease. v Lay Summary Considered as a rare disease of the lung, new remedies and medicines are needed to treat patients with Idiopathic pulmonary fibrosis. This fatal disease is caused by scarring of the tissue inside the lungs. The word 'idiopathic' means that doctors do not know the cause of the lung scarring. Due to dysregulated series of events, the scarred lungs become thick and stiff (fibrotic) making breathing difficult due to replacement of functional lung tissues. Common symptoms of IPF are shortness of breath, continuous dry cough, and enlargement of fingertips. Currently, there is no cure for IPF, and there are very few treatments for patients with IPF with disease worsening over time and eventually leading to death. The goal of this research was to further investigate the roles of Leukotriene A 4 hydrolase and c-Jun N-Terminal Kinase in pulmonary fibrosis. Targeting these proteins could lead to new treatments for IPF. vi Preface While I took over the pulmonary fibrosis project less than three years ago in Laboratory of Dr. Vincent Duronio, the idea of pursuing LTA4H and its anti-inflammatory role in lung disease came through my experience in working with Naegis Pharmaceutical (NaPh). Dr. Dave Burgoyne and Dr. Erin DeBriun’s from NaPh worked on LTA4H enzyme in asthma and COPD. Their encouraging results and the relevance of their research combined with current studies in pulmonary fibrosis, provided a good avenue for me to investigate this phenomenon in IPF lungs. Throughout this thesis, L-proline protocol and related data analysis was adopted from Naegis Pharmaceutica l standard operating protocols (SOP). All transplant lungs were obtained from donors, with approved human ethics protocol H14-02570. Lungs were processed by VGH pathology, with help of Dr. John English and Dr. Robert O’Connor. The biosafety Ethics protocol number for this research was B14-0167. All histology experiments were carried in and supported by Prostate Research Centre of Vancouver (PRC), along with help of Dr. Ladan Fazli and Dr. Alireza Moen in helping with tissue pathology, TMA scoring and providing the support and infrastructure for data analysis. All data analysis and methodologies were adopted from standard operating protocols from PRC’s pathology unit. Chapter 4 of this thesis was based on suggestions and expertise of Dr. Nasreen Khalil for further investigating the role of JNK in IPF. Also, the idea of using Dexamethasone in these experiments was suggested by Dr. Khalil due to its controversial clinical results and in favour of demonstrating it adverse effects in IPF progression. vii All experiments were carried out at Jack Bell Research Center, in the laboratory of Dr. Vincent Duronio, from division of Respiratory Medicine in Department of Medicine. This master was carried through Experimental Medicine Gradate Program part of department of Medicine graduate program. viii Table of Contents Abstract..........................................................................................................................................iii Lay Summary .................................................................................................................................v Preface............................................................................................................................................vi Table of Contents ........................................................................................................................viii List of Figures................................................................................................................................xi List of Abbreviations ..................................................................................................................xiii Acknowledgements .....................................................................................................................xvi Dedication ...................................................................................................................................xvii Chapter 1: Introduction ................................................................................................................1 1.1 Fibrosis............................................................................................................................ 1 1.2 Idiopathic Pulmonary Fibrosis ........................................................................................ 5 1.3 Leukotriene 4A Hydrolase (LTA4H)............................................................................ 12 1.4 C-Jun N-Terminal Kinase (JNK) .................................................................................. 23 Chapter 2: MATERIAL AND METHODS ...............................................................................29 2.1 Materials........................................................................................................................ 29 2.1.1 Chemicals and Reagents ........................................................................................... 29 2.1.2 Peptides, Inhibitors and Proteins............................................................................... 31 2.1.3 Antibodies ................................................................................................................. 31 2.1.4 Instruments................................................................................................................ 32 2.2 Methods......................................................................................................................... 32 2.2.1 Cell Culture ............................................................................................................... 32 ix 2.2.1.1 Processing the Transplant lung from IPF patients ............................................ 32 2.2.1.2 Fibroblast (FBR) preparation from IPF Lungs ................................................. 33 2.2.1.3 Lung tissue and FBR cell preservation ............................................................. 34 2.2.2 Histology Procedure.................................................................................................. 34 2.2.2.1 Paraffin processing of the lung tissues.............................................................. 34 2.2.2.2 Hematoxylin and Eosin Stain (H&E) Stain ...................................................... 34 2.2.2.3 Immunohistochemistry ..................................................................................... 34 2.2.2.4 IHC Scoring Methodology................................................................................ 35 2.2.2.5 Tissue Micro Array ........................................................................................... 35 2.2.3 Immunoprecipitation ................................................................................................. 36 2.2.4 Immunoblotting......................................................................................................... 36 2.2.5 In-cell western blotting ............................................................................................. 37 2.2.6 PGP formation assay in IPF lung fibroblast ............................................................. 38 2.2.6.1 Preparation of reagents and methodology......................................................... 38 2.2.6.2 Stimulation of peptidase activity ...................................................................... 38 2.2.6.3 Detection of L-Proline by ninhydrin ................................................................. 38 2.2.7 Neutrophil isolation from whole blood ..................................................................... 39 2.2.8 Cell proliferation assay (MTS) ................................................................................. 39 2.2.9 Statistical analysis ..................................................................................................... 40 Chapter 3: Role of LTA4H in metabolizing PGP and collagen production. ..........................41 3.1 Rationale and Hypothesis.............................................................................................. 41 3.2 Results ........................................................................................................................... 42 x Chapter 4: Inhibition of c-Jun N-Terminal Kinase I/II (JNK /II) in IPF derived fibroblasts results in-vitro reduction of pro collagen I A formation...........................................................61 4.1 Rationale and Hypothesis.............................................................................................. 61 Chapter 5: DISCUSSION............................................................................................................73 5.1 Conclusion .................................................................................................................... 79 References:....................................................................................................................................81 xi List of Figures Figure 1. 1 – Fibrosis Process and Tissue Regeneration. ............................................................... 4 Figure 1. 2 Wound healing process and pulmonary fibrosis. ......................................................... 6 Figure 1. 3 TGF-β Signaling from Cell Membrane to the Nucleus. ............................................... 8 Figure 1. 4. Metabolism of arachidonic acid to inflammatory lipid mediators ............................ 13 Figure 1. 5. Binding of LTA4 in the active site of LTA4H .......................................................... 17 Figure 1. 6. Crystal Structure of LTA4H and its complex with PGP ........................................... 18 Figure 1. 7. Proposed PGP pathology in lung neutrophilic inflammation. ................................... 21 Figure 1. 8. JNK Signaling Pathway............................................................................................ 24 Figure 3. 1. LTA4H expression is reduced in IPF tissue derived fibroblasts ............................... 44 Figure 3. 2. Fibrotic lungs contain higher level of PGP. .............................................................. 47 Figure 3. 3. Fibrotic lungs contain higher level of PGP. .............................................................. 48 Figure 3. 4. Aminopeptidase activity of the recombinant LTA4H confirms the higher levels of PGP in fibrotic lungs when compared to Normal ......................................................................... 50 Figure 3. 5. LTA4H expression levels are lower in IPF lung homogenized tissues. .................... 52 Figure 3. 6. Tissue micro array scores indicate a lower expression levels of LTA4H in fibrotic tissues. ........................................................................................................................................... 56 Figure 3. 7. Intracellular levels of MMP-9 are lower in fibroblasts derived from IPG lung when compared to normal fibroblast. ..................................................................................................... 58 Figure 3.8. Formation of Pro-Collagen I A treated with N-α-PGP and neutrophil supernatant. . 60 xii Figure 4.1. Expression levels of total JNK in normal and fibrotic tissues.................................... 63 Figure 4. 2. Tissues in early stages of fibrosis detected with higher levels of p-JNK. ................. 68 Figure 4. 3. JNK inhibitor reduces the Collagen I A growth in IPF derived fibroblasts and counteracts the Neutrophil (NP) and Dexamethasone (Dex) Pro Collagen formation. ................ 70 Figure 4. 4. JNK inhibitor decreases the proliferative rate of tissue derived fibroblast in normal fibroblast but most notably in fibrotic fibroblast. ......................................................................... 72 xiii List of Abbreviations AP Aminopeptidase AP-1 Activator protein 1 AT Type II Alveolar type II ATF2 Activating Transcription Factor 2 BAL Bronchoalveolar CCL18 Chemokine ligand 18 CD3 Cluster of differentiation 3 CF Cystic fibrosis COPD Chronic Obstructive Pulmonary Disease CXCR Chemokine receptor Dex Dexamethasone DMSO Dimethyl sulfoxide ECM Extracellular matrix EH Epoxide hydrolase EMT Epithelial mesenchymal transition FBR Fibroblast FBS Fetal bovine serum FFPE Formalin-fixed paraffin-embedded IHC Immunohistochemistry IL-13 Interleukin 13 xiv IL-1β Interleukin 1 beta IL-33 Interleukin 33 IL-6 Interleukin 16 INF-γ Interferon gamma IPF Idiopathic pulmonary fibrosis JIP Jun interacting protein JNK c-Jun kinase LTA4H Leukotriene A 4 hydrolase MAPK Mitogen activated kinase MAP2K Mitogen-activated protein kinase kinase MAP3K Mitogen-activated protein kinase kinase kinase MMP Matrix Metalloproteinases NE Neutrophil elastase NP Neutrophil PBS Phosphate buffered saline PDGF Platelet derived growth factor PE Propyl endopeptidase PGP Proline-Glycine-Proline ROS Reactive oxygen species TGF-β Transforming growth factor beta TIMP Tissue Inhibitor of matrix metalloproteinases TLR Toll like receptor xv TMA Tissue microarray TNF-α Tumor necrosis factor alpha Zn Zinc α-SMA Alpha smooth muscle actin xvi Acknowledgements Behind every individual achievement, there is a team of people who provided help and support. Dear Vince, it’s been over 20 years working in your laboratory and I have had nothing but fantastic experience. I would like to thank you immensely for all these years. You are a great supervisor and friend and hope we can continue this friendship for years to come. Dear Dr. Khalil, thank you for all your support behind this research and believing in me. I am looking forward in all future projects. To my dear colleagues and friends Reza and Rouhollah, Erin and so many to count throughout the years… thanks for your help and friendship. To my children, Alessandro, Ariana and Sofia for giving me the strength and making me strive to be a better person and a caring father. Last but not least to my best friend and wife Teresa, who has been with me in good and bad. I love you very much. xvii Dedication To Opa: You are always in my heart 1 Chapter 1: Introduction 1.1 Fibrosis Fibrosis is often defined as a wound-healing process that has gone out of control. The process is generally defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components comprising mostly of collagen[1]. Repair of damaged tissues is a fundamental biological process that allows the proper replacement of dead or injured cells. Damage to tissues can result from various acute or chronic stimuli, including infections, autoimmune reactions and mechanical injury [2]. Excessive scarring from stimuli can cause destruction of the affected structures such as lung, heart or liver. The repair process typically consist of two distinct stages: regeneration phase, where injured cells are replaced by cells of the same type, removing any evidence of the damage followed by the phase known as fibroplasia, or fibrosis, where normal parenchymal tissue is replaced by connective tissue [1]. The scar is composed primarily of type I collagen, and lesser extent of types III and IV collagens. Also proteoglycans, fibronectin, glycoproteins, and proteins are also prominent [3, 4]. Although initia l ly beneficial, the healing process becomes pathogenic if it continues unchecked, resulting in substantial remodeling of the Extra Cellular Matrix (ECM) and formation of abnormal thick collagen deposition hence permanent scar tissue. This process can lead to progressive distortion of tissue architecture, dysfunction and ultimate failure of fibrotic organs. Many of the key pathological and morphological features of fibrosis are common in the lungs, liver, kidneys, heart, and skin[5, 6]. Myofibroblasts are the key cellular mediator of fibrosis, which when activated serves as the primary collagen-producing cell. A variety of sources including resident mesenchymal cells as well as the epithelial and endothelial cells are involved in generation of myofibroblasts, in 2 processes termed epithelial/endothelial-mesenchymal transition (EMT) [7]. When epithelia l and/or endothelial cells are damaged, they release inflammatory mediators which initiate an anti-fibrinolytic coagulation cascade, triggering blood-clot formation and formation of a transitiona l ECM. Aggregation, clot formation and hemostasis are achieved by platelets that are exposed to ECM components. Platelet degranulation also promotes vasodilation and increased blood vessel permeability, while epithelial and/or endothelial cells produce MMPs, which disrupt the basement membrane, allowing inflammatory cells to be easily recruited to the site of injury [2]. Recruitment and proliferation of leukocytes across the ECM are stimulated by chemokines, growth factors and cytokines. Some of the early responders include macrophages and neutrophils, which eliminate invading organisms, tissue debris, and any dead cells. Macrophages and neutrophils also produce cytokines and chemokines, which are mitogenic and chemotactic for endothelial and epithelial cells, which begin to surround the injured site. As new blood vessels form, epithelial/endothelial cells migrate towards the central part of the wound. Lymphocytes and other cells become activated during this period and begin secreting profibrotic cytokines and growth factors, such as TGFβ, IL-13 and PDGF [8-10]. This process activates resident fibroblasts, EMT cells as well as fibrocytes into α-SMA expressing myofibroblasts as they migrate along the fibrin lattice into the wound. Wound contraction is promoted by the activated myofibroblasts. In the final stage, epithelial and/or endothelial cells divide and migrate over the basal layers to regenerate the damaged tissue, which completes the wound healing process. However, chronic inflammation and repair can trigger an excessive accumulation of ECM components, which leads to the formation of a permanent fibrotic scar. Collagen turnover and ECM remodeling is regulated by various Matrix metalloproteinases (MMPs) and their inhibitors, which include the tissue inhibitors of metalloproteinases (TIMPs). 3 Shifts in synthesis versus catabolism of the ECM regulates the net increase or decrease of collagen within the wound. Fibrosis can occur when for instance the synthesis of new collagen by myofibroblasts exceeds the rate at which it is degraded, such that the total amount of collagen increases over time [7, 11, 12]. Figure 1.1 depicts the process of tissue generation and possible dysregulation of tissue repair to organ fibrosis when persistence injury in endothelial or epithelia l cells occurs. 4 (courtesy of Thomas A. Wynn et. al, [13]) Figure 1. 1 – Fibrosis Process and Tissue Regeneration. Following injury, inflammatory mediators activate the blood clot formation cascade. Leukocytes are recruited by chemokine and growth factors. Leucocytes secrete pro-fibrotic cytokines such as IL-13 and TGF-β , as well MMPs produced by myofibroblast and endothelial cells. The latter, disrupts the basement membrane, which recruits additional chemokines and cytokines that activate neutrophils and macrophages and other important tissue regeneration components. After the inflammatory phase, ECM is produced by myofibroblasts, which are derived from the local mesenchymal cells. While activated myofibroblast contraction stimulates the wound, collagen fibers are secreted and replace the scare tissue. Epithelial and endothelial cells restore the wound by generating the epithelium and endothelium, respectively. Persistence in inflammation and collagen production by excessively activated myofibroblasts can lead to fibrosis and tissue or organ damage. 5 1.2 Idiopathic Pulmonary Fibrosis Idiopathic pulmonary fibrosis represents a chronic and progressive disease with high mortality and limited therapeutic options. Similar to other organ fibrosis, pulmonary fibrosis also occurs due to excessive deposition of extracellular matrix (ECM) proteins resulting in fibrotic remodeling, alveolar destruction, and irreversible loss of lung function [14]. The disease occurs predominantly from middle age onwards, with more than two-thirds of patients being over 60 years old at the time of diagnosis. Men are nearly twice as likely as women to suffer from this lethal disease [15, 16]. As the disease progresses, it leads to ventilation restriction, impaired gas exchange, respiratory symptoms, exercise limitation, poor quality of life and ultimately death[17]. [13]While there is a low occurrence of IPF (2–29 subjects per 100 000), its incidence and mortality rates have increased sharply over the past few decades. It was found that the mortality rate for pulmonary fibrosis increased 28.4% in males and 41.3% in females in the United States from 1992 to 2003 [18-20]. What triggers the fibrogenic processes that initiate and maintain the disease state of fibrotic pulmonary remodeling is still debatable, but extensive research has shown that factors such as infections, use of cigarette, and environmental pollutants and pollutants from variety of occupations can trigger fibrosis [21-24]. As mentioned earlier, fibrogenesis is a perpetual dysregulated connective tissue repair and wound healing process. In lung, alveolar injury initiates the repair process. Components such as fibronectin and collagen are produced which lead to thickening of the alveolar and peribronchial walls and inhibit gas exchange [13, 25]. Figure 1.2 provides a brief summary of epithelial cell injury in lung and its repair process. Fibrosis in lung can occur when any stage of repair mechanism grows of out control, for instance, excess deposition of ECM. 6 (Courtesy of Thomas A Wynn et. al., [13]) Figure 1. 2 Wound healing process and pulmonary fibrosis. Epithelial cell injury in the alveoli results in coagulation and clotting. Pro inflammatory mediators such as neutrophils and macrophages release the inflammatory cytokines. This process will cause the activation, migration and proliferation of the epithelial cell derived fibroblast leading to tissue remodeling known as Epithelial-to-Mesenchymal transition. Resident fibroblasts also different iate into myofibroblasts, which release ECM components. Fibrosis may occur when one the above process is dysregulated. 7 During the initial lung injury phase, activated alveolar epithelial cells and recruited inflammatory cells such as macrophages, neutrophils release TGF-β (transforming growth factor β) a potent fibrogenic growth factor, along with TNF-α (tumor necrosis factor-α), PDGF, IL-1β, IL-6, IL-13, and IL-33 [26]. These growth factors are involved in injury and apoptosis of alveolar epithelial cells, as well as activation, proliferation, invasion, or apoptosis resistance of fibroblasts and myofibroblasts [25, 27]. Among mentioned cytokines studied, TGF-β was found to play the key central role in the pathogenesis of pulmonary fibrosis by promoting the activation, migration, proliferation, and differentiation of epithelial cells, resident fibroblasts and bone marrow fibrocytes to collagen-producing myofibroblasts [28]. TGF-β is involved in the EMT process from the resident epithelia l cells in alveolar both Type I and II cells [29, 30]. It drives EMT via SMAD proteins 2/3-dependent downstream mechanisms and promotes the transition of epithelial cells to fibroblasts through the transcription factors, Zinc finger protein SNAI (known as Snail) and Twist-related proteins TWIST [27, 31, 32]. Inflammation has a modulatory effect on TGF-β mediated pathways, as the proinflammatory cytokines, IL-1β, TNF-α and IFN-γ, were found to enhance TGF-β1–induced EMT via up-regulation of TGF-β receptor type I [11, 33-36]. TGF-β secretion initiates the EMT process by recruiting bone marrow fibrocytes, resident fibroblasts and local epithelial and endothelial cells, which results in production of the highly activated myofibroblasts. These highly activated and contractile myofibroblasts are positive in α-smooth muscle actin (α-SMA). 8 (Courtesy of Joan Massague et.al.,[37]) Figure 1. 3 TGF-β Signaling from Cell Membrane to the Nucleus. TGF-β ligand initiates signaling by binding to its type I and type II receptor serine/threo nine kinases on the cell surface. Type II phosphorylates the receptor I kinase domain, which initiates sequences of signaling through various SMAD proteins. SMAD proteins are categorized as receptor regulated R-SMAD, Co-mediator SMAD (C-SMAD) and inhibitory SMAD (I-SMAD). The activated SMAD complexes are translocated into the nucleus and, in conjunction with other nuclear cofactors, regulate the transcription of target genes such as fibrogenic and pro inflammatory cytokines [37]. Green dots define phosphorylation and red dots define as ubiquitin. 9 The source of α-SMA in these myofibroblasts has been speculated to be from the epithelia l-endothelial cells that have already lost their characteristics due to certain stimuli and now have acquired a mesenchymal- like phenotype by expressing excess α-SMA. These activated α-SMA positive myofibroblast, which are located mostly in the sub-epithelial regions with close proximity to injured hyperplastic alveolar type II cells (ATII cells), are considered to be the key source of excessive deposition of ECM and the pathologic hallmark of IPF [38-41]. It is important to mention that a high proportions of α-SMA positive myofibroblasts, express and synthesize higher levels of type I collagen compared with normal lung fibroblasts [42]. While the initial deposition of (ECM) from myofibroblasts provides a healthy scaffolding for the tissue regeneration and wound healing process, aberrant accumulation of the ECM molecules in the injured alveolus signals for overproduction of the collagen and abnormal lung architecture and remodeling [43]. One important class of molecules that are thought to be involved in pathogenesis of IPF are called matrix metalloproteinases (MMPs) and their respective inhibitors, tissue inhibitors of metalloproteinase (TIMPs). MMPs and TIMPs, mainly derived from macrophages and neutrophils are a family of extracellular endopeptidases defined by conserved pro-domains and catalytic domains with 25 members that are mainly membrane bound or secreted enzymes [44, 45]. These proteases are involved in maintenance of ECM and processes of tissue repair as well as the ECM remodeling. MMPs and TIMPs can either act in a pro- or anti- fibrotic manner, depending on the protease/antiprotease net balance and the micro-environmental tissue context; for instance, the depletion of neutrophils has been associated with an MMP-9/TIMP-1 imbalance in IPF [46-48]. In other studies, it was reported that MMP-1, MMP-7 and MMP9 were substantially increased in broncho alveolar lavage (BAL) of IPF patients [49]. 10 The immune system has shown to have an crucial role in fibrogenesis and IPF. Through interesting findings, it has been implicated that imbalances in the immune system might cause or elevate the pulmonary fibrosis during the tissue repair process. The involvement of skewed T Helper type 1 and 2 cells (Th1, Th2) imbalance has shown to modulate the fibrosis effect during early stages of the IPF [50]. The depletion of cluster of differentiation 3 (CD3) positive T cells attenuated the fibrosis in a bleomycin- induced pulmonary fibrosis model [51]. Alveolar Macrophages have been long understood be involved in IPF pathogenesis. They are a potent source of PDGF, TGF-β1 and MMPs [52]. While their role in fibrosis yet to be fully understood, depending on their polarization and the IPF stage, macrophages exhibited both anti and pro-fibrotic effect. While M1 type macrophages have been shown to induce collagen degradation and depletion of collagen, M2 type macrophages were shown to stimulate the collagen production loop via profibrotic chemokine CCL18 [53, 54]. The presences of certain cytokines such as IL-1β, IL-13, IL-7 and IL-23 are all involved in IPF at various stages of the disease. For instance, analyzed BAL fluid of the IPF patients was observed with an elevated level of TGF- β and IL-1β, a set of pro-fibrotic cytokines, primarily produced by activated macrophage and neutrophils [55]. Other groups have shown that its overexpression in the alveolar epithelial cells can induce pulmonary fibrosis [56]. Neutralizing the IL-17 cytokine also showed reduced collagen production in the bleomycin- induced fibrosis model in mice. Reduction of IL-17 was also associated with delayed neutrophils recruitment to the site of injury thus decreasing fibrosis in fibrotic lung [57]. Chemokines and their respective receptors have benn shown to be involved in various stages of fibrosis. Produced by macrophages and monocytes, chemoattractant protein-1 (CCL2) was increased in the BAL fluid of the IPF patients. A decreased production of MMP-2 and 11 MMP-9, reduced collagen deposition as well as reduced infiltration of macrophages and fibrocytes to the site of injury was observed in CCL2 knockout mice [58]. Elevated thymus levels of chemokine ligand 17 (CCL17) was found in the sputum of the IPF patients and was found to increase the fibrosis through chemokine receptor 4 positive (CXCR4) Th2 cells and alveolar macrophages. These observations have been linked to the ELR+ CXCR 1/2/4 receptors that PGP can mimic and bind to, hence causing a neutrophilia chemotaxis [59]. While neutrophils are thought to play a key role in combating bacterial infection in the respiratory tract, recently they were also shown to be involved in the pathogenesis of certain kinds of pulmonary diseases such as pneumonia, cystic fibrosis and idiopathic pulmonary fibrosis [60, 61]. Like macrophages, neutrophils shape their tissue environment by releasing proteases, oxidants, cytokines, and chemokines. [62, 63]. Increased neutrophils levels in BLAF of patients with IPF, was associated with early mortality [64]. It has been noticed that neutrophil elastase (NE), a proteolytic product, was secreted in excess in IPF patient’s airways. NE is responsible for the breakdown of a variety of ECM proteins as well degrading the collagens (type I-IV), fibronectin and elastin [65]. Asides from serine proteases, neutrophils also produce of MMP1 (collagenase) and MMP9 (gelatinase B) that are directly involved in IPF signaling. Neutrophils also contain and secret LTA4H, a bifunctional enzyme involved in pro and anti-inflammatory process which is also involved in metabolizing the immunogenic tri-peptide PGP, which is a breakdown product of various collagen types but particularly Type I [66, 67]. This topic will be discussed in more detail in the subsequent section of this chapter. While numerous other approaches have been taken by many groups around the world in order to understand this lethal and debilitating disease, the focus of this research was primarily to provide an understanding of: 12 1- An important bifunctional enzyme, Leukotriene A4 Hydrolase (LTA4H), and its novel involvement in IPF by metabolizing tripeptide Proline-Glycine-Proline (PGP), a breakdown product of collagen degradation and a potent neutrophilic chemoattractant. Therefore, the hypothesize of this research is that fibrotic lungs produce large amount of PGP which could exacerbate the inflammation cycle hence causing chronic inflammation and damage to the lung. In addition, Leukotriene A4 Hydrolase (LTA4H), a bifunctional zinc dependent enzyme, has an AP activity that can alleviate the neutrophil chemotaxis 2- The c-Jun N-Terminal Kinase I/II (JNK /II) involvement in IPF derived fibroblasts, and its inhibition which results in-vitro reduction of pro collagen I A formation. Therefore, based on the research conducted here, we hypothesize that fibrotic tissue has elevated levels of activated JNK1/2 which might be a potential factor in cause or progression of IPF. We suggest that inhibition of JNK in lung fibroblasts reduces the in-vitro formation of procollagen JNK inhibitors could serve as potential drugs for IPF treatment. 1.3 Leukotriene 4A Hydrolase (LTA4H) LTA4H is a bifunctional zinc-containing enzyme with both proinflammatory and anti-inflammatory functions. The epoxide hydrolase (EH) site catalyzes the conversion of leukotriene A4 to the proinflammatory mediator and neutrophil attractant leukotriene B4 (LTB4). LTB4 is an extremely pro-inflammatory lipid mediator that can exert and drive the recruitment and activation of an array of cells including neutrophils and thus playing a role in protection against invading 13 Figure 1. 4. Metabolism of arachidonic acid to inflammatory lipid mediators The arachidonic acid cascade generates leukotrienes from membrane phospholip ids. PLA2, phospholipase A2; COX, cyclooxygenase; 5-LOX, 5-lipoxygenase; FLAP, 5-lipoxygenase activating protein; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid. The names of the enzymes are given in the boxes. 14 micro-organisms[68]. In addition to the fatty acid epoxides, LTA4H is also the key precursor for biosynthesis of lipoxins, a family of pro-inflammatory lipid mediators utilizing arachidonic acid and eicosanoid metabolites [69]. In recent years, in addition to its pro-inflammatory EH activity, a secondary anti-inflammatory activity for LTA4H was identified by which it functions as an aminopeptidase (AP) to degrade the tripeptide Pro-Gly-Pro (PGP) [70]. Aminopeptidases are enzymes that catalyze the cleavage of amino acids from the amino terminus (N-terminus) of proteins or peptides and are a zinc-dependent enzyme produced and secreted by immune system components such as macrophages and neutrophils[71]. For over 30 years, it has been understood that degraded collagen fragments, have the capacity to increase the inflammatory response due to injuries as well as facilitating fibroblast migration [67]. PGP is a neutrophil chemoattractant derived from break down of extracellular matrix (ECM) collagen after initial injury via the sequential enzymatic activity of matrix metalloproteinases (MMPs) and propylendopeptidase (PE). Propylendopeptidases are serine proteases that cleave peptide bonds at the carboxylic side of proline residues. The main differences between the PE and serine proteases are structural and specificity, PE are more designated in cleaving proline residues and residues less than 30-100 amino acids, contrary to other serine proteases where they have less limitation in their enzymatic activity with lesser specificity [72]. It has been well understood that in many lung diseases such as COPD and IPF, matrix metalloproteinases such as MMP8 and MMP9 are partly responsible for degrading collagen, an important component of the ECM, followed by PE activity leading to generation of the chemotactic tripeptide PGP. Increased levels of PGP essentially would turn into a loop of chronic inflammation and ultimately chronic lung injury. Increased levels of PE and MMPs were noted in the BAL fluid of the patients with chronic lung inflammation including IPF patients [73]. 15 In physiological conditions, help of enzymatic degradation via enzymes such as LTA4H can prevent accumulation of PGP in the lungs. Although the N-Terminal acetylation, N-α-PGP, or methylation, N-m-PGP, of the PGP which can occurs for instance in the presence of chemica ls such as acrolein present in cigarettes, can enhance an acute immune response and fortify chemotactic potentials which can protect PGP from degradation by LTA4H [74]. LTA4H consists of an N-terminal, catalytic, and C-terminal domains that produces a deep cleft harboring an L-shaped pocket, with a wide polar arm that binds PGP and a narrow hydrophobic extended arm that has been shown to accommodate and bind LTA4 [71]. The elbow of the pocket includes H295, H299, and E318, which coordinate a Zn2++ ion essential for both LTA4H anti and pro inflammatory activities (Figure 1.3.2-A). As per crystal structure analysis of LTA4H it is understood that the epoxy hydrolase reaction strictly depends on the correct alignment of LTA4 in the active site cavity [75]. As the substrate enters, the hydrophobic cavity, the carboxylate group anchors to R563, while Zn2++ and Y383 coordinate the epoxide oxygen (figure 1.3.1 A and B). At the same time Water 1 molecule as shown in Figure 1.3.1, polarized by Zn2++ and E271, donates a proton to promote 1 substitution-elimination reaction (SN1) acid-induced opening of the epoxide ring (Figure 1.3.1 C and D). A carbonium ion is formed, whose positive charge is delocalized over the triene system in a pro-cis configuration stabilized by the L-shape of the tight pocket of the enzyme. At the final step of the reaction, catalytic Water 2 generates a hydroxyl group facilitated by D375 which leads to conversion of LTA4 into LTB4 (Figure 1.3.1 E and F). In order to understand the aminopeptidase activity of LAT4H and obtain its proper crystal structure bound to PGP tripeptide, it was necessary to produce an analogue of the PGP that LTA4H would not break it down during the process of crystallization. Crystal structure of the PGP in the pocket of the LAT4H was obtained using OPB-Pro, which it has been shown in the Figure 1.3.2 C with its difference in 16 structure when compared to PGP. The structure difference exhibits a change of the glycyl nitrogen group to carbon, where proline is cleaved by LTA4H [76]. Comparison of the structures of LTA4H-LTA4 and a previously reported structure of LTA4H complex with PGP tripeptide shows that a single catalytic water, is involved in aminopeptidase cleavage rather than two molecules of water (Figure 1.3.1 E and F). Water is coordinated to both E271 and E296, in addition to H295 and H299 ring intermediates to stabilize the LTA4H-OPG-Pro complex at the binding pocket via Zn2++ molecule interaction with their histidine rings. Water 1 is activated by E296 for tripeptide bond hydrolysis at the N-terminus end of the PGP or OPB-Pro [77]. Thus, in PGP complex, E271 and water remains hydrogen bonded only to the side chain of E296 where AP activity of the LTA4H breaks proline at the glycyl nitrogen of the N-terminus of the PGP tripeptide (Figure 1.3.2 B and C) [77-79]. 17 (Courtesy of Haeggstrom et. al., [68]) Figure 1. 5. Binding of LTA4 in the active site of LTA4H A and B show active site of an L-Shaped pocket where LTA4 binds to the enzyme. C and D show different views of the active site of the complex with LTA4. E and F depict the involvement of catalytic Water 1 and Water 2 and Zn2++ molecule and hydrolysis of LTA4 to an eventual LTB4 molecule [79]. Water 1 is hydrolyzed by help of Zinc and Y383 and E271 which facilitates in turning the epoxide group to hydroxyl group. Catalytic Water 2 with the help of N375 will turn double bond carbon group, located at C12 on LTA4, to hydroxyl group. 18 (Courtesy of Haeggstrom et. al., [75]) Figure 1. 6. Crystal Structure of LTA4H and its complex with PGP A depicts the crystal structure of LTA4H complex with PGP analogue, OPB-Pro. B shows the binding interaction and configuration of the PGP analogue in the L-shaped pocket of the LTA4H. C shows the chemical difference between PGP and its analogue non-degradable OPB-Pro. The analogue structure was used to capture the crystal structure without degradation of the PGP by aminopeptidases activity of the enzyme. Only catalytic Water 1 is involved in PGP breakdown. Water 2 is only involved in LTA4 conversion to LTB4 [17, 74, 76]. 19 Understanding the interaction between LAT4H and PGP has been an important task in unraveling the importance of LTA4H in maintaining the neutrophilic chemotaxis of the collagen breakdown in pathogenesis of lung diseases such as IPF. Focus also has been dedicated into understand the signaling pathway by which PGP is generated and exacerbates the inflammation in an already injured lung and ultimately leading to vicious immunogenic recruitment and fibrotic cycles. As mentioned earlier, the generation of PGP occur after the initial injury to the epithelia l layer of the lung leading to exposure of damaged collagen. The release of PGP is initialed by MMPs followed by propylendopeptidase to complete the process. While high levels of the MMPs and PE were in abundance in serum and sputum of the patients with CF, COPD and IPF, the PGP generation found to be dependent on PE enzyme activity with a 25-fold increase when compared to healthy donors [72]. In addition, it was also observed that these patients had overexpressed PE not only in macrophages and neutrophils but also in their alveolar epithelial cells [80]. In vitro studies also demonstrated that using the sputum of patients affected with cystic fibrosis and pulmonary fibrosis when incubated with collagen type I and II generated significant amounts of PGP when compared to control experiments [80]. As described earlier, the acetylated and methylated form of PGP appears to be resistant to degradation. Therefore, experiments were designed in mice where N-α-PGP administration into the airways of the animal led to chronic neutrophilic recruitment leading to COPD like pathology in the trachea-bronchiolar section of the mouse lungs [74]. Alveolar enlargement, emphysema-like changes and ventricular hypertrophy was also demonstrated in mice when resistant PGP form was administrated to the animals. Using L-20 Arginine-Threonine-Arginine tripeptide (L-RTR), which can impede the collagen breakdown by competing with PGP binding site, was shown to dampen the neutrophilic activation and migrat ion and this exhibited reduced emphysema like symptoms in mice [81-83]. The chemotactic studies in recent years have revealed that chemokine receptors 1/2 (CXCR 1/2) which are present in both macrophages and neutrophils, have shown to bind PGP as well its acetylated / methylated forms leading to adverse chemotaxis. Weathington et al. demonstrated that PGP shares a structural homology to that of Glutamate-Leucine-Arginine positive (ELR+) Chemokines such as CXCL8, which activates CXCR 1/2 upon binding, hence causing a neutrophilic chemotaxis and inflammatory augmentation [84]. While there have been controversies over PGP being able to displace the CXCL8 from its receptor CXCR 1/2, experiments demonstrated that N-α-L-Proline- L -Glycine- L-Proline (LL-N-α-PGP) isomer is the active form of PGP that can compete with ELR+ chemokines and not the N-α-D-(PGP) isomer [85]. While it is very apparent that PGP can induce neutrophilic chemotaxis, which can lead multiple chronic lung diseases such as COPD, CF and emphysema, based on research conducted in this thesis, we propose that PGP generation as the result of collagen degradation can lead to an exacerbation of pulmonary fibrosis when its degradation not controlled by LTA4H. Figure 1.6 depicts and summarizes the connection between the external factors such as cigarettes, lipopolysaccharides (LPS) and bacterial infections in commencing the inflammation, while injury to the epithelium of the lung can start the repair process by activating MMPs and PE pathway. Collagen breakdown from ECM and generation of PGP followed by its alteration via acrolein, can lead to further neutrophil and macrophage recruitment. This cycle will promote the fibroblast to 21 (courtesy of Gagger et. al., [86]) Figure 1. 7. Proposed PGP pathology in lung neutrophilic inflammation. External factors such as cigarette smoke inhalation causes tissue resident macrophages, Neutrophils and epithelial cells to release several mediators, including PE, IL-8, LTA4H and MMP 8/9. 2 and 3- IL-8 attracts neutrophils via binding with CXCR1/2. 4 and 5- MMPs and PE release by neutrophils will lead to collagen cleavage from the ECM to release the tripeptide and neutrophil chemoattractant PGP. While LTA4H is able to inactivate PGP, components released from of cigarette smoke, such as acrolein alters the PGP to its more potent acetylates form, LL-N-α-PGP, which cannot be cleaved by LTA4H. This will lead to a vicious cycle of neutrophilic inflammation which can lead to COPD and possibly pulmonary fibrosis, as the process intends to repair the structural damage by continuous production of collagen [86] . 22 produce more collagen since the signal activating the repair mechanism is on continuously, which ultimately can lead to lung fibrosis or other chronic lung diseases such as COPD and emphysema. 23 1.4 C-Jun N-Terminal Kinase (JNK) JNK belongs to one of three well-characterized mammalian mitogen-activated protein kinase (MAPK) pathways, which transduce extra-cellular signals that can control cell proliferat ion, apoptosis, differentiation and migration [87]. Each of these MAPK pathways are activated via phosphorylation cascades. For instance, JNK is activated via upstream MAP3K family members which phosphorylate and activate MAP2K enzymes, or by interacting with JNK-interacting protein 1 (JIP1), a scaffold protein [88]. The inactivation of JNK can be achieved by dephosphorylation performed by a group of dual specificity serine, threonine or tyrosine protein phosphatases. Activated JNK can phosphorylate serine and threonine residues in specific proteins such as c-Jun a transcription factor, SMAD proteins, a family of proteins involved in TGF-β pathways, and p53, an important tumor suppressor gene [89, 90]. A simplified and general pathway of JNK is shown in figure 1.7, which illustrates its involvement in pro-inflammatory and fibrotic events as well as cell growth and proliferation. Although JNK has been associated with a variety of pathways involving inflammation, cancer, apoptosis and diabetes, however, the focus of this thesis is to focus mainly on its singling pathways revolving around epithelial cell injury and fibrosis. It is important to mention that a JNK pathway has been a subject of experiments in understanding its role in liver and kidney and heart fibrosis. However, studies have not specifically revealed a solid understanding of the JNK role in IPF as well its potential therapeutics in general lung fibrosis [91]. JNK was named after its ability to phosphorylate serine residues at positions 63 and 73 in the N-terminal domain of the c-Jun, a proto-oncogene that is involved in cell cycle progression and apoptosis [92]. 24 (Courtesy of Nikolic-Paterson et. al, [91]) Figure 1. 8. JNK Signaling Pathway MAPK family of proteins is activated via inflammatory cytokines and pro-fibrotic growth factors such as TGF-β, IL-1 and TNF-α. Other factors such as reactive oxygen species (ROS) can also lead to MAP3K activation. Individual members of the MAP3K and MAP2K families plus JNK are held in close proximity by the Scaffolding protein called JIP-1 which facilitates phosphorylation reactions leading to phosphorylation of the activation site on JNK protein. The activated JNK then dissociates from this complex and can promote mitochondria-dependent apoptosis. Active JNK can promote transcription of genes involved in the inflammatory and fibrotic responses through phosphorylation of c-Jun AP-1 function], SMAD3 and ATF2. JNK activation can promote cell death, inflammation and fibrosis. 25 There are three genes in the JNK family, Jnk1, Jnk2 and Jnk3. JNK1 and JNK2 enzymes are expressed in most cells of the body, including the lungs, whereas expression of JNK3 is limited to the brain, heart, and testis [93]. When both Jnk1 and Jnk2 genes are deleted in mice, it causes embryonic lethality, whereas deletion of either gene results in viable and healthy animal [94]. An important aspect of the JNK pathway is that it can be activated in response to a range of stimuli such as proinflammatory cytokines, oxidative stress and pro-fibrotic factors that have been implicated in acute and chronic heart, lung and kidney injury [87]. Therefore JNK signaling may contribute to fibrosis through activation of apoptosis and inflammation pathway aside from its direct contribution to fibrosis in general [95]. There are number of pathways in which JNK interacts with the fibrotic process, such as TGF-β/SMAD pathway. As mentioned earlier, the TGF-β/SMAD signaling pathway plays a central role in the development of a variety of fibrotic pathway [96]. Production of TGF-β and activation of latent TGF-β leads to activation of SMAD2/3/4 complex which can promote in transcription of pro-fibrotic molecules [91]. JNK signaling can enhance pro-fibrotic TGF-β /SMAD signaling through mechanisms as follows. JNK activation can be induced by factors such as angiotensin II, IL-1 and TNF-α, which in turn can increase TGF-β production through activation of AP1 which will promote to increase transcription of TGF-β, hence promoting fibrosis [97]. Residues in the SMAD linker regions can be phosphorylated by JNK, which can directly enhance the SMAD3 transcriptional activity and thus promote a fibrotic pathway [93, 98]. JNK activation can promote neutrophil and macrophage infiltration, which subsequently can promote fibroblast activation and collagen production; however, JNK signaling in fibroblasts needs yet to be understood fully [99]. Studies of cultured fibroblasts have identified a role for JNK signaling in proliferation induced by PDGF and TGF-β, where JNK caused cell proliferation via 26 activation of PDGF-B in pulmonary artery fibroblasts isolated from hypoxic animals when compared to control fibroblasts from control animals [100]. In recent years there has been an interest by many research groups targeting JNK directly using variety of inhibitors to its pro-inflammatory and pro-fibrotic role [94, 101]. Blocking individual enzymes in the upstream MAP2K or MAP3K families has been desirable target to block JNK signaling in fibrosis, although this method could also interrupt the other pathways essential for the cell survival and function [91, 98]. A member of the MAP3K family, TAK1 can be activated by the pro-fibrotic factor TGF-β1, although the Toll-like receptor 4 (TLR4) and the Interleukin 1 (IL-1) and Tumor necrosis factor α (TNF-α) receptors are just as potent in activation of this enzyme, which in turn promote the activation of JNK pathways [102, 103]. There has been great clinical interest in producing a JNK inhibitor that can suppress fibrosis in lung, liver and kidney. Following the demonstration that JNK blockade could suppress renal fibrosis, experiments have shown that JNK inhibitors are successful in alleviating the fibrosis in lung disease [104, 105]. However, JNK inhibitor Tanzisertib also known as CC-930, an inhib itor that is kinetically competitive with ATP in the JNK-dependent phosphorylation of the protein substrate c-Jun, and also a potent inhibitor against all isoforms of JNK, was used in a clinical trial in idiopathic pulmonary fibrosis, but the trial was halted due to acute liver toxicity [105]. Nevertheless, improvements in markers of lung fibrosis were noted which resulted in continued interest in new development of other JNK inhibitor that can combat idiopathic pulmonary fibrosis as well other organ fibrosis. 27 Inhibiting the JNK upstream activating proteins such as JIP1 has also been a subject in clinical trial. Brimapitide, a JNK1/2 inhibitor inhibiting JNK at Threonine183 and Tyrosine185 is currently in clinical trials to be evaluated in models of renal and liver fibrosis [106]. Experiments recently have demonstrated that elevated phosphorylation of JNK1/2 is present in the airway epithelium and pulmonary endothelium from patients with IPF [89, 107]. JNK1-deficient mice (JNK1/2 -/-) were shown to be protected against TGF-β1- and bleomyc in-induced pro-fibrotic gene expression and pulmonary fibrosis [108, 109]. IPF is a disease in which collagen is produced at a very rapid pace and without any regulation and control. Research has demonstrated that collagen content, Col 1A1 mRNA expression, and TGF-β, levels were lower in JNK1/2 -/- mice when compared with controls, suggesting that JNK1/2 and the collagen pathway could be linked [110, 111]. It was also shown that lowered TGF-β1 content in JNK1/2 -/- mice reduced the extent of collagen deposition [112-114]. Recent in vivo studies have demonstrated that JNK1/2 -/- mice fail to induce mesenchymal protein markers such as collagen type 1A1 and α-SMA in airway epithelial cells [27, 115, 116]. Using primary cultures of mouse tracheal epithelial cells, it was shown that JNK1/2 was required for TGF-β1 induced EMT. This information provides another link and importance to the TGF-β pathway and its requirement of JNK1/2 activation in fibrosis process [27, 108]. TGF-β1 administration to wild type mice induced marked reactivity of α-SMA in airway epithelial cells, indicating that epithelial plasticity associated with EMT and this transition was noticeably absent in lung from JNK1/2 -/- mice [27, 117]. The importance of mechanistic link between signaling cascades of TGF-β1 and JNK1/2, has created an opportunity in finding a novel target to JNK in order to control the EMT process, which in turn, could help lowering the fibrotic process in lung and perhaps general fibrosis across other organs. 28 In conclusion, systemic JNK inhibition, designing a specific cell-type delivery of JNK inhibitors as well as targeting different isoforms of JNK protein could be plausible ways to ultimately reduce aberrant collagen production in IPF and other tissue fibrosis. 29 Chapter 2: MATERIAL AND METHODS 2.1 Materials 2.1.1 Chemicals and Reagents Acrylamide Bio-Rad Casein Bio-Rad TEMED Bio-Rad Trypan Blue Bio-Rad Casein Bio-Rad Acetic Acid Fisher Scientific Adenosine 5-Triphosphate Fisher Scientific Ammonium persulphate Fisher Scientific Bovine serum albumin Fisher Scientific Citric acid Fisher Scientific Dexamethasone Fisher Scientific Dextran Fisher Scientific DMEM Fisher Scientific DMSO Fisher Scientific EDTA Fisher Scientific Ethyl alcohol Fisher Scientific glycerol Fisher Scientific glycine Fisher Scientific Hematoxylin Fisher Scientific 30 Hydrochloric acid Fisher Scientific L-Glutamate Fisher Scientific Magnesium Chloride Fisher Scientific Methanol Fisher Scientific Ninhydrin Fisher Scientific Phosphate buffered saline Fisher Scientific Potassium bicarbonate Fisher Scientific Potassium Chloride Fisher Scientific Skim milk Fisher Scientific Sodium azide Fisher Scientific sodium chloride Fisher Scientific Sodium dodecyl sulphate (SDS) Fisher Scientific Sodium fluoride Fisher Scientific Sodium Hydroxide Fisher Scientific Sodium pyruvate Fisher Scientific Toluene Fisher Scientific Tris Fisher Scientific Triton X-100 Fisher Scientific Tween-20 Fisher Scientific Xylene Fisher Scientific β-mercaptoethanol Fisher Scientific Paraffin blocks Fisher Scientific 31 Phosphate buffer saline Fisher Scientific Sodium Citrate Fisher Scientific Ficoll-Hypaque GE HealthCare Supersignal Pierce MTS Solution Promega PMS Solution Promega Protease inhibitor Cocktail Roche Scientific Protein A/G Agarose Roche Scientific Ponceau S Sigma 2.1.2 Peptides, Inhibitors and Proteins L-Proline Caymen Chemicals Human Recombinant LTA4H Caymen Chemicals Proline-Glycine-Proline (PGP) Caymen Chemicals Phospho-JNK 1/2 Inhibitor-SP600125 Calbiochem 2.1.3 Antibodies LTA4H (ab133512, Abcam) Fisher Scientific Phospho-JNK 1/2/3 (NEB 4668) Abcam MMP9 (ab119906) Abcam IRDye 800 (LICOR 680RD) LICOR Systems IRDye 680 (LICOR 680RD) LICOR Systems 32 c-Jun N-Terminal Kinase1/2 (ab112501) Abcam Procollagen 1 A (84336) NEB Procollagen III (83829) Abcam Vinculin (V-9131-100ug) Sigma 2.1.4 Instruments Digital Cell counter Bio-Rad Odyssey cxl Licor Leica SCN-400 Leica Leica Fluorescent Microscope Leica Transfer Apparatus GE Healthcare Elisa Plate EPOCH Reader Fisher Scientific Cell Culture Centrifuge Eppendorf Western Blotting equipment Bio-Rad Power pack 1000 Bio-Rad 2.2 Methods 2.2.1 Cell Culture 2.2.1.1 Processing the Transplant lung from IPF patients Lungs of IPF or normal patients were delivered to the pathology unit at Vancouver General Hospital (VGH) after transplant procedure. The clinical research ethics board of the University of British Columbia approved the usage of patient’s lungs samples for IPF studies in accordance with Human Ethics Protocol number: H14-02570, Human Lung Fibroblasts. Dr. John English incised 33 the lungs at the pathology laboratory in 4 sections: Apex or Superior, Middle, Basal and Lingula. Cut pieces were kept in conical tubes and transported to the laboratory at Jack Bell Research Center. Tissues were named after donor number, date of transplant and location of lung. For instance, LL, Apex #2, January 01, 2017, meant Donor number 2, with Apex region of the Left Lung with transplant day of January 01, 2017. These lung sections were used to derive fibroblasts, produce paraffin embedded blocks for IHC and TMA blocks, and the rest were frozen in liquid nitrogen for future DNA and RNA analysis. 2.2.1.2 Fibroblast (FBR) preparation from IPF Lungs Fresh IPF lung sections were thoroughly minced in small pieces and were placed in the 10 cm tissue culture dishes in DMEM supplemented with 10% fetal calf serum. Cells were cultured at 37oC in 5% CO2 at 95% air humidity, treated with antimitotic reagent at 100g/ml and streptomycin. Media was changed after 2-3 and after 1-2 week(s), tissues were removed, and plate was washed 5x with PBS at 37o C and FBR cells were removed using trypsin EDTA. Cells were spun at 1200 rpm room temperature (RT) and were cultured in DMEM with 10% FBS. This was called passage 1 of the FBR cells. For future experiments, some portion of these cells were frozen in 5% DMSO and 95% FBS and were kept in liquid nitrogen. During the period of this thesis, the number of IPF lungs obtained from VGH pathology was n=5 and normal lungs on hand was n=7. It is important to note lungs used as normal in this thesis were obtained from biopsies or transplants of the patients affected with lung cancer. 34 2.2.1.3 Lung tissue and FBR cell preservation All lung tissues were preserved by placing them directly in cryovial and setting them on ice for 20 minutes before submersing them directly in liquid nitrogen. The Fibroblast cells were washed with phosphate buffered saline (PBS) and placed in cryovial containing 95% FBS and 5% DMSO. Vials were placed in ice for 20 minutes before submersion of the vials in liquid nitrogen. 2.2.2 Histology Procedure 2.2.2.1 Paraffin processing of the lung tissues For the sections kept for histology purposes, tissues were fixed in 10% formalin for 48 hours and all samples were sent to BC Cancer agency for paraffin processing. All paraffin blocks were kept at RT. 2.2.2.2 Hematoxylin and Eosin Stain (H&E) Stain Rehydrated sections obtained from the paraffin block were placed on adhesive slides and were stained in Hematoxylin solution for 20-40 minutes. Slides were washed in water for 1-5 minutes, until sections turned blue. Sections were placed in 70% ethanol containing 1% Hydrochloric acid (HCl) for 5 seconds to remove excess dye and allowing nuclear details to emerge. After 5 minutes of washing with water, slides were placed in Eosin solution for 10 minutes. Slides were washed with water followed by acidification and dehydration with 70% ethanol. 2.2.2.3 Immunohistochemistry Tissue sections slides from the FFPE blocks were deparaffinized by heating at 55ᵒ C for 30 minutes following by three xylene washes, 5 minutes each. Slides were rehydrated by consecutive 35 washing in 100%, 95%, and 80% ethanol and distilled water, 5 minutes each. Using the Ventana Ultra machine (Roche Scientific), the slides were set into the slots and both the primary and secondary antibodies were loaded into the machines. The Ventana machine prepares the antigen retrieval as well washing, blocking and incubation with primary and secondary antibodies. Slides were than scanned using Leica SCN-400 scanner and slides were scored an expert pathologist. 2.2.2.4 IHC Scoring Methodology All sets of IHC slides were scored blindly by a physician and two independent pathologists at Vancouver Prostate Centre. The expressions of proteins were scored in two quantitative aspects; percentage of the intensity of the staining, 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%) multiplied by the number of positive cells. This method was suggested by the pathologist based on the nature of the antibodies and final staining. All scoring methods were left at the discretion of the pathologists. It is important to note that both macrophages and neutrophils and all leukocytes, although some positive in LTA4H count, were not included in the counts. Only positive count of the local tissues were considered for data presented in this thesis. 2.2.2.5 Tissue Micro Array For preparing tissue microarray (TMA) blocks, the most representative area of affected tissues was carefully chosen and marked based on the paraffin blocks based on the (H&E) stained slide. TMAs were assembled using a tissue-array instrument (Beecher Instruments, Silver Spring, Md). After taking up to 6 cores of 0.6 mm tissues from each biopsy specimen embedded in paraffin blocks, Leica microtome (Leica Microsystems, Bannockburn, Ill) was used to cut multiple 4 µm sections. Using regular histology procedures, sections were transferred to adhesive-coated slides. 36 One section of each TMAs was stained with H&E routinely and the others were used for IHC staining and analysis. 2.2.3 Immunoprecipitation The lysate of the IPF and normal FBR cells (1x107) were reconstituted in lysis buffer containing protease and phosphatase inhibitors. After taking 30 µl of the lysate for protein concentration and post immunoprecipitation purposes, 1 ug of the LTA4H antibody was added to the lysate. Tubes were rotated at 4oC overnight. Protein A/G Agarose (20ul packed beads) was added to the samples at 20 µl of and samples were rotated at 4o C for 1 hour. Beads were washed with lysis buffer containing inhibitors and an were placed in 25 µl of lysis buffer or assay dilut ion buffer. 2.2.4 Immunoblotting Fibroblasts were grown to confluence in DMEM and 0.5% bovine serum albumin for 24-48 hours. After stimulation or inhibition, cells were lysed using lysis buffer and whole cell lysates were kept for separation of proteins and kept analysis on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cast gels at desired concentrations (between 8-10%) were prepared or purchased from Bio-Rad laboratories. Samples were mixed with 4x SDS Sample Buffer containing 4% β-mercaptoethanol (Bio-Rad Laboratories) for final 1x concentration, followed by loading on the gel (10-50 µl of samples). Loading of the protein on the gel was adjusted based on the protein concentration of each sample. Proteins were separated based on molecular weight using SDS-PAGE apparatus. Gels were transferred onto a nitrocellulose membrane at 30 V for 90 min. Membranes were blocked by placing them in 5% casein in 37 phosphate-buffered saline containing 0.2% Tween 20 incubating for 1 hour followed by incubation with 1µg of JNK1 or LTA4H antibodies in sodium azide at 4oC overnight. After washing the blots with PBS containing casein blocking buffer, appropriate secondary IRDye(s) (680 nm, or 800 nm) were incubated with the blot. PBS was used to wash the blots and Odyssey CLx was used to detect the signals at 680-800 nm (infrared detection). 2.2.5 In-cell western blotting Fibroblasts cells at 1x105 were cultured in a 96 well tissue culture plate in DMEM and place in 37o C and 5% CO2 incubator overnight. Media was removed carefully by aspiration. Cells were fixed immediately with 150µl of 3.7% formaldehyde (fixing solution) in 1X PBS. After 20 minutes incubation with no shaking, cells were permeabilized and washed five times with 1X PBS containing 0.1% Triton X-100 for 5 minutes per wash. At RT, 200 μL of Triton X-100 washing solution was added to each well and plates were set for 5 minutes on shaker. This procedure was repeated 4 more times. Casein blocking buffer was added to each well and incubated for 1.5 hours at room temperature (RT) with moderate shaking. Primary antibodies were diluted at 1:50 to 1:200 dilutions as per manufacturer recommendations and after removal of the blocking buffer, wells were incubated with primary antibody for 2.5 hours at RT or overnight at 4°C with gentle shaking. Wells were washed five times with 1X PBS + 0.1% Tween 20 for 5 minutes at RT. To each well 200μL of Tween-20 washing solution was added; five washes each for 5 minutes. This was followed by adding 50µl of the appropriate IRDye secondary antibody to each well, between 1:200 to 1:1.200 depending on the efficiency of the antibody, and the plate was incubated for 3 hours at RT. Wells were washed with Tween-20 washing buffer and plates were read using the Odyssey CLx 680 nm or 800 nm. Odyssey Clx is equipped with presetting for In-Cell western blotting, as 38 this method was developed and pioneered by Licor systems. More information can be obtained on www.licor.com. 2.2.6 PGP formation assay in IPF lung fibroblast 2.2.6.1 Preparation of reagents and methodology A stock of 2.5 M Tris-HCl pH 7.5, 2 M potassium chloride (KCl), 50mM proline-glyc ine proline (PGP), 50 mM proline in milliQ purified water and 2.5% ninhydrin solution were prepared for free- L-Proline assay. An effective standard curve for this experiment ranges from 6.25 µM to 800 µM using serial dilutions of free L-Proline. Treatment with Inhibitors was at a final concentration of DMSO at 0.2% or less, due to toxicity. The final concentration of recombinant human LTA4H enzyme in each peptidase well was 0.2 nM diluted in assay dilution buffer. 2.2.6.2 Stimulation of peptidase activity PGP stock was diluted to 1mM in assay buffer for a final concentration of 500 μM. 50 uL of 1 mM PGP was added to the enzyme mixture and mixed by vortexing. Samples were incubated at 37° C for 30 minutes. One extra sample of recombinant enzyme + substrate was prepared and incubated at 37° C for 90 minutes to ensure that all the PGP can be converted by the enzyme which would serve as the control experiment. To stop the reaction 150 μL of glacial acetic acid was added to the tubes. 2.2.6.3 Detection of L-Proline by ninhydrin For standard curve, 150 μL of glacial acetic acid was added to each tube, followed by 150 μL of 2.5% ninhydrin solution. Samples were vortexed in micro centrifuge tubes with lid lock. Samples were placed into heat block (set at ~100°C) for 30 minutes followed by cooling period of 39 ~30 minutes. To extract colour, 350 μL of toluene was added to each reaction followed by 5 seconds vortex and 100 μL of the top phase were aliquoted into a 96 well plate for detection at 520 nm. To analyze, a standard concentration curve of L-Proline was generated and used for data extrapolation. 2.2.7 Neutrophil isolation from whole blood Four ml of Sodium Citrate -Citric Acid-Dextrose (ACD) was added into a 50 ml conical tube and was poured 20 ml of whole blood down the side of the tube. The tube was gently inverted several times to mix and 12 ml (50% of blood volume) of 6% Dextran / 0.9% NaCl solution was added into the ACD/blood mixture and inverted 18-20 times to ensure adequate mixing. The mixture was pipetted into 4 x 15 ml conical tubes (~10ml/tube). Tubes were left to stand at room temperature for 45min - 1hr, for separation to complete. The yellowish supernatant was pipetted into a 50 ml tube and tubes were spun at 1150 rpm for 12 minutes at 4°C, with centrifuge set at no brake. Supernatant was discarded and re-suspended in 12 ml of the ice-cold double distilled water. After 20 seconds, 4 ml of 0.6 M KCl was added to the mixture and diluted with 50 ml of cold PBS, mixed by pipetting and spun at 1300 rpm for 6 minutes at 4oC. This step was repeated 1-2 times, until no red blood cell (RBC) was visible in pallet. Supernatant was discarded, and pellet was suspended in 2.5 ml of HBSS. To determine the cell concentration, we used a digita l hemocytometer (Bio-Rad, Ca) utilizing trypan blue mixture. 2.2.8 Cell proliferation assay (MTS) A fresh mixture containing 100 μl of PMS Solution and 2.0ml of MTS solution in water was prepared, followed by adding 20 ul of the PMS/MTS mixture to a 24 well assay plate 40 containing 1 x 105 of human fibroblast cells from IPF and normal lung in 1ml of the DMEM media with 10% FBS. Plate was incubated in 5% CO2 incubator for 1–4 hours at 37°C. Absorbance was recorded at 490 nm on an ELISA plate reader. Final concentrations of MTS in the assay was 333 μg/ml MTS and 25 μM of PMS. 2.2.9 Statistical analysis Data were analyzed by using Student t-Test or ANOVA statistical test for attaining the significance within the normal and fibrotic groups. The probability values obtained are indicated in the text or the figure legends when statistically significant. 41 Chapter 3: Role of LTA4H in metabolizing PGP and collagen production. 3.1 Rationale and Hypothesis Idiopathic pulmonary fibrosis is a non-neoplastic aggressive pulmonary disease that is characterized by abnormal scar formation in the lung which leads to invasive collagen formation. The chronic deposition of collagen leads to irreversible destruction of lung architecture and honeycombing. This will essentially restrict the proper ventilation and gas exchange and leads to an overall poor quality of life [118, 119]. While the occurrence of IPF is relatively low (5-30 subjects in 100,000), it carries a high mortality rate with this rate increasing sharply in the past decade [120, 121]. While tremendous research has been done recently to further understand the abnormal behaviour of the alveolar epithelial cells transition as well various signaling pathways leading to IPF, an effective therapeutic solution has yet to be introduced to battle this deadly disease. Drugs such as Pirfenidone and Nintedanib have demonstrated some efficacy in slowing down IPF progression, but an eventual lung transplant would be required for the patients affected with IPF[122-125]. In recent studies, the tripeptide Proline-Glycine-Proline has been the focus of research for its role as a neutrophil chemoattractant, specifically in lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD). PGP has also been linked to fibrosis by keeping the inflammation cycle active and essentially leading the fibroblast cell in lung to produce more collagen [74, 126]. LTA4H, is a bifunctional zinc metalloenzyme, that has an aminopeptidase activity which can break down PGP and relieve this artificial inflammation cycle when collagen in scar tissue is no longer required [67, 70, 127]. The purpose of this part of my studies was to 42 provide some preliminary information on PGP production and LTA4H expression levels in fibroblast derived from the normal and IPF lungs. 3.2 Results The fibroblast cells were prepared and derived using a DMEM medium that contained 10%. FBS Lung were obtained from VGH lung transplant recipients with donor’s approval and human ethic approval (refer to material and method section of the thesis). Lung sections were grown for two weeks and proliferative fibroblast were isolated and regrown in fresh media and plates. Based on literature, neutrophils and macrophages are the main LTA4H source in lung [70]. We tested whether the normal and IPF derived fibroblasts also express LTA4H and if there are any significant differences in expression levels (Figure 3.1.A). Results shown in Figure 3.1.A, suggest that the endogenous level of LTA4H is relatively higher in normal fibroblasts than fibroblast from fibrotic lungs. Also, this result might suggest that the pool of available LTA4H from neutrophils and macrophages can also be synergistically affected by fibroblasts’ contribution of the enzyme in regulate PGP break down. The use of Vinculin throughout this thesis is solely for protein content normalization. Based on ANOVA statistical test on the combined results (Figure 3.1B) the levels of the LTA4H expression was indistinguishable in the two types of fibroblasts. 43 A 00.20.40.60.811.21.41.61.82Apex #2 Lingula #3 Superior #2 Basal #4 LT16-020 LT16-024 LT16-027Relative Corrected Optical Density of Signal to Constant Area Vinculin LTA4H 44 B Figure 3. 1. LTA4H expression is reduced in IPF tissue derived fibroblasts A- Fibroblasts produced from various regions of the lung tissues were cultured at 1x106. After trypsinization, cells were lysed, and western blot was performed for detection of total LTA4H expression (1ug/ml). Detection was performed using Licor Odyssey machine; IRDye 680nm for LTA4H and IRDye 800nm for Vinculin as control. In the figure legend, LT16 followed by numbers, refers to fibroblasts derived from normal tissue. The numbering was adopted based on VGH-Pathology unit numbering. B- Result from part A was combined to demonstrate the overall differences in expression levels of LTA4H between normal and fibrotic fibroblasts. The results are representative of multiple experiments of various regions of the normal and IPF affected Lung. Regions include: Apex, Basal, Lingula, Superior of the left lung and Middle, Upper, lower Lobes of the right lungs. 00.511.522.5Normal FibroticAverage Optical Density of Fibrotic and Normal Groups45 The importance of PGP and its stress on the inflammation pathway leading to aggressive collagen production in COPD and cystic fibrosis has been recently well recognized [86]. The tri-peptide PGP is one the common building blocks of collagen, and it appears to be more abundant in Type I and III collagen [128]. Collagen of different types are made out of various combinat ions of the amino acids, with glycine and proline being the major amino acid constituents. Some of the variation to mention are (Gly-Pro-Pro)n, (Gly-Gly-Pro)n or (X-Pro-Gly)n which can be found in almost all types of collagen. However, there has been no solid implications of the latter tripeptides to be involved in any types of inflammation [128, 129]. In lungs, PGP is produced via collagen degradation from wound healing and/or dissolution and clearance of extra fibrosis. It is interesting to note that the rate of collagen synthesis and degradation per day in the lungs are the highest amongst the organs such as heart, skin, skeletal muscle, kidney and liver, which can make lungs a vulnerable organ to fibrosis when abnormalities occur in the process. This might be due to the fact that lung might have great deal of exposure with variant air toxins and epithelial damages might result in constant repair [129, 130]. To assess if there are any differences in PGP formation between the normal and IPF derived fibroblasts, fresh passages of the fibroblasts (passage 1 or 2) were cultured for two and five days, figure 3.1.2 (A and B). Longer passages is not suggested as some FBR can cells act aberrant and grow immortally after 5-6 passages. The supernatants of these cultures were collected and tested for collagen degradation and PGP formation by performing the free L-Proline detection assay. The results suggest that the normal fibroblast from all regions produce significantly lower amount of PGP when compared to the fibrotic lineage of the similar regions (p < 0.5). In addition, the amount of the PGP formation tends to elevate by approximately 20% in fibrotic FBR cells when cultures are compared between 2 and 5 days growth, respectively. There seem to be no change in collagen 46 formation and break down in the normal fibroblasts. This experiment also has demonstrated the aggressive nature of the fibroblasts derived from the fibrotic lungs in collagen production and degradation [26]. Assay was normalized using protein quantification assay. For clarity, figure C was added to demonstrate the regions of the lungs where tissues have been used in this thesis. 47 A B Figure 3. 2. Fibrotic lungs contain higher level of PGP. Fibroblast from different lung sections with normal (N) and fibrotic (F) areas of the same patient were cultured for 2 days (A) and 5 days (B) and supernatants were collected and added to Acetic acid and Ninhydrin, vortexed and boiled to 100o C for 30 min. 350 µl of toluene was added to mixture and reactions were read in 96 well plate at 520nm. The assay detects the free L-Proline proportional to PGP formation in fibroblasts. Background control, Media + PGP was subtracted from the readings. Legends description: BASE N 2 represent the basal part of the lung from Normal patients (N) and number represent the days of culture. Other regions tested are Apex and Superior (SUP). Fibrotic lungs are represented by (F). 012345678BASE N 2 BASE F 2 APEX N 2 APEX F 2 SUP N 2 SUP F 2Relative production of L-Proline Detected after 2 days (nM)0246810BASE N 5 BASE F 5 APEX N 5 APEX F 5 SUP N 5 SUP F 5Relative production of L-Proline Detected acter 5 days (nM)48 C Figure 3. 3. Fibrotic lungs contain higher level of PGP. Fibroblast from different lung sections with normal (N) and fibrotic (F) areas of the same patient were cultured for 2 days (A) and 5 days (B) and supernatants were collected and added to Acetic acid and Ninhydrin, vortexed and boiled to 100o C for 30 min. 350 µl of toluene was added to mixture and reactions were read in 96 well plate at 520nm. The assay detects the free L-Proline proportional to PGP formation in fibroblasts. Background control, Media + PGP was subtracted from the readings. (C) Regions of the right and left lung. Legends description: BASE N 2 represent the basal part of the lung from Normal patients (N) and number represent the days of culture. Other regions tested are Apex and Superior (SUP). Fibrotic lungs are represented by (F). 49 Aminopeptidase activity of the recombinant LTA4H confirms the higher levels of PGP in fibrotic lungs when compared to normal non-fibrotic lung and normal from fibrotic section of the IPF lung. To further investigate the PGP production and the importance of the PGP being the natural substrate for the LTA4H in our experiments, primary fibroblast of early passages were cultured for 5 days and supernatants were collected for in-vitro enzyme assay using the human recombinant LTA4H at 100 nM/assay, as shown in Figure 3.1.3. Following the enzyme assay, the supernatant from the enzymatic assay was used to detect the free-L-Proline formation based on aminopeptidase activity of the recombinant enzyme. According to clinical observations, it has been confirmed that almost in all IPF cases, the fibrosis process commences at the basal region of the lung and as the disease progresses the fibrosis moves upward and towards the Apex and superior part of the lung [14]. Therefore, experiments were always structured to compare the healthy and fibrotic lungs at basal, middle, lingula (in some cases), apex and superior regions. Figure 3.1.3 suggest that generally all fibrotic lung tissues produces significantly larger amount of PGP, hence, higher collagen synthesis and degradation (p < 0.05 using ANOVA statistical test). In addition, the superior part of the fibrotic lung has shown the highest PGP production, which might suggest the initial phases of fibrosis in fibroblasts from the upper region of the IPF lung when compared to fibroblast from normal lungs of the same region. Assay was normalized using protein quantification assay. 50 Figure 3. 4. Aminopeptidase activity of the recombinant LTA4H confirms the higher levels of PGP in fibrotic lungs when compared to Normal PGP assay was performed utilizing culture of fibroblast cells grown for 5 days without change of media. Wells were incubated for 2 hours with the recombinant human LTA4H at 0.25 µg/assay, and supernatant were collected for free L-Proline detection assay. In this experiment, LTA4H peptidase acts on PGP to generate L-Proline. Background control, Media+ LTA4H was subtracted from the readings. Results is a representative of multiple experiments. Results showed significance, using ANOVA statistical test when each normal tissue was compared to its fibrotic tissue of the same region with p < 0.05) 0.005.0010.0015.0020.0025.00BASE N LTA4H BASE F LTA4H APEX N LTA4H APEX F LAT4H SUP N LTA4H SUP F LAT4HRelative production of L-Proline Detected acter 5 days (nM)51 While the levels of PGP were shown to be higher in fibrotic fibroblasts when compared to normal fibroblasts derived from the lung tissues, it was appropriate to investigate the levels of LTA4H in the tissues where the fibroblast were derived from. An immunoprecipitation using the LTA4H antibody from the homogenized tissue of normal and fibrotic region of the same lung were followed by PGP enzyme assay and free L-Proline detection. As demonstrated in Figure 3.4, a comparison between the apex (fibrosis free) and basal region of the same fibrotic lung may suggest that the fibrotic region tend to have significantly less active pool of LTA4H available (p < 0.05). The reason for the less enzyme activity in fibrotic section of the fibrotic tissues when compared to normal lung as well as the normal tissue from the IPF lung could be a topic that needs to be more investigated. As has been recognized in past research, chemoattractant characteristics of the PGP tripeptide has a significant role in neutrophilic inflammation cycles, such as in COPD and CF [70, 131]. The accumulation of PGP as well as acetylated form of PGP that no longer is cleaved by LTA4H stimulates neutrophils via their CXCR1/2 receptors. This interaction will lead to increased collagen break down and more PGP formation by local MMP8/9 and propyl endopeptidases [132]. Therefore, based on these observations, LTA4H could play an important role in degrading PGP production from ECM break down, which can lead to reduction of the neutrophilic inflammation cycle, thus keeping collagen production and ultimately fibrosis in control. 52 Figure 3. 5. LTA4H expression levels are lower in IPF lung homogenized tissues. Fresh lung tissues from normal-non-IPF and normal and fibrotic section of the lung of IPF patients were homogenized for protein extraction followed by immunoprecipitation (IP) of the lysate using LTA4H antibody (1µg/ml, Cell Signaling Technologies). Samples were assayed at 37o C for LTA4H activity in vitro, using PGP as substrate. L-Proline detection assay was performed to measure free L-Proline formation based on peptidase activity of LTA4H presence in the tissues. Control contained PGP+ recombinant LTA4H, 0.15 µg/assay, for maximum activity. Assay was normalized to the protein concentration per IP samples across all conditions. Results are representative of multiple experiments. Results showed significance (p < 0.05) when LTA4H expression levels were compared in normal to fibrotic section of the IPF lung. 0.00%20.00%40.00%60.00%80.00%100.00%120.00%140.00%PGP+LTA4HControlNormal non IPFLungNormal section ofIPF LungFibrotic section ofIPF LungPercentage production of L-Proline Detected53 Tissue micro array (TMA) score indicates lower expression levels of LTA4H in fibrotic tissues. To further investigate the pathology of the normal and fibrotic tissues with respect to expression of LTA4H, a TMA block from all paraffin embedded blocks were prepared. Figure 3.5 A shows the immunohistochemistry of the TMA block probed for LTA4H antibody. This result further confirms the earlier experiments performed on the fibroblasts which demonstrated higher LTA4H expression levels in normal tissues when compared to the IPF lung tissues. The results are based on multiple cores taken from each block in order to provide a better statistical outcome. Figure 3.5 B depicts a sample TMA core taken from the tissues of the normal and IPF alveolar sacs, probed for LTA4H. As shown in Figure 3.5 B, it can be observed that alveolar sacs of the fibrotic lung have scored lower IHC score on LTA4H. The TMA clearly indicates that fibrosis has invaded the crucial areas of the lung, thus preventing neutrophil infiltration, while fibroblast continue producing collagen. Figure 3.5 C represents the combined data of the IHC scores from figure 3.5 A, illustrating the general presence of LTA4H in fibrotic lungs when compared to normal lung. While earlier experiments suggested that fibroblasts could be one source of LTA4H, but overall and based our observations, we suggest that neutrophils and potentially macrophages are the ultimate source of LTA4H that can help reducing the PGP tripeptide and its chemotactic effects in early stages of the lung fibrosis. Also the focus of these experiments were on fibroblast and local tissues rather than other factors such as immune cell [70, 133] 54 A 0.00100.00200.00300.00400.00500.00600.00IHC score of LTA4H on TMA Block, score = intensity x countsLT16-020 N LT16-023 N LT16-023 N BLT16-024 N LT16-028 N LT16-029 N LT16-030 N LT16-051 N Apex-F-#1Apex F #2 Apex RL F #2 Apex-LL F #3Apex RL F #3 Apex C- F -LL- #4 RL UL F #3RL UL F #4 Sup P- F-#2 Sup LL-F-#2Sup C LL-F-#3 Ling F #1 Ling P-F #3Ling C-F #4 RL-ML-F-#2 RL ML- F #3RL ML-F #4 Basal LL F #1 Basal P -LL F- #3Basal C-F-#3 RL LL F #3 RL LL F #455 Normal Lung Alveolar sac IPF Alveolar sac 56 B Figure 3. 6. Tissue micro array scores indicate a lower expression levels of LTA4H in fibrotic tissues. A-Paraffin blocks of the normal and fibrotic lung tissues were prepared. Tissue microarray (TMA) block from various regions of the Formalin-Fixed Paraffin-Embedded (FFPE) blocks were prepared using the TMA spotting machine at Prostate Center of Vancouver (for some samples up to 6 cores were produced on the TMA block for better scoring average). TMA block was used to perform IHC using the automated Ventana system. LL = Left long, RL = right lung, F= Fibrosis. LT16 = Normal non-fibrotic tissues. B- A representative TMA core from normal and fibrotic lungs. Left core represents a normal broncho alveolar region while right core demonstrates the fibrosis of the basal region of an IPF lung. IHC was performed on the TMA section and results were scanned using Leica SCN-400 scanner. Pathologist scored the IHC results using Leica high definition microscope. Arrows indicate the detected IHC signal for LTA4H. C- Results represents a statistically combined average of staining from regions of normal and IPF lung for all patients based on the TMA score of LTA4H. Regions were divided into Normal (any given regions of the normal lung), Apex, Lingula and Basal. 050100150200250300350400Normal Apex Lingula BasalIHC score: positive counts x intensity 57 Intracellular levels of MMP-9 are lower in fibroblasts derived from IPF lung when compared to normal fibroblast. Proteases such as matrix metalloproteinases (MMPs) and propyl endopeptidases (PE) play a key role in ECM remodeling. The combination of both mentioned proteases have shown to produce the largest quantity of the PGP detected in broncho alveolar lavage (BAL) from cystic fibrosis patients [134]. Matrix metalloproteinases are comprised of a large family of members that have constantly been found in the sputum and BAL of individuals affected with COPD and CF [80]. It is important to note that neutrophils are the substantial source of MMPs, such as MMP8 (collagenase 2) and MMP9 (gelatinase B) which have been involved in pulmonary fibrosis [45, 135]. Based on these findings, normal and fibrotic tissue derived fibroblasts were tested for intra and extracellular expression levels of MMP9. Tissues from normal and IPF lungs were minced and placed in tissue culture plates as described above. Tissues were removed after 5 days and fibroblasts derived from the tissues were washed with PBS, trypsinized and re-cultured until 70% confluent. Cells were lysed, and protein was extracted for western blot analysis on intracellular MMP9. Figure 3.6 shows that all regions of the normal lungs have higher intracellular levels of MMP9 when compared to the fibrotic fibroblasts. This result might suggest that the balance between the intra and extracellular levels of MMP9 and in general matrix metalloproteinases, might play an important role in balancing the ECM and collagen degradation and ultimately PGP formation. 58 MMP9 Vinculin Figure 3. 7. Intracellular levels of MMP-9 are lower in fibroblasts derived from IPG lung when compared to normal fibroblast. Normal and fibrotic lung FBR cells were collected, lysed and western blot was performed on the whole cell lysate using MMP-9 antibody (2 µg/ml, Abcam). After incubation with primary antibody, the secondary rabbit IRDye 800 was incubated for 3 hours followed by 5 x washes of Tween-20 in PBS. Signal was detected using LICOR Odyssey Machine at 680 nm for MMP9 and 800 nm for Vinculin. Results were normalized by dividing the densitometry values of MMP9 antibody over Vinculin as control housekeeping antibody. Densitometry was performed on Odyssey and using Image J. 00.050.10.150.20.25Base N Base F Apax N Apax F Supoerior N Superior F Lingula N Lingula FRelative corrected optical density of the signal to constant area59 Fibrosis is a process that ultimately lead to a large deposition of collagen, which alters the structure and function of the affected organ. Therefore, one important goal in fibrosis research would to introduce solutions to reduce abnormal collagen formation. Since in earlier experiment it was demonstrated that fibrotic fibroblasts contain higher levels of PGP, a correlation between these factors and collagen formation had to be obtained to further test the overall hypothesis of the importance of LTA4H in fibrosis. Figure 3.7 shows the Pro Collagen 1A formation after treatment of the normal and fibrotic fibroblasts of various regions of the lung, with acetylated-PGP and neutrophil supernatant for 24 hours. Like macrophages, neutrophils can affect their surrounding environment by releasing proteases, antioxidants, cytokines and chemokines [136]. Neutrophils also contain Neutrophil Elastase (NE), an enzyme that can activate TGF-β and it is shown to have increased levels in the airways of IPF patients. Therefore, utilizing neutrophil supernatant was an important part of this experiment to verify if collagen formation can be enhanced by neutrophil factors in fibrotic fibroblasts. As predicted, both acetylated-PGP and neutrophils were able to enhance the in-vitro pro collagen1 A formation only in fibrotic fibroblasts. This effect was not observed in the normal fibroblasts, leading to the idea that activated fibrotic fibroblasts might have different characterist ics when compared to normal functioning fibroblasts. The results were significant when Basal plus NP was compared to Basal with no treatment (t-test, P < 0.05). 60 Pro Collagen 1A Vinculin NT 10uM Ac-PGP NP NP+AcPGP CTL NT 10uM Ac-PGP NP NP+AcPGP CTL Figure 3.8. Formation of Pro-Collagen I A treated with N-α -PGP and neutrophil supernatant. Normal and fibrotic lung FBR cells were isolated from patient tissues and were plated on 96 well assay plate overnight. Neutrophils were collected from donor blood and cultured in DMEM for 6 hours. In-Cell Western assay was performed after fixing and perforating the cell with paraformaldehyde and Triton X-100, respectively. Wells were blocked with Bio-Rad Casein blocking buffer and incubated with anti-Collagen 1A antibody for 24 hours. Wells were washed with 0.1% Tween-20 in PBS and secondary IRDye 800 was added for 3 hours. Cells were washed 5x with Tween-20 in PBS and the LICOR Odyssey was used to detect the signal and gather data. FBR cells were compared when treated and not treated (NT) with neutrophils supernatant (NP) and Acetylated PGP peptide at 10 µM and 50 µM. Results represent the combined regions for Normal lungs and Apex and basal for IPF patients. The results were significant when Basal plus NP was compared to Basal with no treatment (t-test, P < 0.05). 0.001.002.003.004.005.006.007.00NT 10uM AC-PGP NP Sup NP+ AC-PGP(10uM)IR Detection at 700nm corrected with Vinculin/well at 800nm in fold increaseLT16-031APEX-SUPBASALNormal Apex#2 Basal#2 Basal#3 Basal#4 Middle#2 Apex#3 61 Chapter 4: Inhibition of c-Jun N-Terminal Kinase I/II (JNK /II) in IPF derived fibroblasts results in-vitro reduction of pro collagen I A formation. 4.1 Rationale and Hypothesis C-Jun N-terminal kinases 1/2(JNK) is a mitogen-activated protein kinase family member which is responsive to stress stimuli and might be linked to remodelling and progression in IPF [90]. While the expression of JNK was not different across the IPF and normal patients, however, phosphorylation of JNK at threonine183 and tyrosine 185 was substantially elevated amongst the IPF fibroblasts. In addition, the same fibroblast cells shown to produce more pro-collagen I A, while the utilizing the phospho-JNK (P-JNK) inhibitor 1/2 reduced the collagen formation in the IPF fibroblast cells. Activation of JNK has been shown to be an important mediator in TGF-β1-induced fibrosis in liver [108]. Other findings have also indicated that JNK-/- mice have reduced collagen deposition due to reduced expression of pro fibrotic genes such as MMP7 and MMP9 [137]. These encouraging results with respect to JNK inhibition and fibrosis has provided treatment options for pharmaceutical industry. In this chapter, a commercially available JNK1/2 inhibitor, SP600125, was used to further demonstrate the importance of JNK inhibition in collagen1 A production in IPF derived fibroblast. 62 Expression levels of total JNK in normal and fibrotic tissues- The importance of JNK activation in organ fibrosis and collagen deposition have been well established. While phosphorylation of JNK has been the focus of the research in fibrosis, the expression level of total JNK would also demonstrate whether overexpression of the kinase might have a role in exacerbation of the disease. Histological analysis of the TMA in Figure 4.1 A and combination of the regions in Figure 4.1.B, interestingly indicated that JNK expression was relatively higher in the Normal lung tissues and basal region of the IPF lung when compared to Apex and middle regions. These results might be surprising when correlating the total intracellular JNK levels and hence, its expected elevation of JNK phosphorylation in fibrotic tissues. However, this might also suggest that the expression levels of total JNK might not be a deciding factor in IPF lungs but rather its activation via upstream pathways factors such as TGF-β could be most important. Results were significant when normal tissues were compared to Apax & superior and middle & lingula section of the fibrotic lung. ANOVA statistical test was used to compare the value with p < 0.05. 63 A B Figure 4.1. Expression levels of total JNK in normal and fibrotic tissues A- IHC procedure was performed as per explained in the material and method section of the thesis. B- Results of the regions were combined from figure A to demonstrate the total JNK expression in various regions of the normal and IPF lung. 050100150200250300350400450IHC score: positive counts x intensity 050100150200250300350400Normal Apex & Sup Lingula & Middle BasalIHC score: positive counts x intensity 64 While earlier results indicated higher levels of intracellular JNK in normal tissues and basal region of the IPF lung tissue, it was imperative to verify the JNK phosphorylation amongst all tissues and compare these results to the total JNK expression levels. Figure 4.2 A and B (combined regions) depicts the IHC of p-JNK in the same TMA used in Figure 4.1. All IHC processes were performed as explained in the Material and Method section of the thesis with the help of pathologist and technicians for scoring the IHC. As indicated in Figure 4.2 B, the Apex and Middle/Lingula regions of the lung tend to show significantly higher levels of JNK phosphorylation although their total JNK expression tend to be half of the Normal and Basal regions. Student t-test was performed on Lingula/middle IHC scores compared to normal tissue with a p value below 0.05, however, when normal tissues were compared to Apex region the p was above the significance levels (p = 0.0656) (see Figure 4.1 B). These results indicate that increased level of JNK phosphorylation and not its expression levels, could be a factor in lung fibrosis. Recent studies have shown that JNK phosphorylation was dependent on upstream activation of the TGF-βII pathway [108], which lead to SMAD2/3 phosphorylation by JNK and causing over production of collagen 1A in mice fibroblasts [111, 117]. 65 A B 050100150200250300350400450500IHC Score on p-JNK TMA, Postive counts X Intensity050100150200250300350Normal Apex & Sup Lingula & Middle BasalCombined score of the regions from TMA 66 C Normal Basal 67 Lingula Middle 68 Figure 4. 2. Tissues in early stages of fibrosis detected with higher levels of p-JNK. A- IHC was performed on TMA section of the TMA as explained in material and method section. B- Regions combined in figure A in order better demonstrate regional phosphorylation of JNK in normal and IPF lungs. P < 0.05 when normal tissues compared to Lingula and Middle lung. C- Scanned representative cores of the TMA probed for p-JNK in the fibrotic and normal tissues. Arrows indicate the IHC spots in the tissues. 69 Phospho-JNK inhibitor reduces the Collagen I A formation in IPF derived fibroblasts and counteracts Neutrophil (NP) and Dexamethasone (Dex) Pro Collagen formation. Corticosteroids such as Dex have been widely used in treating IPF, with the belief that they can reduce the progression of the fibrosis [138]. This idea has been very controversial as recent clinical studies and observations have proved otherwise. In one report, Zimmermann et al. have reported that Dex treatment did not reduce the fibrosis, and in fact, after Dex treatment, the mitogenic activity of broncho alveolar fluid for fibroblasts was increased [139], and Budinger et al. have shown that dexamethasone therapy did not prevent the monocyte-derived recruitment of the alveolar macrophages in patient affected with pulmonary fibrosis [140]. Neutrophilic inflammation and their chemotactic factors have been very well studied in progression of IPF. In Figure 4.3, an In-Cell Western analysis of the normal and IPF derived fibroblast at 1 x 104 cells per well was performed. After 24 hours cells were treated with Dex ( 30 µM) and JNK inhibitor (JNKI, 12 µM) with presence or absence of neutrophils. The assay showed that NPs and Dex treatments caused an increase in collagen 1A formation, while JNKI alone reduced the collagen 1 A formation significantly. The pro collagen production caused by neutrophils was also kept in control by JNKI in both superior and basal FBRs. An increase in pro collagen formation was seen in normal FBRs when treated with JNKI which could not be explained. We suspect this might be due to stress induced by JNKI, which in turn activates the normal cells to antagonize the JNKI. In addition, a separate experiment was done using a combination of Dex and NPs which pro collagen levels were similar to NP and Dex treatments alone. This data is not shown in this thesis. 70 Figure 4. 3. JNK inhibitor reduces the Collagen I A growth in IPF derived fibroblasts and counteracts the Neutrophil (NP) and Dexamethasone (Dex) Pro Collagen formation. NP was obtained from 60 ml of donor blood and was isolated and cultured for 4 hours at 4X108/ml and 10 uL of supernatant was added to cultures of fibroblasts on 96 well plate at 2X104 cells/well. JNK Inhibitors (JNKI, 12 µM, Calbiochem) and Dexamethasone (30uM, Sigma) were added to the wells and after 24 hours the In-Cell Western method was performed for measurement of procollagen I A formation. NP and dexamethasone caused procollagen I A expression in fibrotic FBRs, but the inhibitor of p-JNK significantly reduced procollagen I A expression in IPF fibroblasts most in the basal and superior region of the lung. Separately an experiment was done with presence of NP-Dex, which the data is not shown, nevertheless, no effect on procollagen I A formation was noticed. Results were significant for JNKI treatment on superior region for NT, NP, JNKI and NP-JNKI treatment (ANOVA, p < 0.05). 05101520253035404550NT DEX NP JNKI JNKI-NPHIR Detection at 700nm corrected with Vinculin / well at 800nmNORMALSUPBASAL71 Phospho-JNK inhibitor decreases the proliferative rate of tissue derived fibroblast in all fibroblast but most notably in fibrotic fibroblast- While JNKI had shown earlier that it can help reducing the collagen formation, an experiment showing the proliferative rate of the normal and fibrotic derived fibroblast was performed using the MTS assay. Cells were counted, and 1 x 105 cells were seeded in 48 well plates and after 24 hours JNKI (12 µM) and Dex (30 µM) were added to the wells for overnight incubation at 37o C in a CO2 incubator. The proliferation rate of the cells was measured after 48 hours. Figure 4.4 demonstrates that JNKI has significantly reduced the in-vitro proliferation of both superior and basal region of the fibrotic fibroblasts when compared to NT with p < 0.05. However, the proliferation of the fibroblast with Dex treatment did not show any reduction and may have shown some increased proliferation rate. Clinical studies have shown that treatment of the IPF patients with Dex caused a rapid progression of fibrosis in late stages of the disease and eventually led to worsening of the disease [139, 140]. While from our studies, Dex treatment did not show a promising inhibitory effect in the FBR cells, more clinical studies need to be performed for more concrete conclusions. Figure 4.4 B demonstrates the fold change based on the Figure 4.4 A, which clearly indicates that JNKI has been successful in inhibiting the collagen formation. Based on these preliminary yet encouraging in-vitro results, it can be suggested that JNKI could be pursued as a potential clinical inhibitor in controlling fibroblasts' production of collagen. 72 A B Figure 4. 4. JNK inhibitor decreases the proliferative rate of tissue derived fibroblast in normal fibroblast but most notably in fibrotic fibroblast. Fibroblasts from normal section of lung as well as basal and superior regions from IPF donors were cultured in 24 well tissue culture plate. Measurement of fibroblast proliferation was measured by MTS assay in the absence or presence of p-JNK-1/2 inhibitor (12 µM, Calbiochem-420128) or dexamethasone (Dex 30 µM, Sigma). After 48 hours of incubation with the drugs, MTS reagents were added (between 1-3 hours) and plates were read at 495 nm. Control wells consisted of non-treated wells with cultured fibroblasts as well as empty wells with media only, which were taken into calculation for normalization of the results. Figure 4.4 B was produced by converting the Figure A to fold change for better data visualization. Results showed significance in proliferation reduction when JNKI treatments were compared to FBR cells with no treatment (t-test, p < 0.05). Dex increased overall proliferation of the RBF cells. 00.20.40.60.811.21.41.6NT Dex JNKIAbsorbance at 495 nm LT16-031SupBasal0.00.20.40.60.81.01.21.41.6NT Dex JNKIMTS proliferation converted into fold chaneg LT16-031SupBasal73 Chapter 5: DISCUSSION In classical organ fibrosis, it has been well documented that TGF-β is the master switch for initiating the signaling pathway. Therapeutic options such as Pirfenidone, while not fully understood in terms of mechanism of action in fibrosis, have been helpful in antagonizing TGF-β in attenuating IPF progression in some patients, while causing side effects such as skin rash, hepatic dysfunction and gastrointestinal complications [141]. In addition, due to the multifunctional properties of TGF-β, which controls proliferation, differentiation, embryonic development and angiogenesis, it has been difficult so far to directly target this protein for neutralizing its pro-fibrotic role in IPF. For these reasons, other experimental and clinica l approaches have been used to battle IPF and fibrosis in other organs. In recent studies, it has been demonstrated that extracellular matrix collagen breakdown in wound healing and repair processes, produce a variety of collagen derivatives such as PGP tripeptides [80, 84]. Production of PGP has been closely tied to neutrophil chemotactic response which led to significant elevations in inflammation and further self-propagation of the inflammatory pathway in diseases such as COPD, CF, as well as IPF [142]. The importance of PGP levels has influenced clinical studies to correlate its levels as a bio marker in progression of COPD and asthma [143]. While the involvement of PGP in disease related to over-production of collagen has been well demonstrated, its inflammatory effects have been shown to be controlled via a bifunctional enzyme called LTA4H [144]. The L-Proline amino acid located on N-Terminal of PGP the has been reported to be selectively targeted and cleaved by aminopeptidase activity of LTA4H [133]. Current studies have indicated that LTA4H is ubiquitous in mammalian tissue [78, 79]. Based on our studies, it is suggested that in general, normal lung fibroblast cells did express more LTA4H than the fibrotic FBR cells, but the difference of expression levels were not significant 74 between the two cells lines. However, when the fibrotic and normal homogenized tissues were tested for the LTA4H expression levels, the LTA4H expression levels were significantly lower in fibrotic tissues when compared to the normal. This led to the idea that while lung fibroblasts cells do produce LTA4H, but they might not be the essential source of LTA4H in dealing with inflammation caused by PGP. Following on these results, it was important to show whether there were differences in PGP production when comparing fibrotic versus normal lung fibroblasts. When latter fibroblast cells from the same patient’s lung were grown for 2 to 5 days, followed by the L-Proline detection assay, it was concluded that fibrotic cells did produce significantly more PGP than the normal fibroblasts. To further confirm the PGP productions levels, the same normal and fibrotic FBRs were grown for 5 days and the supernatants were tested for L-Proline detection assay using a recombinant human LTA4H enzyme. This experiment further confirmed the previous results by showing that the fibrotic fibroblasts produce higher levels of PGP than normal fibroblasts. Based on these observations, FBRs from the superior and apex region of the fibrotic lungs seemed to be producing more PGP over time than the basal region. We speculate that this production difference might be due to the fact that upper parts of fibrotic lungs are still undergoing an early stage of fibrosis and being overly active in producing factors that help in the progression of the disease. It is understood that the main function of fibroblast cells is to maintain the structural integrity of the connective tissue by continuously secreting ECM proteins such as glycoprote ins, glycosaminoglycan, and collagen. While not exclusive to FBR cells, fibroblasts do have the ability to express matrix metalloproteinase (MMPs) and their inhibitors (TIMPs) to balance the connective tissue production and breakdown [145, 146]. It has been reported that broncho alveolar 75 fluid of IPF patients particularly contained strong levels of MMP9 [147]. In conjunction with these observations, our results indicated that the normal fibroblast cells contained higher levels of intracellular MMP9 when compared to fibrotic FBRs. We speculate that the balance of intra and extracellular levels is important for ECM integrity and exaggerated extracellular levels of MMPs can increase the PGP concentrations levels in the surrounding lung tissues and create more inflammatory responses. Therefore, the higher levels of PGP in fibrotic FBRs might be connected to their extracellular MMP9 as well as secretion of other MMPs, which could be directly linked to ECM degradation and PGP formation. Earlier results indicated that homogenized lungs tissues had higher levels of LTA4H expression when compared to the fibroblasts derived from the same lung tissues. Therefore, a histological approach was used to understand the differences between this observation. The tissue microarray obtained from all patients in our library showed that all regions of the fibrotic lung, such as alveolar sacs, remainder of the in-tact epithelial cells and fibroblasts had considerably lower levels of LTA4H expression when compared to normal tissues. While lower LTA4H expression was expected in fibrotic tissues, in general, the aggressive nature of fibrosis would eventually lead to massive tissue remodeling and lung parenchyma dysfunctionality, further blocking the infiltration of important factors such as macrophages in helping with the resolution of inflammation and abnormal cascade of reactions in IPF. Neutrophils have long been linked to pathogenesis of certain kind of pulmonary diseases such as COPD, CF, asthma and IPF. In general, neutrophils are rare in the lung tissues of the healthy person and only thought to be attracted to lesions by neutrophil chemotactic factors (NCF) [148, 149]. Neutrophils have the capability to generate PGP since they are a major source of MMPs 76 and other enzymes that are involved in ECM degradation, as well as restricting their own recruitment when required [150]. In our studies, neutrophil supernatants increased in-vitro pro-collagen 1A formation in fibrotic fibroblasts when compared to normal cells, while acetylated -PGP, which cannot be targeted by LTA4H, did not exert any further effect on the pro-collagen formation. Based on available literature and our studies, we hypothesize that neutrophils continuously produce signals directing fibroblasts to produce collagen. This will lead to further PGP production via secretion of fibroblast’s and neutrophil’s degradative enzymes, thus continuing the vicious cycle of collagen formation and inflammation. The persistence of this cycle could lead to an exacerbation and further progression of an already diseased IPF lung. In conclusion, current experiments and clinical reports have provided a great proof-of-concept in rationalizing efforts for seeking inhibitors to LTA4H that can be translated into treatment of IPF. The challenge of utilizing the anti-inflammatory role of LTA4H in degrading PGP, still requires the design of more selective epoxide hydrolase (EH) inhibitors which will not interfere with its aminopeptidase (AP) activity. While the inhibition of EH has shown great potential in alleviating diseases such as asthma and COPD, the AP activity of LTA4H has shown prospects in targeting IPF progression. Currently there are many compounds in clinical trials that are targeting specifically the epoxide hydrolase activity of the LTA4H, however they have not shown success in keeping the aminopeptidase activity intact. In addition, better animal IPF models can help the clinical aspect of IPF research when new drugs are being tested for effectiveness in alleviating fibrosis. Current bleomycin- induced lung injury leading to scarring is not considered a true IPF lung model. One suggested future model 77 could be the treatment of lung with short burst of bleomycin followed by PGP addition which should trigger the pathways that normally function in lung leading to fibrosis. In recent years, mitogen-activated protein kinases (MAPK), a family of well characterized signaling proteins which are activated by a variety of stress signals, have been shown to play an important role in fibrosis [87, 151]. One member of the MAPK family is the c-Jun N-Terminal Kinase (JNK) 1/2/3, which has the ability to promote proliferation, differentiation and inflammation, but most importantly has been linked to downstream signaling caused by overexpression of TGF-β in organ fibrosis [96]. Activated MAPKs were significantly increased in lungs of patients with IPF compared with controls [152]. In addition, JNK phosphorylation was shown to cause tissue remodeling in airways of the bleomycin- induced mouse model [105]. Studies have shown that JNK activation in pulmonary fibrosis is related to inhibition of TIMP protein expression. This event could lead to an imbalance between MMP and TIMP proteins, hence affecting particularly type I collagen production [105]. Based on clinical and in-vitro studies on lung fibroblasts, it was observed that TGF-β-induced activation of JNK/SMAD pathway was responsible for inhibition of TIMP-1, leading to matrix accumulation [153]. In our studies, the IHC score on TMA from our patient’s tissue library indicated that the fibrotic tissues contained higher levels of total JNK expression when compared to the normal lung tissues. The basal region of the fibrotic tissue showed a slightly higher level of total JNK expression amongst the fibrotic cells, which could not be explained. While it was important to determine the relative total JNK expression in normal and fibrotic lung tissues, it is the activation of JNK by phosphorylation that has the ultimate importance in impacting cell and tissue physiology and remodeling. Therefore, when the same TMA was scored for phospho specific 78 JNK1/2 antibody, results clearly suggested that superior and middle regions of the fibrotic lungs contained elevated levels of Phospho-JNK (p-JNK) signal when compared to normal tissues, yet they exhibit a lower level of total JNK expression. We speculate that this difference in phosphorylation might be due to the fact that upper and middle regions of the lungs are still undergoing an active stage of fibrosis with dysregulated TGF-β and JNK upstream signaling pathways. As mentioned earlier, neutrophils have shown the capability of inducing fibroblasts to produce collagen as well as helping the ECM degradation [127, 136]. Also, we had observed in earlier experiments that Dex, in contrast to some reports, had no effect in reducing the pro collagen formation. The results suggest that JNKI was able to reduce the pro collagen 1A formation in the fibrotic fibroblast cells and also was able to control neutrophil-induced pro collagen formation. These results combined with current reports are encouraging in showing that JNKI could be a potential candidate in helping IPF patients. While few groups have shown that JNKI can lead to liver dysfunction and toxicity and excessive weight gain in mice, Peter H. Schafer et.al at Celgene Department of Translational Development in New Jersey demonstrated that both JNK1 and JNK2 knockout mice were resistant to pulmonary inflammation and fibrosis, as well as obesity, when the subject animals were fed a high fat diet [105]. JNK is one of the three well-characterized MAPK protein that has the ability to transduce extracellular signals that can control, differentiation, apoptosis, migration as well as prolifera t ion [88]. Upon activation of the JNK by MAKK4 and MAKK7, JNK can phosphorylate serine 63 and 73 and threonine 91 and 93 on c-Jun protein, which will promote interaction of the c-Jun with c-Fos proto-oncogene and AP-1. This complex will act as a transcriptional factor that can trigger apoptosis, inflammation or fibrotic responses. Fibrosis is a dysregulated growth of fibroblast and 79 production of collagen. Since JNK has been shown to be involved in cell proliferation, an MTS assay was setup to observe whether JNK had any effect on growth and proliferation rate of the lung fibroblasts. Therefore, both normal and fibrotic FBR cells were grown to 80% confluency and were treated with JNK 1/2 inhibitor along with dexamethasone as a proliferative control agent, for 48 hours followed by cell proliferation assay. Results of this study indicated that the JNK inhibitor is able to reduce the overall proliferative rate of FBR cells with significant effect on the basal fibrotic fibroblasts cells with an approximate 40% growth reduction. Dexamethasone seemed to slightly elevate the proliferation amongst the FBS cells. This result can be very encouraging in showing that JNK could be a potential target controlling the growth of fibrotic fibroblasts, which could lead to reduction and accumulation of collagen in the ECM. In conclusion, the JNK signaling blockade has shown a clear pharmacological potential in treatment of organ fibrosis in kidney, and liver and recently has shown promising results in lung fibrosis. However, additional work is required to elucidate the mechanistic link between JNK and TGF-β signaling in lung as well as molecular details and significance of JNK isoforms, when designing therapeutics for pulmonary fibrosis. In addition, systemic JNK inhibition, upstream targeting of JNK pathways and cell-type specific delivery of JNK inhibitors could be other avenues that can be investigated for potential clinical use. 5.1 Conclusion Idiopathic pulmonary fibrosis is an irreversible and progressive lung disease with high mortality rate and limited treatments. In past decades, variety of experimental models in animals have been generated to study the mechanisms of IPF. The mouse bleomycin model has been the 80 closest approach in exerting lung epithelial injuries leading scaring and fibrosis. Nevertheless, due to its transient nature and the reversibility of the fibrosis observed in this model, it is unclear whether this model can truly replicate the chronic and progressive form of the disease seen in humans. While epithelial damage, inflammation and repetitive injuries are important initiators of fibrosis, detailed understanding of the cellular, genetic and epigenetic that cause promoting the progression of chronic pulmonary fibrosis is needed for successful therapeutic development. Thus, an integrated approach that targets both key inflammatory cytokines and profibrotic mediators simultaneously could emerge as a successful strategy to treat this fatal disease. Based on current publications and our preliminary studies, a plausible model could be an approach by initial bleomycin administration to cause lung epithelial injury followed by PGP pulses to create a neutrophilic activation of FBR cells leading to abnormal collagen formation and eventually fibrosis. The Bleomycin-PGP fibrotic model, if successful in vivo, can be utilized to further investigate better inhibitors targeting LTA4H’s epoxide hydrolase activity while leaving the aminopeptidase activity functional. In addition, our studies demonstrated that fibrotic tissues contained elevated levels of phosphorylated JNK. In addition, publications have shown that JNK1 knockout mice were resistant to bleomycin- induced fibrosis, showing the importance of JNK involvement as one of the key downstream mediators of pulmonary fibrosis. Therefore, using the bleomycin-PGP model and focusing on LTA4H AP activity and JNK inhibition might provide a synergistic avenue in treating IPF progression. 81 References: 1. Wynn, T.A., Cellular and molecular mechanisms of fibrosis. J Pathol, 2008. 214(2): p. 199-210. 2. Wynn, T.A., Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest, 2007. 117(3): p. 524-9. 3. Wang, Y., P.S. Fan, and B. Kahaleh, Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts. Arthritis Rheum, 2006. 54(7): p. 2271-9. 4. Quan, T.E., S.E. Cowper, and R. Bucala, The role of circulating fibrocytes in fibrosis. Curr Rheumatol Rep, 2006. 8(2): p. 145-50. 5. Pardo, A. and M. Selman, Matrix metalloproteases in aberrant fibrotic tissue remodeling. Proc Am Thorac Soc, 2006. 3(4): p. 383-8. 6. Schuppan, D., M. Ruehl, R. Somasundaram, and E.G. Hahn, Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis, 2001. 21(3): p. 351-72. 7. Willis, B.C., R.M. duBois, and Z. Borok, Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc, 2006. 3(4): p. 377-82. 8. Li, M.O., Y.Y. Wan, S. Sanjabi, A.K. Robertson, and R.A. Flavell, Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol, 2006. 24: p. 99-146. 9. Parsons, C.J., M. Takashima, and R.A. Rippe, Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007. 22 Suppl 1: p. S79-84. 10. Wynn, T.A., IL-13 effector functions. Annu Rev Immunol, 2003. 21: p. 425-56. 11. Kalluri, R. and E.G. Neilson, Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest, 2003. 112(12): p. 1776-84. 12. Zeisberg, E.M., O. Tarnavski, M. Zeisberg, A.L. Dorfman, J.R. McMullen, E. Gustafsson, A. Chandraker, X. Yuan, W.T. Pu, A.B. Roberts, E.G. Neilson, M.H. Sayegh, S. Izumo, and R. Kalluri, Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med, 2007. 13(8): p. 952-61. 13. Wynn, T.A., Integrating mechanisms of pulmonary fibrosis. J Exp Med, 2011. 208(7): p. 1339-50. 14. Katzenstein, A.L. and J.L. Myers, Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am J Respir Crit Care Med, 1998. 157(4 Pt 1): p. 1301-15. 15. Raghu, G., H.R. Collard, J.J. Egan, F.J. Martinez, J. Behr, K.K. Brown, T.V. Colby, J.F. Cordier, K.R. Flaherty, J.A. Lasky, D.A. Lynch, J.H. Ryu, J.J. Swigris, A.U. Wells, J. Ancochea, D. Bouros, C. Carvalho, U. Costabel, M. Ebina, D.M. Hansell, T. Johkoh, D.S. Kim, T.E. King, Jr., Y. Kondoh, J. Myers, N.L. Muller, A.G. Nicholson, L. Richeldi, M. Selman, R.F. Dudden, B.S. Griss, S.L. Protzko, H.J. Schunemann, and A.E.J.A.C.o.I.P. Fibrosis, An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med, 2011. 183(6): p. 788-824. 16. Rosas, I.O. and N. Kaminski, Update in diffuse parenchymal lung disease, 2013. Am J Respir Crit Care Med, 2015. 191(3): p. 270-4. 82 17. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med, 2000. 161(2 Pt 1): p. 646-64. 18. Cottin, V. and J.F. Cordier, Velcro crackles: the key for early diagnosis of idiopathic pulmonary fibrosis? Eur Respir J, 2012. 40(3): p. 519-21. 19. Ley, B. and H.R. Collard, Epidemiology of idiopathic pulmonary fibrosis. Clin Epidemiol, 2013. 5: p. 483-92. 20. Raghu, G., B. Rochwerg, Y. Zhang, C.A. Garcia, A. Azuma, J. Behr, J.L. Brozek, H.R. Collard, W. Cunningham, S. Homma, T. Johkoh, F.J. Martinez, J. Myers, S.L. Protzko, L. Richeldi, D. Rind, M. Selman, A. Theodore, A.U. Wells, H. Hoogsteden, H.J. Schunemann, S. American Thoracic, s. European Respiratory, S. Japanese Respiratory, and A. Latin American Thoracic, An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med, 2015. 192(2): p. e3-19. 21. Fubini, B. and A. Hubbard, Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med, 2003. 34(12): p. 1507-16. 22. Oh, C.K., L.A. Murray, and N.A. Molfino, Smoking and idiopathic pulmonary fibrosis. Pulm Med, 2012. 2012: p. 808260. 23. Ohbayashi, M., S. Kubota, A. Kawase, N. Kohyama, Y. Kobayashi, and T. Yamamoto, Involvement of epithelial-mesenchymal transition in methotrexate-induced pulmonary fibrosis. J Toxicol Sci, 2014. 39(2): p. 319-30. 24. Taskar, V.S. and D.B. Coultas, Is idiopathic pulmonary fibrosis an environmental disease? Proc Am Thorac Soc, 2006. 3(4): p. 293-8. 25. Fernandez, I.E. and O. Eickelberg, New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis. Lancet, 2012. 380(9842): p. 680-8. 26. Kendall, R.T. and C.A. Feghali-Bostwick, Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol, 2014. 5: p. 123. 27. Willis, B.C. and Z. Borok, TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol, 2007. 293(3): p. L525-34. 28. Xu, Y. and S. Lu, Role of WNT1-inducible-signaling pathway protein 1 in etoposide resistance in lung adenocarcinoma A549 cells. Int J Clin Exp Med, 2015. 8(9): p. 14962-8. 29. Bauer, Y., J. Tedrow, S. de Bernard, M. Birker-Robaczewska, K.F. Gibson, B.J. Guardela, P. Hess, A. Klenk, K.O. Lindell, S. Poirey, B. Renault, M. Rey, E. Weber, O. Nayler, and N. Kaminski, A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol, 2015. 52(2): p. 217-31. 30. Bolanos, A.L., C.M. Milla, J.C. Lira, R. Ramirez, M. Checa, L. Barrera, J. Garcia-Alvarez, V. Carbajal, C. Becerril, M. Gaxiola, A. Pardo, and M. Selman, Role of Sonic Hedgehog in idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol, 2012. 303(11): p. L978-90. 31. Liu, X., Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I. Cell Motil Cytoskeleton, 2008. 65(12): p. 935-44. 83 32. Pozharskaya, V., E. Torres-Gonzalez, M. Rojas, A. Gal, M. Amin, S. Dollard, J. Roman, A.A. Stecenko, and A.L. Mora, Twist: a regulator of epithelial-mesenchymal transition in lung fibrosis. PLoS One, 2009. 4(10): p. e7559. 33. Camara, J. and G. Jarai, Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha. Fibrogenesis Tissue Repair, 2010. 3(1): p. 2. 34. Gu, H., E.A. Mickler, O.W. Cummings, G.E. Sandusky, D.J. Weber, A. Gracon, T. Woodruff, D.S. Wilkes, and R. Vittal, Crosstalk between TGF-beta1 and complement activation augments epithelial injury in pulmonary fibrosis. FASEB J, 2014. 28(10): p. 4223-34. 35. Kim, K.K., Y. Wei, C. Szekeres, M.C. Kugler, P.J. Wolters, M.L. Hill, J.A. Frank, A.N. Brumwell, S.E. Wheeler, J.A. Kreidberg, and H.A. Chapman, Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest, 2009. 119(1): p. 213-24. 36. Li, L.C., D.L. Li, L. Xu, X.T. Mo, W.H. Cui, P. Zhao, W.C. Zhou, J. Gao, and J. Li, High-Mobility Group Box 1 Mediates Epithelial-to-Mesenchymal Transition in Pulmonary Fibrosis Involving Transforming Growth Factor-beta1/Smad2/3 Signaling. J Pharmacol Exp Ther, 2015. 354(3): p. 302-9. 37. Shi, Y. and J. Massague, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003. 113(6): p. 685-700. 38. Fernandez, I.E. and O. Eickelberg, The impact of TGF-beta on lung fibrosis: from targeting to biomarkers. Proc Am Thorac Soc, 2012. 9(3): p. 111-6. 39. Khalil, N., T.V. Parekh, R. O'Connor, N. Antman, W. Kepron, T. Yehaulaeshet, Y.D. Xu, and L.I. Gold, Regulation of the effects of TGF-beta 1 by activation of latent TGF-beta 1 and differential expression of TGF-beta receptors (T beta R-I and T beta R-II) in idiopathic pulmonary fibrosis. Thorax, 2001. 56(12): p. 907-15. 40. Eickelberg, O. and G.J. Laurent, The quest for the initial lesion in idiopathic pulmonary fibrosis: gene expression differences in IPF fibroblasts. Am J Respir Cell Mol Biol, 2010. 42(1): p. 1-2. 41. Ramos, C., M. Montano, J. Garcia-Alvarez, V. Ruiz, B.D. Uhal, M. Selman, and A. Pardo, Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol, 2001. 24(5): p. 591-8. 42. Raghu, G., S. Masta, D. Meyers, and A.S. Narayanan, Collagen synthesis by normal and fibrotic human lung fibroblasts and the effect of transforming growth factor-beta. Am Rev Respir Dis, 1989. 140(1): p. 95-100. 43. Kuhn, C. and J.A. McDonald, The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol, 1991. 138(5): p. 1257-65. 44. Giannandrea, M. and W.C. Parks, Diverse functions of matrix metalloproteinases during fibrosis. Dis Model Mech, 2014. 7(2): p. 193-203. 45. Henry, M.T., K. McMahon, A.J. Mackarel, K. Prikk, T. Sorsa, P. Maisi, R. Sepper, M.X. Fitzgerald, and C.M. O'Connor, Matrix metalloproteinases and tissue inhibitor of metalloproteinase-1 in sarcoidosis and IPF. Eur Respir J, 2002. 20(5): p. 1220-7. 84 46. Visse, R. and H. Nagase, Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res, 2003. 92(8): p. 827-39. 47. Beeh, K.M., J. Beier, O. Kornmann, and R. Buhl, Sputum matrix metalloproteinase-9, tissue inhibitor of metalloprotinease-1, and their molar ratio in patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and healthy subjects. Respir Med, 2003. 97(6): p. 634-9. 48. Kim, S.N., J. Lee, H.S. Yang, J.W. Cho, S. Kwon, Y.B. Kim, J.D. Her, K.H. Cho, C.W. Song, and K. Lee, Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicol Res, 2010. 26(3): p. 217-22. 49. Richards, T.J., N. Kaminski, F. Baribaud, S. Flavin, C. Brodmerkel, D. Horowitz, K. Li, J. Choi, L.J. Vuga, K.O. Lindell, M. Klesen, Y. Zhang, and K.F. Gibson, Peripheral blood proteins predict mortality in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 2012. 185(1): p. 67-76. 50. Luzina, I.G., N.W. Todd, A.T. Iacono, and S.P. Atamas, Roles of T lymphocytes in pulmonary fibrosis. J Leukoc Biol, 2008. 83(2): p. 237-44. 51. Sharma, S.K., J.A. MacLean, C. Pinto, and R.L. Kradin, The effect of an anti-CD3 monoclonal antibody on bleomycin-induced lymphokine production and lung injury. Am J Respir Crit Care Med, 1996. 154(1): p. 193-200. 52. Barron, L. and T.A. Wynn, Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages. Am J Physiol Gastrointest Liver Physiol, 2011. 300(5): p. G723-8. 53. Pechkovsky, D.V., A. Prasse, F. Kollert, K.M. Engel, J. Dentler, W. Luttmann, K. Friedrich, J. Muller-Quernheim, and G. Zissel, Alternatively activated alveolar macrophages in pulmonary fibrosis-mediator production and intracellular signal transduction. Clin Immunol, 2010. 137(1): p. 89-101. 54. Stahl, M., J. Schupp, B. Jager, M. Schmid, G. Zissel, J. Muller-Quernheim, and A. Prasse, Lung collagens perpetuate pulmonary fibrosis via CD204 and M2 macrophage activation. PLoS One, 2013. 8(11): p. e81382. 55. Cavarra, E., F. Carraro, S. Fineschi, A. Naldini, B. Bartalesi, A. Pucci, and G. Lungarella, Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs. Am J Physiol Lung Cell Mol Physiol, 2004. 287(6): p. L1186-92. 56. Kolb, M., P.J. Margetts, D.C. Anthony, F. Pitossi, and J. Gauldie, Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest, 2001. 107(12): p. 1529-36. 57. Wilson, M.S., S.K. Madala, T.R. Ramalingam, B.R. Gochuico, I.O. Rosas, A.W. Cheever, and T.A. Wynn, Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent. J Exp Med, 2010. 207(3): p. 535-52. 58. Moore, B.B., J.E. Kolodsick, V.J. Thannickal, K. Cooke, T.A. Moore, C. Hogaboam, C.A. Wilke, and G.B. Toews, CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol, 2005. 166(3): p. 675-84. 59. Belperio, J.A., M. Dy, L. Murray, M.D. Burdick, Y.Y. Xue, R.M. Strieter, and M.P. Keane, The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis. J Immunol, 2004. 173(7): p. 4692-8. 85 60. Nelson, S., B.E. Laughon, W.R. Summer, M.A. Eckhaus, J.G. Bartlett, and G.J. Jakab, Characterization of the pulmonary inflammatory response to an anaerobic bacterial challenge. Am Rev Respir Dis, 1986. 133(2): p. 212-7. 61. Fick, R.B., Jr., R.A. Robbins, S.U. Squier, W.E. Schoderbek, and W.D. Russ, Complement activation in cystic fibrosis respiratory fluids: in vivo and in vitro generation of C5a and chemotactic activity. Pediatr Res, 1986. 20(12): p. 1258-68. 62. Crystal, R.G., P.B. Bitterman, S.I. Rennard, A.J. Hance, and B.A. Keogh, Interstitial lung diseases of unknown cause. Disorders characterized by chronic inflammation of the lower respiratory tract. N Engl J Med, 1984. 310(4): p. 235-44. 63. Janoff, A., Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis, 1985. 132(2): p. 417-33. 64. Xaubet, A., C. Agusti, P. Luburich, J.A. Barbera, M. Carrion, M.C. Ayuso, J. Roca, and R. Rodriguez-Roisin, Interleukin-8 expression in bronchoalveolar lavage cells in the evaluation of alveolitis in idiopathic pulmonary fibrosis. Respir Med, 1998. 92(2): p. 338-44. 65. Hedstrom, L., Serine protease mechanism and specificity. Chem Rev, 2002. 102(12): p. 4501-24. 66. Hardison, M.T., F.S. Galin, C.E. Calderon, U.V. Djekic, S.B. Parker, K.M. Wille, P.L. Jackson, R.A. Oster, K.R. Young, J.E. Blalock, and A. Gaggar, The presence of a matrix-derived neutrophil chemoattractant in bronchiolitis obliterans syndrome after lung transplantation. J Immunol, 2009. 182(7): p. 4423-31. 67. Postlethwaite, A.E., J.M. Seyer, and A.H. Kang, Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides. Proc Natl Acad Sci U S A, 1978. 75(2): p. 871-5. 68. Haeggstrom, J.Z., F. Tholander, and A. Wetterholm, Structure and catalytic mechanisms of leukotriene A4 hydrolase. Prostaglandins Other Lipid Mediat, 2007. 83(3): p. 198-202. 69. Serhan, C.N., Pro-resolving lipid mediators are leads for resolution physiology. Nature, 2014. 510(7503): p. 92-101. 70. Snelgrove, R.J., P.L. Jackson, M.T. Hardison, B.D. Noerager, A. Kinloch, A. Gaggar, S. Shastry, S.M. Rowe, Y.M. Shim, T. Hussell, and J.E. Blalock, A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science, 2010. 330(6000): p. 90-4. 71. Thunnissen, M.M., P. Nordlund, and J.Z. Haeggstrom, Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation. Nat Struct Biol, 2001. 8(2): p. 131-5. 72. Ma, Y., K. Kleinbeck, and W.J. Kao, Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration. Wound Repair Regen, 2011. 19(6): p. 718-26. 73. Haddox, J.L., R.R. Pfister, D.D. Muccio, M. Villain, C.I. Sommers, M. Chaddha, G.M. Anantharamaiah, W.J. Brouillette, and L.J. DeLucas, Bioactivity of peptide analogs of the neutrophil chemoattractant, N-acetyl-proline-glycine-proline. Invest Ophthalmol Vis Sci, 1999. 40(10): p. 2427-9. 74. O'Reilly, P., P.L. Jackson, B. Noerager, S. Parker, M. Dransfield, A. Gaggar, and J.E. Blalock, N-alpha-PGP and PGP, potential biomarkers and therapeutic targets for COPD. Respir Res, 2009. 10: p. 38. 86 75. Haeggstrom, J.Z., Structure, function, and regulation of leukotriene A4 hydrolase. Am J Respir Crit Care Med, 2000. 161(2 Pt 2): p. S25-31. 76. Stsiapanava, A., U. Olsson, M. Wan, T. Kleinschmidt, D. Rutishauser, R.A. Zubarev, B. Samuelsson, A. Rinaldo-Matthis, and J.Z. Haeggstrom, Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor. Proc Natl Acad Sci U S A, 2014. 111(11): p. 4227-32. 77. Arand, M., A. Cronin, M. Adamska, and F. Oesch, Epoxide hydrolases: structure, function, mechanism, and assay. Methods Enzymol, 2005. 400: p. 569-88. 78. Rudberg, P.C., F. Tholander, M. Andberg, M.M. Thunnissen, and J.Z. Haeggstrom, Leukotriene A4 hydrolase: identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates. J Biol Chem, 2004. 279(26): p. 27376-82. 79. Stsiapanava, A., B. Samuelsson, and J.Z. Haeggstrom, Capturing LTA4 hydrolase in action: Insights to the chemistry and dynamics of chemotactic LTB4 synthesis. Proc Natl Acad Sci U S A, 2017. 114(36): p. 9689-9694. 80. Gaggar, A., P.L. Jackson, B.D. Noerager, P.J. O'Reilly, D.B. McQuaid, S.M. Rowe, J.P. Clancy, and J.E. Blalock, A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. J Immunol, 2008. 180(8): p. 5662-9. 81. Overbeek, S.A., S. Braber, P.J. Koelink, P.A. Henricks, E. Mortaz, A.T. LoTam Loi, P.L. Jackson, J. Garssen, G.T. Wagenaar, W. Timens, L. Koenderman, J.E. Blalock, A.D. Kraneveld, and G. Folkerts, Cigarette smoke-induced collagen destruction; key to chronic neutrophilic airway inflammation? PLoS One, 2013. 8(1): p. e55612. 82. Chen, Y.W., J.M. Leung, and D.D. Sin, A Systematic Review of Diagnostic Biomarkers of COPD Exacerbation. PLoS One, 2016. 11(7): p. e0158843. 83. van Houwelingen, A.H., N.M. Weathington, V. Verweij, J.E. Blalock, F.P. Nijkamp, and G. Folkerts, Induction of lung emphysema is prevented by L-arginine-threonine-arginine. FASEB J, 2008. 22(9): p. 3403-8. 84. Weathington, N.M., A.H. van Houwelingen, B.D. Noerager, P.L. Jackson, A.D. Kraneveld, F.S. Galin, G. Folkerts, F.P. Nijkamp, and J.E. Blalock, A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nat Med, 2006. 12(3): p. 317-23. 85. Jackson, P.L., B.D. Noerager, M.J. Jablonsky, M.T. Hardison, B.D. Cox, J.C. Patterson, B. Dhanapal, J.E. Blalock, and D.D. Muccio, A CXCL8 receptor antagonist based on the structure of N-acetyl-proline-glycine-proline. Eur J Pharmacol, 2011. 668(3): p. 435-42. 86. Abdul Roda, M., A.M. Fernstrand, F.A. Redegeld, J.E. Blalock, A. Gaggar, and G. Folkerts, The matrikine PGP as a potential biomarker in COPD. Am J Physiol Lung Cell Mol Physiol, 2015. 308(11): p. L1095-101. 87. Davis, R.J., Signal transduction by the JNK group of MAP kinases. Cell, 2000. 103(2): p. 239-52. 88. Bode, A.M. and Z. Dong, The functional contrariety of JNK. Mol Carcinog, 2007. 46(8): p. 591-8. 89. Hashimoto, S., Y. Gon, I. Takeshita, K. Matsumoto, S. Maruoka, and T. Horie, Transforming growth Factor-beta1 induces phenotypic modulation of human lung 87 fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway. Am J Respir Crit Care Med, 2001. 163(1): p. 152-7. 90. Mooney, L.M. and A.J. Whitmarsh, Docking interactions in the c-Jun N-terminal kinase pathway. J Biol Chem, 2004. 279(12): p. 11843-52. 91. Grynberg, K., F.Y. Ma, and D.J. Nikolic-Paterson, The JNK Signaling Pathway in Renal Fibrosis. Front Physiol, 2017. 8: p. 829. 92. Nihalani, D., D. Meyer, S. Pajni, and L.B. Holzman, Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components. EMBO J, 2001. 20(13): p. 3447-58. 93. Wagner, E.F. and A.R. Nebreda, Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer, 2009. 9(8): p. 537-49. 94. Kumar, A., U.K. Singh, S.G. Kini, V. Garg, S. Agrawal, P.K. Tomar, P. Pathak, A. Chaudhary, P. Gupta, and A. Malik, JNK pathway signaling: a novel and smarter therapeutic targets for various biological diseases. Future Med Chem, 2015. 7(15): p. 2065-86. 95. Wolters, P.J., H.R. Collard, and K.D. Jones, Pathogenesis of idiopathic pulmonary fibrosis. Annu Rev Pathol, 2014. 9: p. 157-79. 96. Meng, X.M., D.J. Nikolic-Paterson, and H.Y. Lan, TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol, 2016. 12(6): p. 325-38. 97. Kim, S.J., P. Angel, R. Lafyatis, K. Hattori, K.Y. Kim, M.B. Sporn, M. Karin, and A.B. Roberts, Autoinduction of transforming growth factor beta 1 is mediated by the AP-1 complex. Mol Cell Biol, 1990. 10(4): p. 1492-7. 98. Velden, J.L., J.F. Alcorn, A.S. Guala, E.C. Badura, and Y.M. Janssen-Heininger, c-Jun N-terminal kinase 1 promotes transforming growth factor-beta1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3. Am J Respir Cell Mol Biol, 2011. 44(4): p. 571-81. 99. Panzhinskiy, E., W.M. Zawada, K.R. Stenmark, and M. Das, Hypoxia induces unique proliferative response in adventitial fibroblasts by activating PDGFbeta receptor-JNK1 signalling. Cardiovasc Res, 2012. 95(3): p. 356-65. 100. Ren, S., B.G. Johnson, Y. Kida, C. Ip, K.C. Davidson, S.L. Lin, A. Kobayashi, R.A. Lang, A.K. Hadjantonakis, R.T. Moon, and J.S. Duffield, LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1. Proc Natl Acad Sci U S A, 2013. 110(4): p. 1440-5. 101. Gautheron, J., M. Vucur, F. Reisinger, D.V. Cardenas, C. Roderburg, C. Koppe, K. Kreggenwinkel, A.T. Schneider, M. Bartneck, U.P. Neumann, A. Canbay, H.L. Reeves, M. Luedde, F. Tacke, C. Trautwein, M. Heikenwalder, and T. Luedde, A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis. EMBO Mol Med, 2014. 6(8): p. 1062-74. 102. Wang, C., L. Deng, M. Hong, G.R. Akkaraju, J. Inoue, and Z.J. Chen, TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature, 2001. 412(6844): p. 346-51. 103. Dai, L., C. Aye Thu, X.Y. Liu, J. Xi, and P.C. Cheung, TAK1, more than just innate immunity. IUBMB Life, 2012. 64(10): p. 825-34. 104. Ma, F.Y., G.H. Tesch, and D.J. Nikolic-Paterson, ASK1/p38 signaling in renal tubular epithelial cells promotes renal fibrosis in the mouse obstructed kidney. Am J Physiol Renal Physiol, 2014. 307(11): p. F1263-73. 88 105. van der Velden, J.L., Y. Ye, J.D. Nolin, S.M. Hoffman, D.G. Chapman, K.G. Lahue, S. Abdalla, P. Chen, Y. Liu, B. Bennett, N. Khalil, D. Sutherland, W. Smith, G. Horan, M. Assaf, Z. Horowitz, R. Chopra, R.M. Stevens, M. Palmisano, Y.M. Janssen-Heininger, and P.H. Schafer, JNK inhibition reduces lung remodeling and pulmonary fibrotic systemic markers. Clin Transl Med, 2016. 5(1): p. 36. 106. Vives, E., P. Brodin, and B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem, 1997. 272(25): p. 16010-7. 107. Khalil, N., Y.D. Xu, R. O'Connor, and V. Duronio, Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem, 2005. 280(52): p. 43000-9. 108. Alcorn, J.F., J. van der Velden, A.L. Brown, B. McElhinney, C.G. Irvin, and Y.M. Janssen-Heininger, c-Jun N-terminal kinase 1 is required for the development of pulmonary fibrosis. Am J Respir Cell Mol Biol, 2009. 40(4): p. 422-32. 109. Wu, Z., L. Yang, L. Cai, M. Zhang, X. Cheng, X. Yang, and J. Xu, Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre transgenic mouse. Respir Res, 2007. 8: p. 1. 110. Eferl, R., P. Hasselblatt, M. Rath, H. Popper, R. Zenz, V. Komnenovic, M.H. Idarraga, L. Kenner, and E.F. Wagner, Development of pulmonary fibrosis through a pathway involving the transcription factor Fra-2/AP-1. Proc Natl Acad Sci U S A, 2008. 105(30): p. 10525-30. 111. Hayashida, T., A.C. Poncelet, S.C. Hubchak, and H.W. Schnaper, TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression. Kidney Int, 1999. 56(5): p. 1710-20. 112. Hocevar, B.A., C. Prunier, and P.H. Howe, Disabled-2 (Dab2) mediates transforming growth factor beta (TGFbeta)-stimulated fibronectin synthesis through TGFbeta-activated kinase 1 and activation of the JNK pathway. J Biol Chem, 2005. 280(27): p. 25920-7. 113. Itoh, S., M. Thorikay, M. Kowanetz, A. Moustakas, F. Itoh, C.H. Heldin, and P. ten Dijke, Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses. J Biol Chem, 2003. 278(6): p. 3751-61. 114. Utsugi, M., K. Dobashi, T. Ishizuka, K. Masubuchi, Y. Shimizu, T. Nakazawa, and M. Mori, C-Jun-NH2-terminal kinase mediates expression of connective tissue growth factor induced by transforming growth factor-beta1 in human lung fibroblasts. Am J Respir Cell Mol Biol, 2003. 28(6): p. 754-61. 115. Kim, K.K., M.C. Kugler, P.J. Wolters, L. Robillard, M.G. Galvez, A.N. Brumwell, D. Sheppard, and H.A. Chapman, Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A, 2006. 103(35): p. 13180-5. 116. McMillan, S.J., G. Xanthou, and C.M. Lloyd, Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the Smad signaling pathway. J Immunol, 2005. 174(9): p. 5774-80. 89 117. Yue, J., B. Sun, G. Liu, and K.M. Mulder, Requirement of TGF-beta receptor-dependent activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (Sapks) for TGF-beta up-regulation of the urokinase-type plasminogen activator receptor. J Cell Physiol, 2004. 199(2): p. 284-92. 118. King, T.E., Jr., A. Pardo, and M. Selman, Idiopathic pulmonary fibrosis. Lancet, 2011. 378(9807): p. 1949-61. 119. Meltzer, E.B. and P.W. Noble, Idiopathic pulmonary fibrosis. Orphanet J Rare Dis, 2008. 3: p. 8. 120. Nathan, S.D., O.A. Shlobin, N. Weir, S. Ahmad, J.M. Kaldjob, E. Battle, M.J. Sheridan, and R.M. du Bois, Long-term course and prognosis of idiopathic pulmonary fibrosis in the new millennium. Chest, 2011. 140(1): p. 221-229. 121. Selman, M., T.E. King, A. Pardo, S. American Thoracic, S. European Respiratory, and P. American College of Chest, Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med, 2001. 134(2): p. 136-51. 122. Azuma, A., T. Nukiwa, E. Tsuboi, M. Suga, S. Abe, K. Nakata, Y. Taguchi, S. Nagai, H. Itoh, M. Ohi, A. Sato, and S. Kudoh, Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 2005. 171(9): p. 1040-7. 123. Schaefer, C.J., D.W. Ruhrmund, L. Pan, S.D. Seiwert, and K. Kossen, Antifibrotic activities of pirfenidone in animal models. Eur Respir Rev, 2011. 20(120): p. 85-97. 124. Inomata, M., K. Kamio, A. Azuma, K. Matsuda, N. Kokuho, Y. Miura, H. Hayashi, T. Nei, K. Fujita, Y. Saito, and A. Gemma, Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis. Respir Res, 2014. 15: p. 16. 125. Staab-Weijnitz, C.A., I.E. Fernandez, L. Knuppel, J. Maul, K. Heinzelmann, B.M. Juan-Guardela, E. Hennen, G. Preissler, H. Winter, C. Neurohr, R. Hatz, M. Lindner, J. Behr, N. Kaminski, and O. Eickelberg, FK506-Binding Protein 10, a Potential Novel Drug Target for Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med, 2015. 192(4): p. 455-67. 126. Lin, M., P. Jackson, A.M. Tester, E. Diaconu, C.M. Overall, J.E. Blalock, and E. Pearlman, Matrix metalloproteinase-8 facilitates neutrophil migration through the corneal stromal matrix by collagen degradation and production of the chemotactic peptide Pro-Gly-Pro. Am J Pathol, 2008. 173(1): p. 144-53. 127. Ozaki, T., H. Hayashi, K. Tani, F. Ogushi, S. Yasuoka, and T. Ogura, Neutrophil chemotactic factors in the respiratory tract of patients with chronic airway diseases or idiopathic pulmonary fibrosis. Am Rev Respir Dis, 1992. 145(1): p. 85-91. 128. Dabrowski, J., A. Stachow, S. Jablonska, and M. Walski, [Ultrastructural and biochemical study of collagen in scleroderma]. Reumatologia, 1978. 16(1): p. 103-5. 129. McAnulty, R.J. and G.J. Laurent, Collagen synthesis and degradation in vivo. Evidence for rapid rates of collagen turnover with extensive degradation of newly synthesized collagen in tissues of the adult rat. Coll Relat Res, 1987. 7(2): p. 93-104. 130. Traub, W., A. Yonath, and D.M. Segal, On the molecular structure of collagen. Nature, 1969. 221(5184): p. 914-7. 131. Beeh, K.M. and J. Beier, Handle with care: targeting neutrophils in chronic obstructive pulmonary disease and severe asthma? Clin Exp Allergy, 2006. 36(2): p. 142-57. 90 132. de Kruijf, P., H.D. Lim, S.A. Overbeek, G.J. Zaman, A.D. Kraneveld, G. Folkerts, R. Leurs, and M.J. Smit, The collagen-breakdown product N-acetyl-Proline-Glycine-Proline (N-alpha-PGP) does not interact directly with human CXCR1 and CXCR2. Eur J Pharmacol, 2010. 643(1): p. 29-33. 133. Wells, J.M., P.J. O'Reilly, T. Szul, D.I. Sullivan, G. Handley, C. Garrett, C.M. McNicholas, M.A. Roda, B.E. Miller, R. Tal-Singer, A. Gaggar, S.I. Rennard, P.L. Jackson, and J.E. Blalock, An aberrant leukotriene A4 hydrolase-proline-glycine-proline pathway in the pathogenesis of chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 2014. 190(1): p. 51-61. 134. Gaggar, A., A. Hector, P.E. Bratcher, M.A. Mall, M. Griese, and D. Hartl, The role of matrix metalloproteinases in cystic fibrosis lung disease. Eur Respir J, 2011. 38(3): p. 721-7. 135. Corbel, M., S. Caulet-Maugendre, N. Germain, S. Molet, V. Lagente, and E. Boichot, Inhibition of bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor batimastat. J Pathol, 2001. 193(4): p. 538-45. 136. Obayashi, Y., I. Yamadori, J. Fujita, T. Yoshinouchi, N. Ueda, and J. Takahara, The role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis. Chest, 1997. 112(5): p. 1338-43. 137. van der Velden, J.L., S.M. Hoffman, J.F. Alcorn, J.E. Tully, D.G. Chapman, K.G. Lahue, A.S. Guala, L.K. Lundblad, M. Aliyeva, N. Daphtary, C.G. Irvin, and Y.M. Janssen-Heininger, Absence of c-Jun NH2-terminal kinase 1 protects against house dust mite-induced pulmonary remodeling but not airway hyperresponsiveness and inflammation. Am J Physiol Lung Cell Mol Physiol, 2014. 306(9): p. L866-75. 138. Shi, K., J. Jiang, T. Ma, J. Xie, L. Duan, R. Chen, P. Song, Z. Yu, C. Liu, Q. Zhu, and J. Zheng, Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-beta, Smad3 and JAK-STAT pathway. Int J Clin Exp Med, 2014. 7(9): p. 2645-50. 139. Dik, W.A., M.A. Versnel, B.A. Naber, D.J. Janssen, A.H. van Kaam, and L.J. Zimmermann, Dexamethasone treatment does not inhibit fibroproliferation in chronic lung disease of prematurity. Eur Respir J, 2003. 21(5): p. 842-7. 140. Lacronique, J.G., S.I. Rennard, P.B. Bitterman, T. Ozaki, and R.G. Crystal, Alveolar macrophages in idiopathic pulmonary fibrosis have glucocorticoid receptors, but glucocorticoid therapy does not suppress alveolar macrophage release of fibronectin and alveolar macrophage derived growth factor. Am Rev Respir Dis, 1984. 130(3): p. 450-6. 141. Carter, N.J., Pirfenidone: in idiopathic pulmonary fibrosis. Drugs, 2011. 71(13): p. 1721-32. 142. O'Reilly, P.J., P.L. Jackson, J.M. Wells, M.T. Dransfield, P.D. Scanlon, and J.E. Blalock, Sputum PGP is reduced by azithromycin treatment in patients with COPD and correlates with exacerbations. BMJ Open, 2013. 3(12): p. e004140. 143. Pillai, S.G., D. Ge, G. Zhu, X. Kong, K.V. Shianna, A.C. Need, S. Feng, C.P. Hersh, P. Bakke, A. Gulsvik, A. Ruppert, K.C. Lodrup Carlsen, A. Roses, W. Anderson, S.I. Rennard, D.A. Lomas, E.K. Silverman, D.B. Goldstein, and I. Investigators, A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet, 2009. 5(3): p. e1000421. 144. Snelgrove, R.J., Leukotriene A4 hydrolase: an anti-inflammatory role for a proinflammatory enzyme. Thorax, 2011. 66(6): p. 550-1. 91 145. Lemjabbar, H., P. Gosset, E. Lechapt-Zalcman, M.L. Franco-Montoya, B. Wallaert, A. Harf, and C. Lafuma, Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment. Am J Respir Cell Mol Biol, 1999. 20(5): p. 903-13. 146. Fukuda, Y., M. Ishizaki, S. Kudoh, M. Kitaichi, and N. Yamanaka, Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases. Lab Invest, 1998. 78(6): p. 687-98. 147. Selman, M., V. Ruiz, S. Cabrera, L. Segura, R. Ramirez, R. Barrios, and A. Pardo, TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? Am J Physiol Lung Cell Mol Physiol, 2000. 279(3): p. L562-74. 148. Hopkins, H., T. Stull, S.G. Von Essen, R.A. Robbins, and S.I. Rennard, Neutrophil chemotactic factors in bacterial pneumonia. Chest, 1989. 95(5): p. 1021-7. 149. Hunninghake, G.W., J.I. Gallin, and A.S. Fauci, Immunologic reactivity of the lung: the in vivo and in vitro generation of a neutrophil chemotactic factor by alveolar macrophages. Am Rev Respir Dis, 1978. 117(1): p. 15-23. 150. Crooks, S.W., D.L. Bayley, S.L. Hill, and R.A. Stockley, Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4. Eur Respir J, 2000. 15(2): p. 274-80. 151. Weston, C.R. and R.J. Davis, The JNK signal transduction pathway. Curr Opin Genet Dev, 2002. 12(1): p. 14-21. 152. Yoshida, K., K. Kuwano, N. Hagimoto, K. Watanabe, T. Matsuba, M. Fujita, I. Inoshima, and N. Hara, MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis. J Pathol, 2002. 198(3): p. 388-96. 153. Hall, M.C., D.A. Young, J.G. Waters, A.D. Rowan, A. Chantry, D.R. Edwards, and I.M. Clark, The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1. J Biol Chem, 2003. 278(12): p. 10304-13.
Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Reduction of neutrophilic inflammation in idiopathic pulmonary fibrosis via metabolism of n-alpha-pgp by leukotriene A4 hydrolase
Text