UNIVERSITY OF CALGARY. The Role of Neutrophil MMP-9 in the Development of Fibrosis in Hypersensitivity Pneumonitis. Abrar Mohammad Alansary A THESIS

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1 UNIVERSITY OF CALGARY The Role of Neutrophil MMP-9 in the Development of Fibrosis in Hypersensitivity Pneumonitis by Abrar Mohammad Alansary A THESIS SUBMITED TO THE FACULTY OF GRADUATE STUDIES IN PARTICULAR FULFILMENT OF THE REQUIRMENTS FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF IMMUNOLOGY CALGARY, ALBERTA FEBRUARY, 2013 Abrar Mohammad Alansary 2013

2 Abstract Hypersensitivity pneumonitis (HP) is a pulmonary disorder caused by repeated inhalation of a variety of organic antigens, which can lead to chronic fibrosis and respiratory failure. In our laboratory we use a model of experimental HP (EHP) involving repeated exposure of mice by oropharyngeal aspiration to Saccharopolyspora Rectivirgula antigen (SR Ag), the main causative agent of farmer's Lung. In EHP, pulmonary fibrosis develops after three weeks of SR Ag exposure and continues increasing up to week five. Previously it has been shown in our laboratory that neutrophils are critical for the development of fibrosis in this model. My hypothesis is that neutrophils and their products, specifically MMP-9, play a role in the development of fibrosis in HP. In this work, I demonstrated that active MMP-9, derived from neutrophils, is present in fibrotic areas of the lung in EHP. GM6001, a broad-spectrum inhibitor of MMP-1, -2, -3, -8, and -9, administrated intraperitoneally to mice at week two and three in a three week SR Ag protocol showed reduced neutrophil infiltration to the lung but had no effect on fibrosis. These results were difficult to interpret due to the multiple MMPs inhibited, so in order to specifically examine the role of MMP-9, we exposed MMP-9-deficient mice to SR Ag for five weeks and compared their response to that of wild type C57BL/6 mice. MMP-9-deficient mice showed no difference in the number of neutrophils in bronchoalveolar lavage (BAL) fluid compared to C57BL/6 mice, but fibrosis was significantly attenuated. To extend our studies, we used in vitro cell culture of human cells. In vitro human lung fibroblasts (HLF) were treated with neutrophil supernatant containing active MMP-9 for 48 h. We could not demonstrate any effect on fibroblast activation/differentiation as assessed by α- smooth muscle actin (α-sma) expression in fibroblasts. However, these results are inconclusive ii

3 as we have needed higher concentrations of MMP-9, longer treatment times or other co-stimuli. In addition, other indices of fibroblast activation were not examined. In conclusion, the work presented in this thesis provides novel insight into the significant role of neutrophil MMP-9 in the development of fibrosis in EHP. iii

4 Acknowledgements First of all, I would like to thank my graduate supervisors Dr. Margaret Kelly and Dr. David Proud for the support, supervision, and guidance throughout this project. I have learned lots of lessons and techniques. I would like to express my deepest appreciation to my committee members Dr. Wee Yong and Dr. Donna-Marie McCafferty for their time and guidance. I would also like to acknowledge members of Dr. Margaret Kelly s and Dr. David Proud s laboratories: Carol, Kim, Cora, Suzanne, Shahina, and Silvia, for the advice, help, and support. I would also like to thank all the training and help provided to me by the Smriti Agrawal from Dr. Wee Yong s laboratory and Janet Ngu from Dr. Paul Fedak s laboratory. In addition, I thank Kuwait University for supporting me during this project. Finally, I would like to express my appreciation to my family and parents who have always been there for me and for all the unconditional love. Thank you for being supportive. Also, to my husband and lovely daughters, thank you for your patience. iv

5 Table of Contents Abstract. ii Acknowledgments.... iv Table of contents... v List of Figures and Illustrations.. vii List of symbols, Abbreviations and Nomenclature.... ix CHAPTER ONE: INTRODUCTION Hypersensitivity pneumonitis Pathology of Hypersensitivity pneumonitis Pathogenesis of hypersensitivity pneumonitis Role of neutrophils in hypersensitivity pneumonitis Pulmonary fibrosis Matrix metalloproteinases (MMPs) Matrix metalloproteinases (MMPs) Matrix metalloproteinase Experimental hypersensitivity pneumonitis (EHP) Overall objective Hypothesis Aims CHAPTER TWO: MATERIALS AND METHODS Material and Reagents Methods Animals Experimental hypersensitivity pneumonitis (EHP) model Bronchial alveolar lavage (BAL) Hematoxylin and eosin (H&E) satin Soluble collagen levels (Sircol Assay) Immunohistochemistry to detect intracellular MMP Immunofluorescence to detect intracellular MMP-9 and MPO In Situ zymography Culture of human lung fibroblasts Human lung fibroblast stimulation with neutrophil supernatant Cell viability assay Measurement of MMP-9 in neutrophil supernatant Measurement of active MMP-9 in neutrophils supernatant RNA isolation, DNase treatment and Real time PCR RNase isolation DNase treatment cdna RT-PCR Detection of intracellular α-sma in cultural human fibroblasts by in-cell western Statistical analysis.. 46 v

6 CHAPTER THREE: PUMONARY FIBROSIS IS ATTENUATED IN THE ABSENCE OF MMP-9 IN EHP BUT NEUTROPHIL INFLUX IS INDEPENDENT OF MMP Results Active MMP-9 in the fibrotic areas of the lung in EHP Neutrophils influx to the lung is reduced in mice treated with GM GM6001had no effect on pulmonary fibrosis induced by SR antigen MMP-9 deficiency had no effect on inflammatory influx induced by SR antigen Pulmonary fibrosis induced by SR antigen exposure was significantly reduced in MMP-9-deficient mice compared to C57BL/6 mice Discussion.. 62 CHAPTER FOUR: NEUTROPHIL SUPERNATANT CONTAINING MMP-9 DOES NOT INDUCE THE EXPRESSION OF α-smooth MUSCLE ACTIN (α-sma) IN HUMAN LUNG FIBROBLASTS Hypothesis Results Establishing optimal culture medium Neutrophil supernatants induced expression of collagen I mrna in human lung fibroblasts Neutrophil supernatants induced expression of α-sma mrna in human lung fibroblasts A pooled supernatant from stimulated neutrophils contain active MMP Neutrophil supernatant does not induce the expression of α-sma in human lung Fibroblasts Discussion.. 78 CHAPTER FIVE: GENERAL DISCUSSION AND FUTURE WORK General Discussion and Future Work Limitations. 91 REFERENCES 93 vi

7 Lists of Figures and Illustrations Figure 1.1 Diagrammatic representation of the lung interstitium. 11 Figure 1.2 Disruptions in normal wound healing contributes to fibrosis development in the lung. 15 Figure 1.3 Domain structure of MMP-9 19 Figure1.4 Chronic SR Ag exposure in WT for three weeks. 24 Figure 1.5 Figure 1.6 Soluble collagen from the right lungs (RL) in chronic SR Ag exposure in WT mice. 26 Anti-neutrophil (anti-gr-1) antibody administrated to mice (IP) during last 2 weeks of 3 weeks SR Ag exposure. 27 Figure 2.1 Chronic SR antigen administration protocol and IP GM6001 for the last two weeks. 34 Figure 2.2 Chronic SR antigen administration protocol. 36 Figure 3.1 Gelatinase activity in fibrotic lung in mice treated with SR Ag for three weeks. 49 Figure 3.2 MMP-9 produced by neutrophils in lung sections from EHP with chronic SR Ag exposure. 51 Figure 3.3 Inflammatory cell response in the BAL fluid of C57B/6 mice exposed to SR antigen for three weeks and IP GM6001 for the last two weeks and vehicle. 54 Figure 3.4 Murine lungs after chronic SR Ag exposure for three weeks and IP GM6001 or vehicle for the last two weeks. 55 Figure 3.5 Fibrotic response of C57BL/6 exposed to SR Ag and GM Figure 3.6 Inflammatory cells response in the BAL fluid of C57BL/6 and MMP-9- vii

8 deficient mice exposed to SR antigen or PBS for five weeks. 59 Figure 3.7 Total soluble collagen from homogenized right lung in C57BL/6 and MMP-9-deficient mice after exposure to SR antigen or PBS for five weeks. 61 Figure 4.1 Neutrophils isolated form human blood under light microscopy. 68 Figure 4.2 Human lung fibroblasts viability in 10% serum and without serum DMEM medium. 69 Figure 4.3 Collagen I mrna expression in primary human lung fibroblasts 71 Figure 4.4 The expression of α-sma mrna in primary human lung fibroblasts 73 Figure 4.5 Activated neutrophil supernatant pool does not modify the expression of α- SMA in primary human lung fibroblasts. 77 viii

9 List of Symbols, Abbreviations, and Nomenclature Symbol Definition Degree α Alpha β Beta γ Gamma κ Kappa µ Micro Ag Antigen BAL Bronchoalveolar lavage BALF Bronchoalveolar lavage fluid CD Cluster of differentiation CTGF Connective tissue growth factor DMEM Dulbecco s modified Eagle medium DMSO Dimethyl sulfoxide ECM Extra cellular matrix EHP Experimental hypersensitivity pneumonitis ELISA Enzyme-linked immunosorbent assay EMT Epithelial mesenchymal transition FBS Fetal bovine serum GM-CSF Granulocyte-macrophage colony-stimulating factor Gro Growth related oncogene Gr-1 Granulocyte receptor-1 H&E Haematoxylin and eosin HBSS Hanks balanced salt solution HLF Human lung fibroblast HP Hypersensitivity pneumonitis HRP Horseradish peroxidase IFN Interferon IHC Immunohistochemistry IL Interleukin IP intraperitoneal IPF Idiopathic pulmonary fibrosis KO Knockout MMP Matrix metalloproteinase MPO Myeloperoxidase mrna Messenger RNA OD Optical density PBS Phosphate buffered saline PMN Polymorphonuclear PSR Picrosirius red RT Room temperature SMA smooth muscle actin SR Saccharopolyspora rectivirgula TIMP Tissue inhibitor matrix metalloproteinase TGF-β Transforming growth factor beta TNF Tumor necrosis factor ix

10 VEGF WT Vascular endothelial growth factor Wild type x

11 Chapter One: Introduction 1

12 1.1 Hypersensitivity pneumonitis Hypersensitivity pneumonitis (HP), also known as extrinsic allergic alveolitis, is a pulmonary disorder caused by repeated inhalation of variety of organic and inorganic antigens. Although various inciting antigens (Ags) may elicit HP, the pathogenesis of the disease that ensues is the same. The most common manifestations are known as Farmer s lung, Bird Fancier s, or Hot tub lung. A pulmonary inflammatory process ensues, which can lead to progressive fibrosis. Once fibrosis develops, 5-year mortality rate for patients has been reported to be as high as 60% (1). HP often affects patients in the prime of their productive years and can progress to permanent sequelae, with significant social and economic consequences. The incidence of HP, based on population-based studies has been estimated to be 1/100,000 of the general population, and up to 50/100,000 in farmers (2 4). There is evidence that HP is often under-diagnosed or misclassified, and new inciting Ags and environmental exposure are still being described (5). Pulmonary fibrosis in HP is an important predictor of mortality. Once fibrosis is present, it is often progressive, despite removal of the inciting Ag and treatment with corticosteroids, suggesting that the immune response has become autonomous with lung transplant being the only cure (5,6). The Canadian Centre for Occupational Health and Safety estimates that 2-10% of the Canadians farmworkers are affected with at least half developing permanent lung damage and giving up farming (6,7). 1.2 Pathology of hypersensitivity pneumonitis Particulate matter less than 3µm in diameter, inhaled into the distal bronchial tree and alveoli of the lung, are capable of inducing HP (8). The inciting Ags can be derived from fungal, bacterial, 2

13 or animal proteins, or reactive chemical sources. Saccharopolyspora rectivirgula is the actinomycete that induces farmer s lung and most studies of HP use Ags derived from this source, referred to here as SR (9). However, it still remains unclear which SR components are responsible for the disease. The cells that initially respond to SR Ag in vivo have not yet been identified, but SR can directly activate neutrophils, bronchial epithelial cells, and macrophages, and there is evidence that mast cells play an important role in experimental hypersensitivity pneumonitis (EHP) and HP itself (10 12). HP can be divided clinically into three forms: acute, subacute, and chronic stages (13). In the acute phase, chills, fever, sweating, cough, dyspnea, headache, and nausea develop after a heavy exposure to Ag. These symptoms peak 4-24 h after exposure, and completely resolve, usually within h upon removal from Ag exposure (8,14 16). The subacute form develops gradually upon continuous exposure to Ag over weeks or months (9). The subacute form is characterized by cough and progressive dyspnea (5). Most patients in the acute and subacute phase appear to respond well after complete cessation of exposure to the provoking Ag, although corticosteroid treatment is often needed (17). The chronic form of HP is defined by the presence of pulmonary fibrosis with honeycomb change in areas and is characterized by persistent cough, dyspnea, fatigue, and weight loss (7,9,10). Most studies of HP have focused on the inflammatory component of the disease and have largely ignored the fibrotic manifestations. A generally held paradigm is that Th1 cytokines suppress, while Th2 cytokines promote, fibrosis (18). However, there is conflicting evidence as to whether a dominant lymphocyte phenotype is present in HP. There is some evidence for both a Th1 (high interferon gamma (IFN-γ)), or Th2 (high IL-4) phenotype in animal models, and in patients with HP (19,20). The CD4/CD8 T cell ratio in the bronchoalveolar lavage (BAL) fluid increases with 3

14 the development of pulmonary fibrosis (21). The uncertain role for Th1 or Th2 lymphocytes in EHP could be explained by a major role of the more recently described Th17 pathway (22,23). IL-17A is a major orchestrator of sustained neutrophilic mobilization to the lung through the induction of cytokines (IL-1β, IL-6, and TNF-α) and chemokines (CXCL8 and CXCL1) from lung epithelial cells, endothelial cells, macrophages, and fibroblasts (24 27). IL-17A has been shown to play a significant role in EHP, with IL-17A being required for the development of inflammation in EHP (22). Th17 cells and IL-17 levels are increased in the lungs of mice treated exposed to SR, compared with the control mice (22,23). IL-17ra-/- mice showed a delay in the clearance of the pathogen, increased lung inflammation, collagen deposition, and fibrosis, suggesting a regulatory role of these cell subsets (5,28). 1.3 Pathogenesis of hypersensitivity pneumonitis The pathogenesis of HP is complex. The immune mechanisms underlying HP are poorly understood. In HP, inhaled Ag can bind to pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs) on innate immune cells and trigger PRR-signaling pathways. SR Ag is recognized by TLR2 but not TLR4, TLR5, or TLR7 (29). The SR Ag can directly activate protein kinase D (PKD1), mitogen activated protien kinases (MAPKs), and the transcription factor NF-κB. These events can lead to the expression and production of proinflammatory cytokines and chemokines in bronchial epithelial cells, as well as alveolar macrophages and neutrophils (12). The SR Ag also can directly activate alveolar macrophages, bronchial epithelial cells, and neutrophils, producing various cytokine and chemokines, such as IL-6, and - 23, IFN-γ, keratinocyte-derived chemokine (KC), macrophage inflammatory proteins 1α (MIP- 4

15 1α) and -2, monocyte chemotactic protein-1 (MCP-1), IP-10, and RANTES, that are critical for the recruitment of leukocytes and the development of Th17 response and HP (12). Farmer s lung disease is reported to be the consequence of a type III hypersensitivity reaction in combination with a type IV hypersensitivity reaction. There is more evidence for type IV hypersensitivity (cell-mediated immune reaction), such as the histology of lymphocytic interstitial infiltrate with granuloma formation and signs of macrophages and lymphocytes activation (30). Type III hypersensitivity reaction (humoral mechanism), predominates early in the development of HP, with antibodies being produced by plasma cells in response to the inciting Ag (31). These antibodies are usually of an IgG isotype, but may also be of IgM or IgA isotypes (16,31). HP is characterized by the proliferation of CD8 cytotoxic lymphocytes and production of IgG from plasma cells stimulated by CD4 T cells. In the acute phase, soluble Ags form immune complexes with circulating IgG that triggers complement activation cascade or Ab-specific FcR-signaling pathways (5,29,32,33). Activation of the complement cascade by Ag enhances vascular permeability through C3a, as well as recruitment and activation of macrophages and neutrophils by C5a (34 36). Activated alveolar macrophages secrete a variety of inflammatory mediators, including IL-8, a chemokine that plays a role in attracting neutrophils to the lung within hours after Ag inhalation (37,38). Migration of neutrophils to the site of inflammation into the lung can lead to direct damage of bronchiolar and alveolar epithelium by proteases, such as matrix metalloproteinases-9 (MMP-9), and by reactive oxygen species (ROS). Neutrophils activated by C5a release lysosomal enzymes that can damage the lung tissue (35). Although several observations, including the presence of antibodies against Ags in 40-64% of patients, support a role for type III immune complex hypersensitivity reactions in some cases, other aspects of HP remains unexplained (39,40). For instance, histological findings 5

16 do not reflect an immune-complex mediated disorder. The presence of precipitating antibodies to inciting Ags is only indicative of exposure and not the presence of the disease. Antibodies to Ags in HP cannot serve as a specific marker for the disease. HP can occur in the absence of antibodies; in which case cytotoxic delayed hypersensitivity involving CD8 T cell is required (41,42). Type IV hypersensitivity reactions are thought to predominate with continuous Ag exposure. In HP patients exposed to the inciting Ags, neutrophils predominate in the BAL fluid within the first 4-48 h, while monocytes and T lymphocytes compose the majority of the cells at later time points (30). Monocytes accumulate in the lung and mature into macrophages. Macrophages secrete chemokines and cytokines that first attract neutrophils to the site of inflammation. Activated pulmonary macrophages increase the expression of FCγRIII receptors on their surface and stimulation of these receptors lead to production of IL-1 and TNF-α that will cause fever and other acute phase reactions (16,32,41). IL-1 and TNF-α selectively stimulate Th1 cells to produce IFN-γ (43). Macrophages have been shown to secrete more IL-1 and TNF-α in HP patients compared to patients with idiopathic pulmonary fibrosis (IPF) (32). Activated macrophages also secrete chemokines, such as IL-8, MIP-1α, and RANTES (37,38,44). IL-12 and MIP-1α secreted from activated macrophages promote the differentiation of lymphocytes into a Th1 phenotype, which, in turn, produce (IFN-γ) that has been shown to be involved in the formation of granulomas in a mouse model of HP (45). In the subacute phase of HP, macrophages develop into multinucleated giant cell sand granulomas in the lung (41,46). The factors that regulate transformation of monocytes into multinucleated giant cells that make up granulomas remain unclear. Goblet cell metaplasia and peribronchiolar inflammation with aggregates of lymphocytes and plasma cells (bronchiole-associated lymphoid tissue (BALT)) 6

17 also develop during the subacute phase (47,48). In the chronic phase of HP, fibrosis develops in the subpleural and peribronchiolar areas of the lung with scattered multinucleated giant cells and poorly formed granulomas (19,47). Early collagen formation by activated fibroblasts occurs and the extracellular matrix (ECM) surrounding the granulomas becomes rich in the proteoglycan versican (49). Thickening of the alveolar basement membrane has been observed in patients with HP, and it is possible to find intra-alveolar clusters of loose connective tissue, called buds, attached to alveolar walls (50). These foci contain small numbers of macrophages, fibroblasts, and myofibroblast (51). Activated macrophages increase the expression of TGF-β, a potent stimulator of angiogenesis and fibrosis (52). Alternatively activated macrophages (M2) play a critical role in the pathogenesis of IPF, enhancing the activity of fibroblasts by providing profibrogenic factors, favouring cell growth, collagen production, and tissue repair (53,54). M2 macrophages trigger a viscous circle between alveolar macrophages and fibroblasts by releasing IL-1 receptor antagonist and CCL18, which promote collagen deposition and fibrotic progression of IPF (55). Patients with pulmonary fibrosis showed elevated levels of Th2 cytokines (e.g., IL- 4) which upregulate the production of chemokines (e.g., CCL17, CCL18 and CCL22), that are linked to alternative activation of macrophages. Increased expression of CD206 by alveolar macrophages, as a patho-mechanism in pulmonary fibrosis, suggest that pulmonary fibrosis is at least partially driven by M2/Th2 process (56). Mast cell numbers are increased in HP patients and they can also promote fibrosis (57). Most of the mast cells in HP have the characteristics of connective tissue type mast cell, rather than the mucosal type of mast cell, and being associated with fibrosis, might differ in function from mast cells activated in asthma (58). Natural killer (NK) cells are increased in HP patients and appear to exert a protective function (59). In a mouse model of HP, NK cell-depleted mice challenged with SR showed increased cellular recruitment 7

18 in the BAL fluid and increased fibrosis compared to control mice (60). Neutrophils also contribute to the development of fibrosis. Neutrophil numbers positively correlated with the degree of fibrosis as well as with increased mortality (61). Regulatory cytokines, such as IL-10, are also secreted to dampen down the inflammatory responses (62,63). The chronic phase seems to be Th2-biased immune response (64). Much of the details about the immunophathogensis of HP are still obscure. Most of the information about these mechanisms has been derived from animal models and from biopsies from patients. Although varying degrees of inflammation can accompany fibrosis, the lymphoplasmacytic interstitial infiltrate seen in HP is usually small or doesn t exist, and granulomas are usually absent in non-hp-associated cases (47). Despite a typical finding of HP on histology, a clear history of exposure can only be obtained in 37-64% of the patients (39,65). Therefore, in many instances the diagnosis is considered a challenge, and depends on the interpretation of the findings at biopsy (47). The development of the disease depends on the amount, nature, intensity, and duration of the exposure to the inciting Ag. It can also depend on the susceptibility of the host immune response, the site of interaction in the respiratory system, and the level of dysregulation of the cellular and humoral immune response over time (41). It involves both genetic and/or environmental risk factors, such as the exposure to HP Ags. Patients with HP have a greater production of the cytokine TNF-α, and express the TNF A2 allele, a genotype associated with high production of TNF-α (66). Tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) has also been shown to decrease the susceptibility of patients to HP (67). Moreover, major histocompatibility complex (MHC) polymorphisms has been shown to contribute to the development of HP (5). Patients with HP have an increase in the frequency of HLA-DRB1*1305 and HLA-DQB1*0501 alleles and a decrease in the frequency of the HLA-BRB1*0802 allele (68). 8

19 1.4 Role of neutrophils in hypersensitivity pneumonitis Relatively few studies have been conducted on the role of neutrophils in pulmonary fibrosis and only one of these examined chronic HP, showing that neutrophil numbers in the lung positively correlated with the degree of fibrosis in patients with hypersensitivity pneumonitis as well as with increased mortality (61). Neutrophils are regarded as critical pulmonary innate immune cells as they are rapidly recruited from the blood into the lung after injury (69,70). In a study of EHP, neutrophils amplified the immune response by releasing IFN-γ, a critical factor for the formation of granulomas in mice exposed to SR Ag (70). Neutrophils generate a wide array of ROS, proteases, including neutrophil elastase (NE) and matrix metalloproteinases-8 (MMP-8) and -9, and antimicrobial peptides. Together, these mediators, promote endothelial and epithelial injury, which are implicated in triggering a repair response with fibrosis (71,72). Peptide fragments from human elastin degraded by neutrophil elastase cause chemotaxis of human blood monocytes and fibroblasts, and cause neutrophil accumulation at the site of injury (91). ROS also activate MMP-9, a pro-fibrotic protease described previously. Neutrophils also secrete hepatocyte growth factor (HGF), shown to be elevated in patients with pulmonary fibrosis and can promote angiogenesis by producing vascular endothelial growth factor (VEGF) (74,75). 1.5 Pulmonary fibrosis Pulmonary fibrosis is characterized by epithelial and endothelial damage, inflammation characterized by neutrophil and lymphocyte infiltration, and increased deposition of ECM proteins, including collagen (76,77). Lung function is sensitive to chronic fibrosis due to disruption of gas exchange, which lead to a high mortality (77 79). The alveolar-capillary barrier is composed of epithelial cells, endothelial cells, and their respective basement membranes. The 9

20 two basement membranes fuse, and ordinarily there is a very little interstitium present in the alveolar walls (Figure 1.1). 10

21 A Type II epithelium Capillary Interstitium Basement membrane Endothelial cells Type I epithelium B Type II epithelium Myofibroblasts Collagen deposition Endothelial cells Capillary Interstitium Basement membrane Type I epithelium Figure 1.1 Diagrammatic representation of the lung interstitium. The lung interstitium in normal lung (A) and in fibrotic lung (B). 11

22 The normal repair process following lung injury proceeds in four distinct stages; a clotting/coagulation phase, an inflammatory phase, a fibroblast migration/proliferation phase, and a remodelling phase that restores the tissue architecture (Figure 1.2) (79). In the clotting/coagulation phase, platelets aggregate to prevent blood and fluid loss and degranulate, promoting blood vessel dilation and increasing recruitment of inflammatory cells, such as neutrophils, macrophages, eosinophils, and lymphocytes to the site of injury (76,80). Neutrophils are the dominant inflammatory cells recruited to the site of injury in the early stages, followed by monocytes that differentiate into macrophages (80). Inflammatory cells produce a variety of cytokines and chemokines that amplify the response, and trigger activation and recruitment of fibroblasts to the site of injury (79). During the remodelling stage, myofibroblasts secrete ECM, deposit collagen, and promote wound contraction; while epithelial and endothelial cells regenerate the damaged tissue (79). Fibroblasts can also activate TGF-β, a central profibrotic cytokine and can attract inflammatory cells (81). Fibroblasts may contribute to neutrophil accumulation in the lung during inflammation by enhancing their survival (25). They can release IL-8, a neutrophil chemoattractant, in response to injury. Eventually the scar is resorbed, the myofibroblasts undergo apoptosis and the tissue is restored to its original state. Chronic fibrosis is believed to be due to an abnormal repair process following injury and it is controversial whether ongoing inflammation is required or if the process become autonomous (82,83). The understanding of the complex mechanisms involved in the pathogenesis of fibrosis continues to evolve. The initial hypothesis of fibrosis development, suggest that chronic inflammation was the underlying cause of pulmonary fibrosis. However, this hypothesis was subsequently called into question as the measures of tissue inflammation correlated poorly with the severity or outcome of pulmonary fibrosis. Moreover, the use of immunosuppressive drugs 12

23 have been unhelpful for pulmonary fibrosis, and may in fact be harmful (84,85). Several concepts have led to the development of modified and alternative hypotheses. One competing hypothesis proposed that the pathogenesis of fibrosis is the consequence of epithelial injury followed by abnormal wound healing. Following alveolar-capillary barrier injury a normal repair process begins, and the epithelium and endothelium are re-established to their normal spatial orientation on the basement membrane. However, in some instances, the loss of alveolarcapillary barrier basement membrane integrity may lead to fibrosis rather than re-establishing normal lung architecture. If the alveolar-capillary barrier is replaced by scar tissue, with a loss of the alveolar structure and persistent inflammation, this can lead to loss of the basement membrane integrity and the alveolus will collapse and lose its structure (86,87). Failure of reepithelialization and reendothelialization also will destroy the integrity of the basement membrane and lead to fibrosis. Furthermore, TGF-β is necessary, but not sufficient, to promote sustained fibrosis. Overexpression of TGF-β led to fibrosis in the peribronchiolar region of the lung with extension to the adjacent lung parenchyma, but when TGF-β production was turned off after peribronchiolar fibrosis had developed, the fibrosis was completely reabsorbed with the return to normal lung architecture (88). Moreover, persistent injury and/or irritant are necessary for the propagation of fibrosis. The alternative mechanism of fibrosis is that tissue injury with the presence of TGF-β induce epithelial cells to undergo transition to a mesenchymal phenotype, the fibroblasts/myofibroblasts that develop subsequently contribute to fibroblast proliferation, a process called epithelial mesenchymal transition (EMT) (89,90). EMT processes and bone marrow derived fibrocytes are cellular mechanisms in the regulation of fibrosis. The classical concept is that injury can activate resident fibroblasts to differentiate into myofibroblasts that express constituents of the ECM. Another theory is that circulating fibrocytes from the bone 13

24 marrow are mesenchymal progenitor cells that home into the site of injury, and differentiates into fibroblasts/myofibroblasts that contribute to the generation of ECM during fibroblast proliferation (91,92). Alveolar type II pneumocytes can serve as progenitor cells for epithelial repair, but in response to TGF-β they can differentiate to fibroblasts/myofibroblasts with the generation of ECM (93). In our model of EHP, persistent inflammation with the disruption of the basement membrane integrity and increase in the ECM deposition may be the cause of pulmonary fibrosis. However, further studies are needed to understand the pathogenesis of fibrosis. 14

25 Injury Inflammatory mediators IL-8 Gro-α ENA-78 Neutrophil Lymphocyte Epithelial cell Platelet activation Macrophage IL-1β TGF-β TNF-α IL-13 Repair and resolution ECM Myofibroblast Resident fibroblast BM fibrocytes EMT Smooth muscle Pericytes Fibrosis Figure 1.2 Disruptions in normal wound healing contributes to fibrosis development in the lung. At the site of injury, epithelial cells release inflammatory mediators that trigger platelet activation and blood clot formation. Inflammatory cells (e.g., neutrophils, macrophages, and T cells) are recruited, and when activated they secrete profibrotic cytokines (e.g., IL-1β, TNF, IL- 13, and TGF-β). Activated myofibroblasts release ECM. In the final remodelling and resolution phase, activated myofibroblasts promote wound contraction and repair. However, if the repair process is dysregulated at any stage, or the lung-damaging stimulus persists, fibrosis can develop. BM= bone marrow. Adapted from (Wynn TA. Integrating mechanisms of pulmonary fibrosis. J. Exp. Med. 2011; 208(7): ) (79). 15

26 1.6 Matrix metalloproteinases (MMPs) Matrix metalloproteinases MMPs belong to the metzincin superfamily of metalloproteinases, together with astacins, a protein with a disintegrin and metalloproteinase domain (ADAMs), and ADAM with a thrombospondin-like motifs (ADAM-TS) proteases (94). The MMP family of enzymes compromises 25 related, but distinct, vertebrate gene products (95). MMPs are either secreted or anchored to the cell surface. Based on their structural and functional characteristics, MMPs have been classified into six different subgroups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs (96). With the exception of MMP-7, -23, -26, MMPs are composed of a conserved domain structure comprising a pro-domain, catalytic domain, hinge region, and hemopexin domain (95,97). The pro-domain contains a conserved cysteine residue in the sequence PRCXXPD (where X is any amino acid) that coordinates with zinc in the active-site to inhibit catalysis (95,97). The catalytic domain contains a zinc ion (Zn 2+ ) in the active site that is ligated to three conserved histidine residues in the sequence HEXXHXXGXXH (95,97,98). MMPs are synthesized as a pro-enzyme (or zymogen) and converted to active proteinases by proteolysis of the pro-domain or modification of the cysteine thiol group in the pro-domain (95). MMPs are activated when the pro-domain is destabilized or removed and the active site becomes available to cleave substrates (97). The activity of MMPs is regulated at the levels of gene expression, compartmentalization, pro-enzyme activation and substrate availability (95). MMPs are essential for repair and remodelling after injury. Imbalance between MMPs and their respective TIMPs can result in pulmonary fibrosis (99,100). MMPs are thought to play a significant role in the pathogenesis of pulmonary fibrosis. Although the exact mechanisms by which MMPs contribute to fibrosis are not well characterized, they are 16

27 thought to include combinations of cleavage of matrix proteins, activation of cytokines and growth factors, and effects on cell surface receptors (94). Expression of MMPs is upregulated in lung fibrosis, indicating a potential role of MMPs in the dynamic nature of fibrosis and remodelling within the lung (78). MMPs associated with fibrosis, include MMP-2, -3, -7, and -9 (98, ) Matrix metalloproteinase-9 MMP-9 (type IV collagenase or gelatinase B) is a member of a subgroup of MMPs that contain three fibronectin type II-like repeats within their catalytic domain, resulting in a higher binding affinity to gelatin and elastin (78). It also contains a type V collagen like domain that is heavily glycosylated and has been suggested to have an effect on substrate specificity (Figure 1.3) (98). The MMP-9 gene is located on human chromosome 20q , a region that has been associated with bronchial hyperresponsiveness (103). In neutrophils, MMP-9 is stored in the tertiary granules, and is secreted upon stimulation of neutrophils during inflammation, amplifying tissue damage (61,104). MMP-9 is produced in response to various forms of stimulation, by neutrophils, bronchial epithelial cells, Clara cells, alveolar type II cells, smooth muscle cells, endothelial cells, and fibroblasts (98). Proinflammatory cytokines, such as TNF-α and IL-1β stimulate the release of MMP-9 in many cell types (105). MMP-9 pro-enzyme is activated by ROS and by proteases, such as MMP-3 and MMP-2 (95,97,98,106). MMP-9 activity is inhibited by α2-microglobulin and by TIMP-1 (98). MMP-9 plays a role in cell migration and invasion. It facilitates the migration of Clara cells and other bronchiolar cells into the regions of alveolar injury (107). MMP-9, through the degradation 17

28 of the basement membrane and ECM, facilitates fibroblast invasion of the alveolar space, inhibits epithelial regeneration, and activates TGF-β (108,109). Secreted TGF-β is maintained as a latent complex where it non-covalently interacts with a latency-associated peptide (LAP). The latent form of TGF-β can be sequestered within the ECM by covalent attachment between LAP and latent TGF-β binding protein (LTBP). TGF-β activity is regulated by proteolytic release of the large latent complex from the ECM and the dissociation of LAP from TGF-β (110). MMP-9 can directly activate TGF-β via the cleavage of LAP (111). The soluble form of LTBP can also be cleaved via MMP-9 (112). MMP-9 can also facilitates the recruitment of inflammatory cells such as macrophages, mast cells, neutrophils, and lymphocytes (113,114). MMP-9 facilitate neutrophils recruitment to the site of injury through the cleavage of six-amino acid peptides from IL-8, increasing the chemoattractant activity for neutrophils ten-fold (115,116). It can also cleave cytokines including Gro-α, ENA-78, CXCL6, IL-1β and CXCL11 (117,118). MMP-9 induces the release of ECM bound pro-angiogenic factors, including VEGF and TNF-α (119). In addition, MMP-9 has been shown to plays a role in EMT. Enhanced expression of MMP-9 cooperate with the transcription factor Snail to facilitate EMT through the loss of E- cadherin (120). 18

29 A B C D E F Figure 1.3 Domain structure of MMP-9. MMP-9 has an amino(n)-terminal signal anchor (A); a pro-domain (B); a catalytic domain with three fibronectin type II-like repeats (C); a hinge region (D); a type-v-collagen-like domain (E); and a Hemopexin-like domain (F). Adapted from (95). 19

30 MMP-9 levels are increased in several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and IPF (98). MMP-9 is elevated in the BAL fluid of IPF patients, consistent with the presence of increased numbers of neutrophils (113,121,122). Furthermore, MMP-9 levels in BAL fluid in patients with cystic fibrosis were significantly higher compared to control (123). Neutrophils containing MMP-9 are increased in the lungs of HP patients, and correlate with the degree of fibrosis (61). MMP-9 has been found in myofibroblasts associated with fibroblastic foci in the lungs of IPF patients, and MMP-9 gene and protein expression is elevated in both human and experimental lung fibrosis (121, ). MMP-9 inhibits the rate of wound closure in the trachea and the inflammatory response is delayed in MMP-9-deficient mice (126). In bleomycin-induced pulmonary fibrosis, increased gelatinase activity, associated with the disruption of alveolar epithelial basement membrane, has been reported (127). Moreover, in a bleomycin-induced pulmonary fibrosis model of guinea pigs, neutrophils and MMP-9 increased in the BAL fluid after cigarette-smoke exposure, suggesting that tobacco smoke creates a fibrotic response in this model (128). 1.7 Experimental hypersensitivity pneumonitis (EHP) Animal models of pulmonary fibrosis have provided important insights into the pathophysiology of human disorders and have been used as an initial model to test the safety and efficacy of new therapies (129). Animal models of pulmonary fibrosis can be induced by several methods, including bleomycin, fluorescein isothiocyanate (FITC), irradiation, silica, and viral vectors (130). The bleomycin murine model of pulmonary fibrosis is well characterized model in use today, but its usefulness is limited by dose-dependent pulmonary toxicity (130,131). The 20

31 bleomycin model has been shown to induce lung injury as well as fibrosis in a variety of experimental animals including mice, rats, hamsters, rabbits, guinea pigs, and dogs, but pulmonary fibrosis does not develop in all animals and the time frame for fibrosis development is relatively long; fibrosis is first observed at week two with interstitial fibrosis becoming more severe at up to week twelve ( ). The FITC-induced model of pulmonary fibrosis involves administration of FITC that will cause acute lung injury and increased mortality (135). In the FITC model, areas in the lung can be visualized where deposition occurs via immunofluorescence imaging for the characteristic green color of FITC (130). However, the FITC solution must be prepared fresh every day before injection and this model is not clinically relevant (130). Irradiation-induced pulmonary fibrosis can be accomplish by a single dose exposure to Gy of total body irradiation, which result in pulmonary fibrosis as early as 20 week post exposure (136). The C3H/HeJ and CBA/J mice are classified as fibrosis resistant (137,138). This model is clinically relevant. However, the length of time to develop fibrosis is long, thus the housing of mice would be of a high cost. In the silica model of pulmonary fibrosis, silica is not readily cleared from the lung, thus the fibrotic stimulus is persistent (130). The fibrosis can be identified as fibrotic nodules that develop in areas of silica deposition. Aerosolization requires special equipment that is not readily available in all institutions. In the silica model of pulmonary fibrosis, fibrosis requires 60 day, as indicated by some studies, which results in high cost per experiment (139). In the transgenic models of pulmonary fibrosis, vectors are not benign carriers and can actually pose serious health problems (130). However, the use of viral vector-directed transgenic models has proven useful in studying individual gene products on the initiation of experimental pulmonary fibrosis, such as TGF-β, IL-1β, TNF-α, and GM- CSF have been employed ( ). 21

32 Models of HP have been described in rabbits, calves, primates, guinea pigs, mice and rats. Animal models of HP involve pulmonary exposure to an inciting Ag via tracheal instillation, nasal instillation, or aerosol inhalation. Most of the work in animal models of HP has used SR Ag, the predominant Ag responsible for farmer s lung disease. Although most mouse strains response similarly to SR Ag, DBA/2 mice express less pulmonary inflammation compared to C57BL/6 animals ( ). Our laboratory has developed an EHP model. Our model of EHP uses C57BL/6 mice, which is the genetic background used to develop most gene-deficient animals. Moreover, C57BL/6 mice are widely used to study fibrosis, as they are more prone to fibrosis. The mouse is the animal of choice for most studies of EHP for several reasons. These include the fact that pathological features of EHP in the mouse model closely resemble features seen in human HP, the relatively short time frame needed for development of disease, cost and ease of use, and the ability to utilize genetic approaches, such as gene-deficient mice and transgenic animals. The murine model that is used in our laboratory is based on a model that is widely accepted but has mainly been used to study inflammation (147,148). We introduce SR Ag into the mouse lung in an accurate and quantitative manner, by oropharyngeal aspiration (149,150). The alveolar pulmonary inflammatory response was assessed in BAL fluid. Prior data from our laboratory showed a persistent alveolar neutrophil response with bronchiolocentric lymphoplasmacytic inflammation. A patchy peripheral and peribronchiolar fibrosis develops after three weeks (Figure 1.4). Elevated soluble collagen levels support the histology findings (Figure1.5). As well as being present in the BAL fluid, neutrophils can be consistently identified in the fibrotic areas. Subsequently our laboratory systematically depleted neutrophils during the final two weeks of a three week exposure to SR Ag and found that neutrophils depletion was accompanied by attenuated pulmonary fibrosis (Figure 1.6). Thus, preliminary data from our 22

33 laboratory indicates a central role of neutrophils and their products in the development of fibrosis in EHP. An important consideration when using an animal model is that it is reflective of the clinical disease (130). SR Ag is derived from the major causative agent of farmer s lung and antibodies to it are found in the serum of up to 80% of affected patients ( ). No current animal model can recapitulate all of the manifestations of the human disease, but our model does reproduce several important feature of HP, including the type of inflammatory influx, the presence of multinucleated giant cells, granulomas, and peribronchiolar and subpleural fibrosis (13,130). 23

34 A B C D E F 24

35 Figure 1.4 Chronic SR Ag exposure in WT for three weeks. H&E stain. Normal lung (A and B). Lymphoid aggregate and plasma cells (arrows, C). Subpleural neutrophils (short arrows, D) and multinucleated giant cells (long arrows, D). Neutrophils in red infiltrating fibrotic area (Picrosirius red stain, short arrows, E). Polarized light, specific for collagen (F). A 100x. B 400x. C: left 400x and right 100x. D: left 200x and right 400x. E 200x. F 200x. Data are from Dr. Kelly s laboratory. 25

36 8000 * Soluble collagen ug/rl WK 0 WK 1 * * WK 2 WK 3 WK 5 Figure 1.5 Soluble collagen measured in homogenized right lungs (RL) in chronic SR Ag exposure in C57BL/6 mice. Total soluble collagen in homogenized RL of mice was measured by Sircol assay. n=6, * denote p<0.05, data expressed as mean ± SEM. WK = week. WK 0 = untreated C57BL/6 mice and WK 1, 2, 3, and 5 = SR Ag administrated to C57BL/6 mice for 1, 2, 3, and 5 weeks respectively. Data are from Dr. Kelly s laboratory. 26

37 A Neutrophils (10 3 ) BAL fluid 2000 * * PBS SR Ag Anti-Gr-1 Ab B Soluble collagen ug/rl PBS * SR Ag * Anti-Gr-1 Ab Figure 1.6 Anti-neutrophil (anti-gr-1) antibody administrated to C57BL/6 mice during last two weeks of a three weeks SR Ag exposure. Anti-Gr-1 antibody was administrated to the mice (4mg/kg) by IP injection. Total BAL fluid neutrophils (A) and soluble collagen levels (B) at the end of three week time point. n=5, * denote p<0.05, data expressed as mean ± SEM. The data are from Dr. Kelly s laboratory. 27

38 1.8 Overall objective Building on the data demonstrating a role for neutrophils in the development of fibrosis in EHP, the overall objective of this thesis was to determine if products produced by neutrophils, in particular MMP-9, play a role in the pathogenesis of fibrosis in HP. 1.9 Hypothesis Products produced by neutrophils, specifically MMP-9, play a critical role in the development of fibrosis in HP Aims Aim 1: Conduct in vivo murine studies to determine the effect of MMPs in the fibrotic process using the broad spectrum MMP inhibitor GM6001 in EHP. Aim 2: Conduct in vivo murine studies to investigate the role of MMP-9 in fibrosis using MMP- 9 deficient mice. Aim 3: Conduct in vitro cell culture studies to study the effects of supernatants from activated neutrophils on human lung fibroblast phenotype. 28

39 Chapter Two: Materials and Methods 29

40 2.1 Materials and Reagents Materials and suppliers used: Eppendorf tips were obtained from VWR (Mississauga, ON, Canada); Tubes were obtained from MaxBioChem (Calgary, AB, Canada); Haematoxylin and eosin (H&E) stain were obtained from Leica microsystems (Concord, ON, Canada); Cell culture flasks and plates were obtained from Corning Life Science (Lowell, MA, USA); SR Ag was obtained from Greer Laboratories (Lenoir, NC, USA); GM6001 drug was obtained from Millipore (Billerica, MA, USA); Optimal cutting temperature solution (OCT) was obtained from VWR (Mississauga, ON, Canada); 3-3 diaminobenzidine (DAB) tablets and urea hydrogen peroxide tablets were obtained from Sigma-Aldrich (Oakville, ON, Canada); Matrix metalloproteinase-9 (MMP-9) antibody was obtained from R&D Systems (Minneapolis, MN, USA); Myeloperoxidase (MPO) antibody was obtained from Dako (Burlington, ON, Canada); CD45 antibody was obtained from BD Pharmingen (Mississauga, ON, Canada); Laminin antibody was obtained from Millipore (Billerica, MA, USA); α-smooth muscle actin (α-sma) antibody was obtained from Sigma-Aldrich (Oakville, ON, Canada); Avidin and Biotin were obtained from Vector laboratories (Burlington, ON, Canada); Hank s balanced salt solution (HBSS) and Dulbecco s modified Eagle medium (DMEM) were obtained from Invitrogen (Burlington, ON, Canada); Dulbecco s PBS was obtained from Invitrogen (Burlington, ON, Canada); RNase/DNase free double distilled water was obtained from Invitrogen (Burlington, ON, Canada); DQ fluorescence quenched gelatin was obtained from Molecular Probe (Burlington, ON, Canada); Gelatin was obtained from Sigma-Aldrich (Oakville, ON, Canada); Isoflurane was obtained from (MTC Pharmaceuticals, Cambridge, ON, Canada); Lympholytepoly was obtained from Cedarlane, (Hornby, ON, Canada); GM-CSF was obtained from R&D Systems (Minneapolis, MN, USA); f-met-leu-phe (fmlp) was obtained from Sigma-Aldrich 30

41 (Oakville, ON, Canada); Human active MMP-9 fluorescence assay kit was obtained from R&D Systems (Minneapolis, MN, USA); Phase lock gel tube was obtained from VWR (Mississauga, ON, Canada); TRIzol was obtained from Invitrogen (Burlington, ON, Canada); DNase I was obtained from Ambion (Carisberg, CA, USA); Superscript III Reverse Transcriptase was obtained from Invitrogen (Burlington, ON, Canada); α-sma primers and probes were obtained from Applied Biosystems (Foster city, CA, USA); Taqman master mix was obtained from Applied Biosystems (Foster city, CA, USA); In-cell western assay kit was obtained from LI- COR (Lincoln, Nebraska, USA); Odyssey Blocking Buffer was obtained from LI-COR (Lincoln, Nebraska, USA). 2.2 Methods Animals Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and were maintained in the Mouse Single Barrier Unit at the University of Calgary. Mice were rested for at least one week before treatment at 5-6 weeks of age. MMP-9-deficient mice on a C57BL/6 mice background were a kind gift from Dr. Wee Yong. They were treated at 5-6 weeks of age. MMP-9-deficient mice are viable, fertile, and generally healthy. They have shorter long bones (i.e. tibia and femur) and a lengthened zone of hypertrophic cartilage compared to C57BL/6 mice due to delayed apoptosis (154). Our data showed normal cell counts in the BAL fluid and blood in MMP-9-deficient mice compared to C57BL/6 mice. All experiments were approved by the University of Calgary Health Sciences Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care. 31

42 2.2.2 Experimental hypersensitivity pneumonitis (EHP) model The SR Ag was prepared once every week for the three SR administrations per week. SR Ag was administrated for three consecutive days per week for a period of three weeks. The SR Ag (160µg/40µl/dose) was introduced to the mice by an oropharyngeal aspiration technique (149,150). Mice were lightly anesthetized with isoflurane and suspended from their front teeth. The tongue was gently pulled to one side and the SR Ag was introduced into the distal trachea via pipette. The alveolar pulmonary inflammatory response was assessed by examining bronchial alveolar lavage (BAL) fluid. Following BAL, the lungs were removed; the right lung was snap frozen to evaluate soluble collagen levels using the Sircol assay, whilst the left lung was inflated and fixed for 24 h with formalin, then processed for histological analysis. In some groups of mice, the left lung was inflated with OCT solution (50% OCT solution in PBS) and stored at - 80 C for frozen sections. GM6001 was initially dissolved in DMSO (0.75% DMSO in PBS) (400mg/ml), and subsequently diluted in PBS to a final concentration of 3mg/ml, the maximum concentration that was recommended by the manufacturer (Millipore). GM6001 was introduced to the mice by intraperitoneal (IP) injection on the last two weeks of a three weeks treatment with SR Ag and again 24 h before harvest (Figure 2.1). The IP injection was introduced to the mice after lightly anesthetising them with isoflurane, to reduce stress, and drug administration was followed by intratracheal administration of SR Ag. We introduced the GM6001 to C57BL/6 mice for three weeks instead of five in order to determine if there was an effect in inhibiting MMPs whilst fibrosis was developing. The study included four separate groups of mice: C57BL/6 mice exposed to intratracheal SR Ag with IP injections of either PBS, vehicle (DMSO) alone, or 32

43 GM6001 dissolved in DMSO, and a group of C57BL/6 mice which were exposed to intratracheal PBS with IP PBS. 33

44 Week 1 Week 3 Week 2 Week 4 C57BL/6 day SR Ag GM6001 IP injection Harvest Figure 2.1 Chronic SR Ag administration protocol and IP GM6001 for the last two weeks. SR Ag was administrated to C57BL/6 mice by oropharyngeal aspiration on three consecutive days each week, for a period of three weeks. GM6001 (3mg/ml) was introduced through an IP injection in the last two weeks and 24 h just before harvesting. Control mice were injected with the vehicle (DMSO) diluted as for the drug. Mice injected with PBS were also used as controls. Mice were harvested at day four after the last SR Ag exposure, n=6 in each group. 34

45 To selectively examine the role of MMP-9 in the pathogenesis of EHP, we performed studies comparing MMP-9-deficient mice with wild type C57BL/6. SR Ag or PBS was introduced to each group of mice by oropharyngeal aspiration for five weeks. This time was selected as this is when maximal fibrosis is observed. The study included four separate groups of mice: C57BL/6 or MMP-9-deficient mice each treated with SR Ag and controls for both strains treated with PBS. Mice were harvested at day four after the last SR Ag exposure (Figure 2.2). 35

46 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 MMP-9 KO mice day or C57BL/6 SR Ag Harvest Figure 2.2 Chronic SR Ag administration protocol. SR Ag was introduced to MMP-9- deficient or C57BL/6 mice by oropharyngeal aspiration technique on three consecutive days each week, for a period of five weeks. Control mice were treated with PBS for five weeks. Mice were harvested at day four after the last SR Ag exposure, n=9. 36

47 2.2.3 Bronchoalveolar lavage (BAL) The trachea was exposed and cannulated using a blunt 18-gauge needle. The BAL fluid was collected by injecting and withdrawing 1ml Dulbecco s PBS into the mice lung to a total of 10ml. Total BAL fluid cells were counted using a standard hemocytometer. Cytospin slides were prepared from BAL fluid (100µl/slide) using a Shandon cytocentrifuge (Thermo Scientific, Ottawa, ON, Canada) (450 x g, 6 min), and stained with hematoxylin and eosin (H&E) for differential cell counts. Differential cell counts were performed on 400 cells. Slides were coded for blinded analysis. Supernatant was collected from BAL fluid after centrifugation (1000 x g, 10 min), and stored at -80 C for later analysis Hematoxylin and eosin (H&E) stain Formalin fixed sections were deparaffinized through three changes of xylene for 2 h, dehydrated through three changes in isopropanol (1 min in each), and washed gently in a running tap water. Sections were washed with distilled water, placed in Gills haematoxylin for one minute, and washed in gently running warm tap water. Sections were placed in eosin for 30 sec and washed in running tap water subsequently. Subsequently, sections were dehydrated, through three changes in isopropanol, cleared through three changes of xylene, and mounted using permount. Frozen sections were fixed using 95% ethanol for 15 min Soluble collagen measurements (Sircol Assay) The right lung was thawed, washed with Dulbecco s PBS, dried gently, and place in homogenizer tubes on ice. Dulbecco s PBS (1ml) was added to the lung in the homogenizer tube and homogenized for 15 sec. The homogenate was transferred to a 5ml tube and washed with 37

48 1ml of acid pepsin solution (0.2mg/ml in 1.0N acetic acid) to a final concentration of 0.1mg/ml acid pepsin in 0.5M acetic acid. The homogenate was rocked at 4 C with acid pepsin overnight. The next day, the sample was centrifuged (400 x g, 10 min, 4 C) to obtain a clarified extract. Collagen standards (6.25μg, 12.5μg, 25μg, 50μg, and 100μg) were prepared in 0.5M acetic acid at room temperature. The sample supernatants were transferred to 2ml eppendorf tubes, and acid neutralizing reagent was added at a ratio of 100µl/ml clarified extract. For each sample, 100μl was pipetted into respective 1.7ml tubes. Sircol Dye Reagent (1ml) was added to the sample and standards but not the blank (alkali reagent). The tubes were agitated at room temperature for 30 min, followed by centrifugation (16000 x g, 10 min) to recover the collagen-dye complex. The supernatant was discarded and 1ml alkali reagent was added to all tubes. The collagen bound dye pellet was solubilized by vortexing thoroughly. Collagen bound dye was measured by microplate reader at 540nm. All measurements were in triplicate and collagen concentrations were obtained from the standard curve. Data are expressed as soluble collagen µg/right lung (RL) Immunohistochemistry to detect intracellular MMP-9 Sections were cut from frozen blocks at 5µm on Fisher plus slides, air-dry at room temperature overnight up to 72 hr, and fixed using 95% ethanol for 15 min. Sections were washed with PBS for 5 min. Endogenous peroxidase was blocked with 0.03% H 2 O 2 for 5 min. Normal serum (5% serum in PBS) was then applied for 30 min. Avidin/Biotin block was applied with 2% normal serum for 15 min and rinsed briefly with buffer. Antibody against MMP-9 (2µg/ml, goat antimouse) was applied overnight at room temperature. After washing in PBS for 5 min, secondary link antibody against MMP-9 (rabbit anti-goat) was applied (5μg/ml) for 45 min. The slides were then washed with PBS for 5 min and rinsed with distilled water. DAB chromogen solution (0.7mg/ml DAB tablet and 0.67mg/ml urea hydrogen peroxide tablet dissolved in distilled water) 38

49 was applied for 5 min. Sections were then rinsed with water, placed in Gills haematoxylin for one minute, and washed in gently running warm tap water. Subsequently, sections were dehydrated, through three changes in isopropanol, cleared through three changes of xylene, and mounted using permount. Control slides with no primary antibody or no secondary antibody added were used Immunofluorescence to detect intracellular MMP-9 and MPO Sections were cut from paraffin blocks at 5µm on Fisher plus slides and deparaffinized through changes in xylene, isopropanol, and water. Endogenous peroxidase was blocked with 3% H 2 O 2 for 10 min. Normal serum (5% serum in PBS) was then applied for 30 min. Avidin/Biotin block was applied with 2% normal serum for 15 min and rinsed briefly with buffer. Antibodies against MMP-9 (2µg/ml, goat anti-mouse) or MPO (0.8µg/ml, rabbit anti-human) were applied overnight at room temperature. After washing in PBS for 5 min, secondary antibody linked to a fluorescent marker against MMP-9 (Alexa Fluor 594 chicken anti-goat) or MPO (Alexa Fluor 488 chicken anti-rabbit) was applied (10μg/ml) for 45 min. The slides were then washed with PBS for 5 min. Sections were rinsed with distilled water before being mounted using DAPI. The control slides were mice treated with PBS. Control slides with no primary antibody or no secondary antibody were also used In Situ zymography Frozen sections were thawed at room temperature for 10 min. DQ gelatin (10µg/ml) solution was prepared from DQ fluorescence quenched gelatin and gelatin in 1:0.5 ratio, respectively. DQ 39

50 gelatin solution was applied to sections and incubated in a humid chamber for 4 h at 37 C. Sections were washed three times in PBS and fixed with methanol for 10 min at -20 C. Sections were blocked with 1% BSA in PBS for 30 min at room temperature then washed three times with PBS before applying the primary antibody. Antibody against laminin (2µg/ml, rabbit anti-mouse) or CD45 (4µg/ml, rat anti-mouse) was applied to the sections overnight at 4 C, followed by three washes with PBS. The secondary antibody linked to a fluorescent marker against laminin (Alexa Fluor 345 goat anti-rabbit) or CD45 (Alexa Fluor 594 goat anti-rat) was applied (5μg/ml) for 1 h, washed three times with PBS, and sections were mounted with Gelvetol. The control slides were mice treated with PBS. Control slides with no primary antibody were also used Culture of human lung fibroblasts Primary human lung fibroblasts (HLF) were derived from the airways of normal non-smoking human lungs that were not used for transplantation (International Institute for the Advancement of Medicine, Jessup, PA). Airways were dissected free of parenchyma and a portion of airway was washed with F-12 containing 10% fetal bovine serum (FBS), penicillin (100U/ml) and streptomycin (100μg/ml), and amphotericin B (25μg/ml). This airway section was dissected into smaller pieces (1-3mm), that were placed in 24-well culture plates in DMEM containing 10% FBS (1ml/well), and cultured at 37 C, 5% CO 2 until fibroblast outgrowths occur (10-14 days). The original tissue plugs were then removed and fibroblasts cells were removed from the plates using TrypLE select (Invitrogen, Burlington, ON, Canada) and passaged into T-75cm 2 tissue culture flasks. HLF cells were grown to confluence, lifted with TrypLE select, and initially frozen for 24 h at -80 C in L-15, 20% FBS, 10% DMSO, and subsequently transferred to liquid nitrogen (N 2 ) for later use. HLF cells were resuspended in DMEM containing 10% FBS, 40

51 penicillin (100 U/ml) and streptomycin (100μg/ml), and cultured at 37 C in T-175cm 2 until they reached confluence. Cells were washed with 5ml HBSS, lifted using 5ml TrypLE select, and centrifuged (311 x g, 8 min, 22 C) in 50ml 10% FBS/DMEM. Cells were plated (1 million cells/ml/well) on 6 well plates or (2,500 cells/150µl/well) on 96 well plates and grown in 10% FBS/DMEM. The media was changed every second day. Cells were used at passages Human lung fibroblast stimulation with neutrophil supernatant Neutrophils were isolated from blood collected from five healthy human donors (4 males and 1 female, years old) by density centrifugation using Lympholyte-poly, according to the manufactures instructions. The whole blood (10ml) collected in anticoagulant heparin tubes, was carefully layered on top of a 5ml lympholyte-poly solution in a 15ml tube, and centrifuged (450 x g, 30 min, 22 C). After centrifugation, neutrophils were isolated using a Pasteur pipette. Isolated neutrophils were washed three times in 50ml Dulbecco s PBS and centrifuged (311 x g, 8 min, 22 C) after each wash. Isolated neutrophils were resuspended in Dulbecco s PBS (1million cells/ml) and stimulated with GM-CSF (4ng/ml) for 30 min at which time the bacterial peptide f-met-leu-phe (fmlp) (10-7 M) was added for the next 30 min. Stimulated neutrophils were centrifuged (311 x g, 8 min, 22 C) and supernatants were collected. Neutrophil supernatants were either used immediately or stored at -80 C. Neutrophil supernatants were added to human fibroblast cells at different dilutions (1/3 and 1/10) in duplicates for 48 h at 37 C. In initial RT-PCR pilot experiment, neutrophil supernatants from individual donors were used. It became clear in these initial experiments however, that studying individual neutrophil supernatants on fibroblasts from different donors introduced a large number of variables that could affect reproducibility of responses. For in-cell western assay studies, therefore, it was 41

52 decided to use a pooled supernatant from three neutrophil donors that could then be used as a single source of MMP-9-containing neutrophils supernatant to be tested on individual fibroblast donors. For the RT-PCR experiments, neutrophil supernatants were removed after 48 h and total cellular RNA was harvested using TRIzol. For in-cell western assay experiments, neutrophil supernatants were removed after 48 h and cells were immediately fixed with formalin. Control wells were treated with media Cell viability assay MTT (Triazolyl blue tetrazolium bromide) was dissolved in HBSS (1mg/ml) (solution was kept in the dark). Neutrophil supernatant was removed from human lung fibroblast cells grown on a 6 well plate and MTT solution was added to the each well (1ml per duplicate) and incubated in the dark for min at 37 C, until purple color developed. The plate was aspirated and DMSO was added to each well (1ml/well). From each well, 200µl were transferred into a 96 well plate for reading. The plate was read on the spectrophotometer at 570nm. The assay measures the activity of mitochondrial succinyl dehydrogenase, an index of cell metabolism and viability Measurement of MMP-9 in neutrophil supernatant MMP-9 was measured in the pooled neutrophil supernatant by ELISA (R&D Systems, Minneapolis, MN, USA). In the assay, all incubations were performed at room temperature. Sample and standard dilutions were done using ELISA diluent (0.1% BSA in 1% PBS PH 7.4). ELISA was performed using a 96 well plate coated with monoclonal anti-human MMP-9 (1µg/ml, MAB936) antibody diluted in PBS and incubated overnight. The plate was washed the next day three times with ELISA wash buffer (0.05% Tween-20 in PBS PH 7.4), followed by 42

53 incubation in blocking buffer (1x PBS, 1% BSA, 5% sucrose) for 1 hr. The plate was washed, and the supernatant and MMP-9 standards (911-MP, pg/ml) were loaded in triplicate and incubated for 2 hr. The plate was washed and incubated with biotinylated anti-human MMP- 9 (0.15µg/ml, BAF911) for 2 hr after which the plates were again washed then incubated in the dark with streptavidin peroxidase (1µg/ml) for 20 min followed by color reagent A (hydrogen peroxide) and reagent B (tertramethylbenzidine) (20 min). The reaction was stopped using 2N H 2 SO 4 and the plate was read on a spectrophotometer at 450nm. The concentration was determined through the linear portion of the standard curve. Data are expressed in ng/ml Measurement of active MMP-9 in neutrophil supernatant A fluorescence assay to measure active MMP-9 was performed according to the manufacturer s instructions. Neutrophil supernatant and standards were applied to plates pre-coated with a monoclonal antibody specific for human MMP-9 for 2 h on a microplate shaker (500rpm). The plate was washed and the activation reagent (APMA) was added to standards and selected samples for 2 h at 37 C in a humidified environment, protected from light. The control was PBS which was substituted for supernatant in two duplicate wells. The plate was washed and incubated overnight with the fluorogenic substrate molecule at 37 C in a humidified environment, protected from light. Active MMP-9 cleaves the peptide linker between the fluorescent quencher and molecule, resulting in a fluorescent signal that is proportional to the MMP-9 activity of the enzyme in the sample, which is measured using a spectrofluorometer and quantified by the standard curve. 43

54 RNA isolation, DNase treatment and Real-time PCR RNA isolation Supernatants were collected after stimulating cells for 48 hr, and total cellular RNA was harvested using 0.5ml TRIzol per well. TRIzol was pipetted up and down to lyse cells. The cell lysate was transferred into phase lock gel tubes. Chloroform (200µl/ml TRIzol) was added with vigorous mixing for 15 sec and the phase lock tubes were centrifuged (12,000 x g, 10 min, 4 C). The top translucent aqueous layer containing the RNA was transferred into RNA/DNA free labeled tubes. The RNA was precipitated by centrifugation (12,000 x g, 10 min, 4 C) after the addition of isopropanol (500µl/ml TRIzol) and the supernatant was removed without disrupting the RNA pellet. The pellet was washed with 75% ethanol (1ml) and centrifuged (7500 x g, 5 min, 4 C). The supernatant was removed and the precipated pellet was left to air-dry for about 15 min. The pellet was reconstituted with 20µl RNase/DNase free double distilled water. Quantification of RNA was determined using Nanodrop apparatus at 260nm. The purity of RNA was determined by monitoring 260nm/230nm (organic compounds) and 260nm/280nm (nucleic acid: protein) ratios DNase treatment DNase treatment was performed using DNase I. RNA was diluted in RNase/DNase free double distilled water to a final concentration of 10µg/ml, and incubated with 2µl DNase buffer and 2µl DNase I for 30 min at 37 C. Samples were centrifuged (10,000 x g, 1 min) after adding 5µl DNase inactivation reagent. The supernatant was removed and added to RNase/DNase free labeled tubes. RNA quantification and purity was determined again using the Nanodrop apparatus. 44

55 cdna cdna was generated using superscript III Reverse Transcriptase. DNase treated RNA (1µg) was incubated with 1µl oligo(dt) and 1µl dntp mix (10mM each) for 5 min at 65 C. Samples were incubated on ice for at least 1 min and centrifuged briefly to collect any condensation. Superscript III RT (200U/µl, 1µl), 5x first standard buffer (4µl), 0.1M DTT (1µl), and RNase OUT (40U/µl, 1µl) were added to the tube and incubated for 60 min at 50 C. Generated cdna samples were frozen immediately at -20 C or used directly for PCR amplification RT-PCR Collagen I and α-sma primers and probes were synthesized by Applied Biosystems and were designed, when possible, to span an intron. A master mix was prepared with Taqman master mix, respective forward and reverse primers and probe, and RNase/DNase free double distilled water according to the manufacturer s instructions. In each well, cdna (1µl) and the master mix (22µl) were added, for a total volume of 23µl per well. GAPDH mrna levels were assessed using GAPDH assay on demand for each sample. The control used was 1µl RNase/DNase free double distilled water and 22µl of the master mix consisting of the primer of interest or GAPDH. Samples were run in triplicate and controls in duplicate on every plate. The data were analyzed using the comparative ΔΔCT method (155) Detection of intracellular α-sma in cultural human fibroblasts by in-cell western The in-cell western assay detects target proteins in fixed cells directly in their cellular context using target-specific primary antibody and infrared dye-labeled secondary antibodies. The fluorescent signal from each well was quantified. In-cell western assay was performed according 45

56 to the manufacturer s instructions. All the incubations were performed at room temperature, unless otherwise indicated. The primary and secondary antibodies were diluted in Odyssey Blocking Buffer (1:1, Odyssey Blocking Buffer: 1x PBS). Human lung fibroblast cells were grown in a 96 well plate, stimulated with neutrophils supernatant (1/3 dilution) for 48 h, and fixed with fixing solution (10%, 3.7% formaldehyde in 1x PBS) for 20 min. human lung fibroblast cells were permeabilized by washing with 0.1% Triton X-100 five times. Cells were blocked with Odyssey Blocking Buffer for 1 h with moderate shaking. Cells were incubated overnight with α-sma, primary antibody (4µg/ml) at 4 C, with gentle shaking. The plate was washed five times with Tween Washing Buffer (1x PBS, 0.1% Tween 20). The fluorescentlylabeled secondary antibody, IRDye 800 (0.5µg/ml) for control wells and IRDye 800 with Sapphire700 stain (0.5µg/ml) and DRAQ5 stain (5M) for sample wells were incubated with gentle shaking for 1 h. The plate was washed again with Tween for 5 min with gentle shaking. The wash buffer was removed and the plate was scanned immediately with detection in both 700 and 800nm channels using the Odyssey system (LICOR). The negative control in two duplicate wells had no primary antibody, but had the fluorescently-labeled secondary antibody, and IRDye Statistical analysis Data were expressed as ± standard error of the mean (SEM). Comparisons between groups with normally distributed data were made using analysis of variance (ANOVA) with Tukey s post hoc analysis. Comparisons between non-parametric data were made using Kruskal-Wallis ANOVA with Dunnett post hoc analysis. All statistical tests were two-tailed and p values < 0.05 were considered significant. 46

57 Chapter Three: Pulmonary fibrosis is attenuated in the absence of MMP-9 in EHP but neutrophil influx is independent of MMP-9 47

58 3.1 Results Active MMP-9 is present in the fibrotic areas of the lung in EHP To determine if active gelatinases (i.e. MMP-9 and MMP-2) are detectable in areas of pulmonary fibrosis in animals with EHP, we utilized in situ zymography. Frozen lung sections from mice treated with SR Ag or PBS were examined. Laminin shows the structure of the lung. Fibrotic areas of the lung showed destroyed laminin. Active gelatinases present cleave fluorescently labeled gelatin applied to the sections and fluorescein isothiocyanate (FITC) is visualized (Figure 3.1.a). Inflammatory cells in the lung were detected with immunohistochemistry for CD45, a marker positive for inflammatory cells (Figure 3.1.b). Fluorescence indicating the presence of active gelatinase was only present in fibrotic areas and CD45 colocalized in the fibrotic areas as shown in Figure 3.1.c. Therefore, only inflammatory cells present within fibrotic areas were positive for active gelatinases in the lung sections. 48

59 A B C D 50µ Figure 3.1 Gelatinase activity in fibrotic lung in mice treated with SR Ag for three weeks. Increased gelatinase activity was detected by cleavage of gelatin labelled with FITC, green color (A). Inflammatory cells were identified with anti-cd45, red color (B). The two colors are merged in panel (C), and the yellow color indicates colocalization of active gelatinase with CD45 cells in the fibrotic areas. Lung sections from mice treated with PBS showed no gelatinase activity (green color), or anti-cd45, red color (D). Representative of n=5. 49

60 The inflammatory cells producing gelatinase activity in areas of fibrosis had the morphological characteristics of neutrophils. Neutrophils or polymorphonuclears (PMNs) stain pink with H&E stain and contain nucleus divided into two to five lobes. Because neutrophils do not produce MMP-2 (gelatinase A) but are a rich source of MMP-9 (gelatinase B), these data indicate that neutrophil MMP-9 activity is present in fibrotic regions of the lung in EHP. Fibrotic sections of EHP lungs of the lung were stained with antibody to MMP-9. Only neutrophils were positive for MMP-9 in these areas (Figure 3.2.a and b). To confirm this, sections were double stained with an antibody to MMP-9 and the neutrophil marker, MPO (Figure 3.2.c). Although detection of MMP-9 by this antibody does not discriminate between active and inactive MMP-9, it confirmed that only neutrophils were positive for MMP-9 in the lung sections. Although macrophages can be positive for MPO, we did not see any positive macrophages. As before, all cells colocalized with MPO and MMP-9 had the morphological characteristics of neutrophils (Figure 3.2.c). Taken together with the results from in situ zymography, this double-staining confirmed that all cells secreting active MMP-9 were neutrophils (Figure 3.2). 50

61 A B C i ii iii D Figure 3.2 MMP-9 produced by neutrophils in lung sections from EHP with chronic SR Ag exposure. A and B: Immunohistochemistry for MMP-9 (3,3 -Diaminobenzidine (DAB) chromogen, brown color, hematoxylin counter stain for nuclei) in a fibrotic area of the lung from 51

62 a mouse exposed to SR Ag for three weeks. Positive cells have the morphology of neutrophils; other inflammatory cells present do not stain for MMP-9 (A, 400X and B, 1000X). C: Immunofluorescent double stain for MMP-9 and MPO. Immunofluorescence for MPO (Alexa 488, green) (C.i), and MMP-9 (Alexa 594, red) (C.ii). Nuclei are stained with DAPI (blue). In the merged image, MPO and MMP-9 colocalize (C.iii). Lung sections for control mice (treated with PBS) showed no staining for either MPO or MMP-9 (D). C and D 600x. 52

63 3.1.2 Neutrophil influx to the lung is reduced in mice treated with GM6001 As we have demonstrated in the previous experiment, using in situ zymography and immunofluorescence double staining that active MMP-9 derived from neutrophils were present in fibrotic areas of the lung in EHP; we wanted to investigate the effect of inhibiting MMPs in the development of fibrosis using GM6001. C57BL/6 mice treated with SR Ag and IP GM6001 showed a significant reduction in the neutrophil and lymphocyte influx into the lung compared to C57BL/6 mice treated with SR Ag and IP vehicle (DMSO) (p<0.001) (Figure 3.3 a and c, respectively). By contrast, there was no significant difference in the number of macrophages in the BAL fluid between C57BL/6 mice treated with SR Ag and IP GM6001 and the C57BL/6 mice treated with SR Ag and IP vehicle (Figure 3.3.b). Mice treated with SR Ag and IP vehicle contained large aggregates of lymphocytes around airways (Figure 3.4.a) and neutrophils in areas of fibrosis (Figure 3.4.b) as seen previously in mice treated with SR Ag alone (Figure 1.4). Mice treated with SR antigen and IP GM6001 in contrast showed few lymphocyte aggregates and neutrophils in fibrotic areas (Figure 3.4.c and d respectively), consistent with the changes seen in the BAL fluid. 53

64 Neutrophils (10 3 ) BAL fluid A 2000 *** PBS ORP *** *** SR ORP IP PBS *** ORP: PBS SR SR SR IP: PBS PBS Vehicle GM6001 SR ORP IP Vehicle SR ORP IP GM6001 B Macrophages (10 3 ) BAL fluid 4000 *** PBS ORP *** ORP: PBS SR SR SR IP: PBS PBS Vehicle GM6001 SR ORP IP PBS SR ORP IP Vehicle SR ORP IP GM6001 C Lymphocytes (10 3 ) BAL fluid 400 *** *** PBS ORP ** ORP: PBS SR SR SR IP: PBS PBS Vehicle GM6001 SR ORP IP PBS SR ORP IP Vehicle SR ORP IP GM6001 Figure 3.3 Inflammatory cell response in the BAL fluid of C57BL/6 mice exposed to SR Ag or PBS for three weeks and IP GM6001, vehicle or PBS for the last two weeks. Neutrophil, macrophage, and lymphocyte cell counts in the BAL fluid (A, B and C, respectively). Control mice were injected with IP PBS or vehicle (DMSO). n=5 in PBS ORP group, n=7 in SR ORP IP PBS group, n=7 in SR ORP IP Vehicle group, n=6 in SR ORP IP GM6001 group, data are shown as mean ±SEM, ** and *** denote p<0.01 and p<0.001, respectively, using one-way ANOVA with Tukey s post-hoc test. ORP= oropharyngeal aspiration. 54

65 A B C D Figure 3.4 Murine lungs after chronic SR Ag exposure for three weeks and IP GM6001 or vehicle for the last two weeks. Formalin fixed lung sections stained with H&E. Mice treated with SR Ag and IP vehicle (DMSO) contained large aggregates of lymphocytes around airways (A) and neutrophils were associated with fibrotic areas (B). Mice treated with SR Ag and IP GM6001 showed few lymphocyte aggregates (C) and few neutrophils in the fibrotic areas (D). Each picture is representative of 5 animals in PBS ORP group, 7 animals in SR ORP IP PBS group, 7 animals in SR ORP IP Vehicle group, and 6 animals in SR ORP IP GM6001 group. A, B, and D 40x. C 200x. 55

66 3.1.3 GM6001 had no effect on pulmonary fibrosis induced by SR Ag Total soluble collagen in the right lung was measured to provide a quantitative measurement of collagen production in the lungs of each group of mice. Collagen levels in the right lung were not significantly reduced in C57BL/6 mice treated with SR Ag and IP GM6001 compared to the C57BL/6 mice treated with SR antigen and IP vehicle (Figure 3.5). 56

67 Soluble Collagen ug/rl PBS ORP ** *** ORP: PBS SR SR SR IP: PBS PBS Vehicle GM6001 SR ORP IP PBS SR ORP IP Vehicle SR ORP IP GM6001 Figure 3.5 Fibrotic response of C57BL/6 mice exposed to SR Ag or PBS for three weeks and IP GM6001, vehicle or PBS for the last two weeks. Total soluble collagen in homogenized right lung (RL) of mice was measured by Sircol assay. n=5 in PBS ORP group, n=7 in SR ORP IP PBS group, n=7 in SR ORP IP Vehicle group, n=6 in SR ORP IP GM6001 group, data are shown as mean ±SEM, ** and *** denote p<0.01 and p<0.001, respectively, using one-way ANOVA with Tukey s post-hoc test. ORP= oropharyngeal aspiration. 57

68 3.1.4 MMP-9 deficiency had no effect on inflammatory influx induced by SR Ag To selectively examine the role of MMP-9 in the development of fibrosis in EHP, we introduced SR Ag to MMP-9-deficient mice. C57BL/6 and MMP-9-deficient mice treated with SR Ag exhibit a significant increase in neutrophils, macrophages, and lymphocytes, cell counts compared to their control, C57BL/6 and MMP-9-deficient mice treated with PBS, respectively (Figure 3.6). MMP-9-deficient mice treated with SR Ag had no significant difference in their BAL fluid neutrophil, macrophage and lymphocyte counts, compared to C57BL/6 mice (Figure 3.6). 58

69 A Neutrophils (10 3 ) BAL fluid C57BL/6 PBS ORP ** ** ORP: PBS SR SR PBS C57BL/6 MMP-9 -/- C57BL/6 SR ORP MMP-9 KO SR ORP MMP-9 KO PBS ORP B Macrophages (10 3 ) BAL fluid C57BL/6 PBS ORP ** *** ORP: PBS SR SR PBS C57BL/6 MMP-9 -/- C57BL/6 SR ORP MMP-9 KO SR ORP MMP-9 KO PBS ORP C Lymphocytes (10 3 ) BAL fluid C57BL/6 PBS ORP *** * ORP: PBS SR SR PBS C57BL/6 MMP-9 -/- C57BL/6 SR ORP MMP-9 KO SR ORP MMP-9 KO PBS ORP Figure 3.6 Inflammatory cells response in the BAL fluid of C57BL/6 and MMP-9-deficient mice exposed to SR Ag or PBS for five weeks. C57BL/6 and MMP-9-deficient mice neutrophils, macrophages, and lymphocytes cell count in the BAL fluid (A, B and C, respectively). n=9 in C57BL/6 PBS ORP group, n=10 in C57BL/6 SR ORP group, n=9 in MMP-9 KO SR ORP group, n=7 in MMP-9 KO PBS ORP group, data are shown as mean ±SEM, *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively, using one-way ANOVA with Kruskal-Wallis post-hoc test. ORP= oropharyngeal aspiration. 59

70 3.1.5 Pulmonary fibrosis induced by SR Ag exposure was significantly reduced in MMP-9- deficient mice compared to C57BL/6 mice Soluble collagen from the lung homogenates was significantly increased in both C57BL/6 and MMP-9-deficient mice treated with SR Ag compared to respective control animals treated with PBS (Figure 3.7). However, MMP-9-deficient mice treated with SR Ag had significantly reduced collagen levels compared to C57BL/6 mice (p<0.001) (Figure 3.7), indicating a role of MMP-9 in the pathogenesis of fibrosis in EHP. C57BL/6 mice showed no significant differences in soluble collagen compared to MMP-9-deficient mice treated with SR Ag (Figure 3.7). 60

71 Soluble collagen ug/rl C57BL/6 PBS ORP *** *** * ORP: PBS SR SR PBS C57BL/6 MMP-9 -/- C57BL/6 SR ORP MMP-9 KO SR ORP MMP-9 KO PBS ORP Figure 3.7 Total soluble collagen from homogenized right lung in C57BL/6 and MMP-9- deficient mice after exposure to SR Ag or PBS for five weeks. Total soluble collagen in homogenized right lung (RL) of mice was measured by Sircol assay. n=10 in C57BL/6 PBS ORP group, n=10 in C57BL/6 SR ORP group, n=9 in MMP-9 KO SR ORP group, n=7 in MMP- 9 KO PBS ORP group, data are shown as mean ±SEM, * and *** denote p<0.05 and p<0.001, respectively, using one-way ANOVA with Tukey s post-hoc test. ORP= oropharyngeal aspiration. 61

72 3.2 Discussion Several MMPs are markedly upregulated in lung fibrosis (78). Imbalance between MMPs and their respective TIMPs have been linked to several pathological processes, including pulmonary fibrosis (99,100). MMP-9 has been shown to be upregulated in IPF (156). MMP-9 is increased in the BALF of IPF patients in association with increase in neutrophil numbers (113,121,122). Similarly, chronic HP patients have been shown to have increased neutrophil numbers with increased expression of MMP-9 which correlate with areas of fibrosis (61). Neutrophils are the predominant pulmonary cellular source of MMP-9 (157). Moreover, data from our laboratory have shown that neutrophils were present in the BAL fluid in EHP, and depleting them systematically during the final two weeks of the three week exposure to SR Ag, attenuated pulmonary fibrosis (Figure 1.6). In the current chapter, therefore, we tested the hypothesis that MMP-9 plays a role in the development of fibrosis in a murine model of EHP. We began by demonstrating that active MMP-9, derived from neutrophils exclusively is present in fibrotic areas of the lung. These data support, but do not prove, the concept that MMP-9 may play a role in the development of fibrosis in our model of EHP. As an initial approach to examine the role of MMPs in the pathogenesis of EHP, we used GM6001, a broad-spectrum inhibitor of MMP-1, -2, -3, -8, and -9. GM6001 was administrated intraperitoneally to mice at week two and three in a three week SR Ag protocol. Inflammatory cell numbers and the degree of fibrosis were assessed. Treatment with GM6001 significantly reduced neutrophil and lymphocyte infiltration into the lung but had no effect on numbers of macrophages recruited to the lung. These data suggests that one or more of the enzymes MMP-1, -2, -3, -8, or -9 play a significant role in the recruitment of neutrophils and lymphocytes, but not macrophages, to the lung in EHP. From these experiments, however, we were unable to make any inferences about 62

73 which of these MMPs may be important regarding the cellular source of the enzymes involved. In addition, fibrosis was not attenuated in C57BL/6 mice treated with the GM6001, compared to the vehicle (DMSO). Because GM6001 affects multiple MMPs and we wanted to selectively examine the role of MMP-9 in the development of fibrosis in EHP, we used MMP-9-deficient mice. Since five weeks has more collagen production than three weeks, we exposed the mice to SR Ag for five weeks, in order to maximize our chances of seeing a difference between the MMP-9-deficient mice and C57BL/6 mice. MMP-9-deficient mice treated with SR Ag for five weeks showed no significant differences in the numbers of neutrophils, macrophages, and lymphocytes in BAL fluid compared to wild-type C57BL/6 mice. Additionally, examination of inflammation in the lung tissue sections showed no obvious differences between the degree of inflammation induced by SR Ag in MMP-9-deficient and wild-type mice. Interestingly, MMP-9-deficient mice exposed to SR Ag for five weeks showed a significant decrease in fibrosis compared to wild-type animals. Since we demonstrated that MMP-9 was only produced by neutrophils in our model (Figure 3.2), these data provide a strong evidence of the significant role of neutrophil MMP-9 in pulmonary fibrosis in EHP. It also confirms that MMP-9 is not necessary for neutrophils influx into the lung in our model of EHP. The lack of reduction of neutrophil numbers in MMP-9- deficient mice exposed to SR Ag would suggest that the reduction in neutrophils observed upon treatment with GM6001 must be due to MMPs other than MMP-9. In summary, the data presented in this chapter describe the novel observation that deficiency of MMP-9, which is produced by neutrophils in our model, results in a significant reduction in fibrosis in EHP, despite numbers of neutrophils and other inflammatory cells being unaffected. This suggests a central role for MMP-9 in the pathogenesis of fibrotic responses in EHP and is in 63

74 keeping with the study done in hypersensitivity pneumonitis (12). They have shown that in the lungs of HP patients, neutrophils were loaded with MMP-9 which was correlated with pulmonary fibrosis (see chapter five for more details) (61). The focus of the following chapter is to correlate our findings from mouse model with the human disease. 64

75 Chapter Four: Neutrophil supernatant containing MMP-9 does not enhance the expression of α-smooth muscle actin (α-sma) in human lung fibroblasts 65

76 4.1 Hypothesis Our laboratory has previously shown that neutrophils are associated with areas of fibrosis in EHP and that systemic neutrophil depletion reduced fibrotic responses. My work on EHP, (described in the preceding chapter), has shown that, in addition, neutrophil-derived active MMP-9 was associated with areas of pulmonary fibrosis and that mice deficient in MMP-9 had significantly attenuated fibrosis, despite no reduction in neutrophil influx. These observations led to our hypothesis that neutrophil products may directly activate human lung fibroblasts to differentiate into a myofibroblast phenotype, marked by increase expression of collagen I and α-sma. Myofibroblasts are key cells in the development of fibrosis due to their ability to proliferate and produce ECM. 4.2 Results Establishing optimal culture medium Neutrophils were isolated from the blood of healthy human donors (Figure 4.1). Purified neutrophils were stimulated with GM-CSF (4ng/ml) and fmlp (10-7 M) for 1 h. To test for optimal culture conditions of neutrophil supernatants with lung fibroblasts, supernatant from activated neutrophils was added, at dilutions of 1/3 and 1/10, to primary human lung fibroblasts. We examined the effect of the supernatant on the viability of fibroblasts from two individual donors cultured in DMEM medium without serum, and in DMEM medium containing 10% fetal bovine serum (FBS). Viability was assessed using the MTT assay. After a 48 h treatment of primary human lung fibroblasts with activated neutrophil supernatant, fibroblasts cultured in DMEM containing 10% FBS showed no decrease in cell viability (Figure 4.2.a). By contrast, fibroblasts grown in DMEM without serum showed reduced cell viability after 48 h treatment 66

77 with activated neutrophil supernatant (Figure 4.2.b). Accordingly, all further experiments were conducted using fibroblasts cultured in DMEM containing 10% FBS. 67

78 Figure 4.1 Neutrophils isolated form human blood under light microscopy. Sections were stained with Diff-Quick. Representative of n=3. 400X. 68

79 A 140 % Viability media media + GM-CSF/fMLP 1/3 neutrophil supernatant 1/10 neutrophil supernatnat B 120 % Viability media media + GM-CSF/fMLP 1/3 neutrophil supernatnat 1/10 neutrophil supernatnat Figure 4.2 Human lung fibroblasts viability in 10% serum and without serum DMEM medium. Stimulated neutrophil supernatant were added to human lung fibroblasts in DMEM medium containing 10% serum compared (a) or in DMEM medium without serum (b) at different dilutions (1/3 or 1/10) for 48 h. Cell viability was compared to the control (media) (n=2 for each group). 69

80 4.2.2 Neutrophil supernatants induced variable expression of collagen I mrna in human lung fibroblasts In an initial analysis of mrna expression, different primary human lung fibroblasts treated with two different individual activated neutrophil supernatants showed marked variability in the expression of collagen I mrna in primary human lung fibroblasts compared to the control (media) (Figure 4.3). Each fibroblast donor defines an n. The expression of collagen I was measured using real-time PCR. 70

81 A. Neutrophil donor Fold Increase media media + GM-CSF/fMLP 1/3 neutrophil supernatant 1/10 neutrophil supernatant B. Neutrophil donor 2 20 Fold Increase media GM-CSF/fMLP 1/3 neutrophil supernatant 1/10 neutrophil supernatant Figure 4.3 Collagen I mrna expression in primary human lung fibroblasts. Human lung fibroblasts were treated with media (control), GM-CSF and fmlp, and neutrophil supernatants stimulated with GM-CSF and fmlp at different dilutions (1/3 or 1/10) for 48 h. Neutrophil supernatants from neutrophils donor 1 (A) and neutrophils donor 2 (B) showed variation in the expression of collagen I in different primary human lung fibroblasts. A (n=3) and B (n=2). 71

82 4.2.3 Neutrophil supernatants induced variable expression of α-sma mrna in human lung fibroblasts Different primary human lung fibroblasts were treated with neutrophil supernatants from two different donors. Primary human lung fibroblasts showed marked variability in the expression of α-sma mrna after treatment with different activated neutrophils supernatant, measured by real-time PCR (Figure 4.4). 72

83 A. Neutrophil donor 1 6 Fold Increase media media + GM-CSF/fMLP 1/3 neutrophil supernatnat 1/10 neutrophil supernatnat B. Neutrophil donor 2 Fold Increase media media + GM-CSF/fMLP 1/3 neutrophil supernatnat 1/10 neutrophil supernatant Figure 4.4 The expression of α-sma mrna in primary human lung fibroblasts. Human lung fibroblasts were treated with media (control), GM-CSF and fmlp, and neutrophil supernatants stimulated with GM-CSF and fmlp at different dilutions (1/3 or 1/10) for 48 h. Neutrophil supernatants from neutrophils donor 1 (A) and neutrophils donor 2 (B) showed variation in the expression of α-sma mrna in different primary human lung fibroblasts. A (n=3) and B (n=2). 73

84 4.2.4 A pooled supernatant from stimulated neutrophils contains active MMP-9 From our initial mrna expression analysis experiments, it became apparent that using supernatants from individual neutrophil donors and individual fibroblast donors induced marked variability in signals being studies. Since our hypothesis is that neutrophil supernatants containing MMP-9 may alter fibroblast phenotype and activation, it was decided to create a pool of neutrophil supernatant by combining supernatants from three different neutrophil donors, and to characterize the expression of MMP-9 in this pool which would then be used on lung fibroblasts in hope it would reduce variability. Each fibroblast donor then defines an n. Total MMP-9 protein levels were measured in the pooled neutrophil supernatant by ELISA (Table 4.1). To determine if the pooled neutrophil supernatant contained active MMP-9, a selective fluorogenic substrate assay was performed. APMA, an activation reagent for MMPs, was added to the standards and selected samples of the activated neutrophil supernatant, while the rest of the activated neutrophil supernatant were tested for MMP-9 activity without the addition of APMA. The neutrophil supernatant pool was shown to contain active MMP-9 (Table 4.1). By relating active enzyme to total immunoreactive MMP-9 determined by ELISA, we calculated that about 20% of the total immunoreactive MMP-9 protein was active enzyme. Addition of APMA increased the level of activated MMP-9, such that approximately 60% of immunoreactive MMP-9 was active (Table 4.1). It is unclear if all immunoreactive MMP-9 was capable of being activated, or if the portion not activated by APMA was complexed to inhibitors. 74

85 Neutrophils supernatant pool MMP-9 (ELISA) Activated MMP-9, APMA (MMP-9 activity assay) No APMA added (MMP-9 activity assay) 118.4ng/ml (~100%) 56.4ng/ml (~60%) 24.8ng/ml (~20%) Table 4.1 MMP-9 from activated neutrophil supernatant pool contains active MMP-9. Neutrophils supernatant pool was analyzed for total MMP-9 protein expression by ELISA. Active MMP-9 with the MMP activator, APMA, and without APMA was measured in the activated neutrophil supernatant by MMP-9 fluorogenic substrate assay. 75

86 4.2.5 Neutrophil supernatant does not increase the expression of α-sma protein in human lung fibroblasts To detect the expression of α-sma protein in fibroblasts, we used an in-cell western assay. The in-cell western assay is an immunocytochemical quantitative assay. It detects the target protein in fixed cells directly in their cellular context using target-specific primary antibody and infrared dye-labeled secondary antibodies. The use of infrared fluorescence labeled secondary antibodies reduces interference from cells and increases the sensitivity of the assay. It is also considered a rapid assay, as there is no requirement for lysate preparation, gel loading, electrophoresis, and membrane transfer, as in conventional western blots. However, the assay is entirely dependent upon the specificity of the primary antibody, and in-cell western assay does not show the molecular weight of the target protein, as in conventional western blots. In our present studies using antibody to α-sma, fluorescent signal from each well was quantified. Data obtained showed that α-sma was constitutively expressed in primary human lung fibroblasts. Activated neutrophil supernatant pool did not cause a significant increase in the expression of α-sma above control levels (Figure 4.5). 76

87 A B C D % induction of -SMA 150 ns media ns media + GM-CSF/fMLP 1/3 neutrophil supernatant Figure 4.5 Activated neutrophil supernatant pool does not modify the expression of α-sma in primary human lung fibroblasts. Normalization of a 96-well plate was performed with DRAQ5 and Sapphire700 Cell Stains using the 700nm channel (A). The detection of α-sma was performed using the 800nm channel with α-sma primary antibody and IRDye 800CW secondary antibody (B). The two colors are merged in panel (C), yellow color. The expression of α-sma was not induced in primary human lung fibroblasts treated with neutrophil supernatant pool (D), n=4, data are shown as mean ±SEM, using one-way ANOVA with Kruskal-Wallis post-hoc test. 77