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 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.