Immune responses during lung infections must be tightly regulated in order to permit pathogen eradication while maintaining organ function. Although mechanisms involve complex networks of cytokines, the interferon (IFN) response has been shown to be an important driver of lung inflammation. Type I IFNs consist of a group of structurally similar cytokines that are produced during virus infection and are an integral part in regulating the immune response. However, in response to certain stimuli, type I IFNs have also been found to be central in the initiation of lung inflammatory responses by inducing the recruitment and activation of immune cells and thus may contribute to disease severity. Another cytokine that has been associated with chronic lung inflammation is the gp130 cytokine, Oncostatin M. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines associated with eosinophilia and alternatively activated (AA/M2) macrophage accumulation. Here we demonstrated that C57Bl/6 mice deficient of the type I interferon receptor (IFNAR1-/-), were less responsive against a suboptimal dose of AdOSM at day 7 post infection compared to AdOSM-treated wild-type. We observed a significant reduction in OSM mRNA and protein levels in AdOSM-treated IFNAR1-/- mice, compared to treated wild-type, which resulted in significant attenuation in OSM-induced inflammatory cell infiltration, epithelial hyperplasia, and alveolar wall thickening. Furthermore, IL-6 overexpression (as a comparator gp130 cytokine), induced lymphocyte accumulation in IFNAR1-/- mice, but at significantly lower levels than AdIL-6-treated wild-type. These results demonstrate that cross talk between IFNAR and gp130 cytokine signaling were required for maximal AdOSM- and AdIL-6-mediated pulmonary inflammation. We also observed that IFNAR1 deficiency directly and negatively regulated OSM-mediated responses in vitro. OSM-induced pSTAT3 levels were consistently lower in murine and human IFNAR1-deficient fibroblasts, compared to OSM-stimulated wild-type cells. This was associated with diminished OSM-induced IL-6 and MCP-1 production from IFNAR1-/- fibroblasts. Furthermore, we found that the combination of OSM and IFN-α led to increased IL-6 production from C57Bl/6 and BALB/c-derived Mouse Lung Fibroblasts (MLFs) then when either cytokine was used alone suggesting that these two cytokines can work in concert. Our findings are the first to suggest that IFNAR signaling participates in OSM-mediated responses in vitro and is required for maximal AdOSM-induced pulmonary inflammation in vivo. / Thesis / Master of Science (MSc)

Excessive tissue fibrosis in various lung diseases contributes to decline in lung function and subsequent morbidity and mortality. Mechanisms involve complex networks of molecules such as cytokines that are not clearly worked out in conditions such as Idiopathic pulmonary fibrosis (IPF). Furthermore, pulmonary virus infection has been linked to exacerbations of IPF. Previous studies have demonstrated that transient pulmonary over-expression of Oncostatin M (OSM) leads to increased extracellular matrix (ECM) accumulation, Th2-skewed cytokines and Arg1+ M2-like macrophage accumulation in mouse models. OSM can also robustly induce interleukin-33 (IL-33), an IL1 family cytokine or alarmin, both in vivo and in vitro mouse lung systems. Since others have shown that soluble IL-33 exacerbates bleomycin-induced lung fibrosis in mouse models and is associated with Th2-type lung diseases, IL-33 may mediate OSM effects on ECM and Arg1+ macrophage-like cell accumulation. The main hypothesis in this thesis is that OSM can induce IL-33 expression and Arg1+ cells, that OSM can potentiate IL-33 release from virally-infected epithelial cells, and that OSM can prime lungs to subsequent influenza infection and exacerbate pathology. Results demonstrated that OSM induced robust up-regulation of pulmonary IL-33 and Arg1 mRNA and protein expression in vivo, in comparison to another gp130 cytokine, IL-6. However, IL-6 was required for OSM-induced arginase-1 expression in vivo, but not IL-33 expression in vivo. OSM-induced Arg1 expression was also dependent upon IL-33 presence as demonstrated in IL-33-/- animals. This finding implicates a role for both IL-33 and IL-6 in mediating OSM-induced Arg1+ macrophage-like cell accumulation within the lung. Additionally, results showed that a respiratory Influenza A virus infection in vivo alone induced a time-dependent increase in OSM and IL-33 (Day 4), however reduced IL-33 by 7-days post-infection. Influenza infection in AdOSM-primed mice and led to decreased IL-33 expression and eosinophilic infiltration within the lung 5-days post-influenza infection. Collectively, these results demonstrate that OSM can drive Th2-associated pathology correlated to increased IL-33 and Arg1 expression. Contrary to expectations, influenza A virus infection led to a reduction in OSM-induced Th2-phenotype in vivo. Further exploration into the OSM-IL-33 pathway will provide insight into innate immune mechanisms of lung inflammation, virus infection and control of ECM accumulation. / Thesis / Master of Science (MSc)

Resistin-like molecule alpha (RELMα) is a secreted protein implicated in murine models of allergen-induced asthma, bleomycin-induced pulmonary fibrosis, and helminth infection. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines, eosinophil and alternatively activated (AA/M2) macrophage accumulation. In AdOSM-treated C57Bl/6 and BALB/c mice, we observed RELMα mRNA and protein markedly induced. RELMα is recognized as a marker of AA/M2 macrophages, and we observed by chromogenic in situ hybridization that RELMα mRNA co-expresses with the macrophage marker CD68, and RELMα mRNA was also highly induced in columnar airway epithelial cells upon AdOSM treatment. Assessing IL-6 as a comparator gp130 cytokine, AdIL-6 induced RELMα at significantly lower levels, however maximal induction of RELMα by AdOSM in C57Bl/6 mice required IL-6, assessed in IL-6–/– mice. Maximal induction of RELMα by AdOSM also required IL-33 in C57Bl/6 mice but not in BALB/c mice, assessed in IL-33–/– mice. We investigated functions of RELMα in response to OSM, in RELMα–/– mice. Inflammatory cell infiltration and Th2-associated cytokine responses were not altered in RELMα–/– in comparison to wildtype mice. However, RELMα-deficiency resulted in less accumulation of CD206+ AA/M2 macrophages, IFNγ+ Th1 cells in the lung, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, TIMP1, and reduced parenchymal alpha smooth muscle actin. RELMα–/– mice also showed less airway epithelial hyperplasia, increased epithelial cell damage/death (assessed morphologically) and increased LDH and soluble CK18 in response to AdOSM. Our findings suggest that RELMα does not modulate Th2 cytokines, but does participate in matrix deposition, airway remodelling mechanisms, and protection from inflammation-induced damage due to OSM-overexpression in lungs of C57Bl/6 mice. / Dissertation / Master of Science (MSc)