Background: Azithromycin polarizes macrophages into an alternative phenotype and promotes a regulated immunity. Arginase is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. Hypothesis: Decreases in inflammation in response to Pseudomonas aeruginosa (PA) pneumonia achieved by polarizing macrophages to an alternative phenotype is dependent upon the production of arginase. Methods: Requirement of arginase was examined by pharmacological inhibition using S-(2-boronoethyl)- l-cysteine (BEC) or l-norvaline and by infecting arginase-1 conditional knock-out mice (Arg1flox/flox;Lyz2-cre (Arg1Δm)) with PA intratracheally. Arg1ΔM and control Arg1flox/flox mice were then dosed with azithromycin daily via oral gavage beginning four days prior to infection. Analysis of weight loss in addition to characterization of inflammatory cells and cytokine production via flow cytometry was performed. Macrophages were then stimulated with LPS and polarized with IL4/13, IFNγ, or azithromycin plus IFNγ. Western blot for signaling mediators, p65 translocation assay, and immunofluorescence were performed. Results: Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory response compared to the Arg1flox/flox mice. Arg1Δm mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to the Arg1flox/flox mice. Conversely, global arginase inhibition resulted in greater weight loss along with greater neutrophil and macrophage infiltration compared to Arg1Δm mice. BEC and l-norvaline treated mice had higher numbers of lymphocytes in their lymph nodes with variable effects on airway lymphocyte counts. Azithromycin treatment comparably reduced the acute inflammatory responses in both Arg1Δm and Arg1flox/flox mice. To evaluate this mechanism, we show in vitro that azithromycin decreases NF-κB activation by preventing p65 nuclear translocation and by decreasing STAT1 activation in a concentration-dependent manner. These effects were reversed with IKKβ inhibition. Conclusions: Myeloid arginase is essential for control of inflammatory responses in PA pneumonia with potentially different effects of other cellular sources demonstrated with global arginase inhibition. Azithromycin reduces excessive inflammation even in the absence of arginase, potentially through a cross-inhibitory mechanism involving STAT1 and NF-κB pathways through IKKβ.

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)