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Hypersensitivity Pneumonitis (HP) is a lung disease caused by repeated inhalation of environmental antigens leading to inflammation, tissue scarring, and some loss of lung function. This pathology is believed to be due to the increased IL-17A, a cytokine secreted predominantly by a subset of T cells, Th17 cells, that induces recruitment of inflammatory cells such as neutrophils and leads to pathology in this disease. The thermophile Sacharopolyspora rectivigula (SR) causes HP in humans, and we have used a murine model of HP, exposure to SR, to study the molecular mechanism of disease development. Using novel IL-17A-GFP reporter mice, our preliminary data suggests that the high levels of IL-17A induced in response to SR are produced in part by CD4+ T cells and not by neutrophils. The Tec family tyrosine kinase Itk is a pharmaceutical target and regulates T cell activation and cytokine production, including Th2 cytokines and IL-17A in conventional Th17 cells. Mice lacking Itk are therefore resistant to developing Th2 cytokine driven allergic lung inflammation. However our experiments indicate that mice lacking Itk develop HP. Histology from these mice indicates an increase in inflammatory cells in lung airways as well as deterioration of lung tissue architecture. Mice lacking Itk also have significant levels of IL-17A mRNA expression in the lung, and an increase in the number of CD4+ IL-17A producing cells. Furthermore, the lack of Itk signaling led to the absence of IL-17A producing [gamma][delta] T cells in the early stage of HP, which recover over the later stage of disease. This phenomenon directly coincides with the emergence of IL-17A producing CD4+[gamma][delta] T cells in the lungs of Itk-/- mice exposed to SR. We conclude that Itk regulated signals are not critical for the production of IL-17A in response to SR, and Itk may therefore differentially regulate the production of IL-17A in different types of T cells. We also show that the inhibition of Itk kinase signaling can be inhibited by targeting allele sensitive Itkas. The block in kinase activity leads to decrease response of response in the BAL and lungs. We investigate the relationship between Th17 cell and T regulatory T cells in an IL-17A driven disease as it relates to Itk signaling. We find that Itk signaling is not altering the proportion or numbers of T regulatory cells. Additionally, we show that iNKT cells are a possible source of IL-17A and this primarily independent of Itk signal as well. Lastly, we investigate the role of IL4Ra signaling and indirectly innate memory phenotype (IMP) cells in HP. Our data shows IL-4 signaling as well as IMP cells play a protective role in the BAL during HP. Furthermore, IL-4Ra and IMP cells are partially responsible for the number of CD4+ T cells seen in HP, which is independent of Itk-/- mice ability to make IL-17A cytokine in SR induced HP. SR induced HP. ! Together, you data show Itk mediate differential immune responses in

Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by repeated inhalation of environmental antigens. The disease is characterized by alveolitis and granuloma formation; however, some patients develop chronic HP (CHP), a restrictive lung disease characterized by fibrosis. Previous studies revealed that neutrophils are recruited into the lung via a MyD88- dependent pathway and regulate disease severity through cytokine production. Toll-like receptors (TLRs) 2 and 9 recognize conserved molecular patterns present in bacteria, and signal through the adaptor molecule MyD88. The goal of my project is to investigate the role of TLRs 2 and 9 in the pathogenesis of HP during the acute phase, granulomatous phase, and chronic phase of the disease. Using the S. rectivirgula (SR) animal model of HP, our studies indicated that individually, TLRs 2 and 9 contributed to neutrophil recruitment. We generated TLR2/9 double knockout (TLR2/9−/− ) mice to determine the extent to which TLRs 2 and 9 cooperate in neutrophil recruitment during HP. During the acute response, TLR2/9−/− mice exposed to SR demonstrated a significant reduction in neutrophil recruitment after a single exposure to SR compared to C57BL/6 mice (WT). The reduction in neutrophils was associated with a reduction in the expression of the neutrophil chemokine CXCL2 and inflammatory cytokines TNFα and IL-6 in the BALF. Disease severity in HP is associated with granuloma formation and a Th17 response. After 3 weeks exposure to SR, our results demonstrate a decrease in IL-17 and IL-22 mRNA expression and a corresponding decrease in the percentage of Th17 cells in TLR2/9−/− mice compared to WT and single knockout (SKO) mice. The decrease in Th17 cells in TLR2/9−/− mice was not associated with a significant increase in T regulatory (Treg) cells or a switch to a Th1 response. In addition, TLR2−/− and TLR2/9−/− mice have significantly fewer activated CD4+ Th cells. Interstitial macrophages from TLR2/9−/− mice have decreased costimulatory molecule and MHCII expression suggesting they are deficient in T cell activation. However, TLR2−/− , TLR9−/− , and TLR2/9−/− still form granulomas in the lung. To determine the extent to which TLRs 2 and 9 contributed to the development of CHP, WT and TLR2/9−/− mice were intranasally exposed to SR three times/week for 15 weeks. WT and TLR2/9−/− mice developed a neutrophilic and lymphocytic alveolitis, but TLR2/9−/− mice had a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid compared to WT exposed mice. Th17 cells, but not Th2 cells were detected in the lungs of mice exposed to SR for 14 weeks, suggesting CHP is not associated with a switch to a Th2 type immune response. WT and TLR2/9−/− mice had significantly reduced static compliance compared to WT unexposed mice. To determine whether the restrictive lung defect was associated with fibrosis, we visualized collagen in the lung by histology using Masson’s trichrome stain. Measuring collagen staining by positive pixel analysis suggested TLR2/9−/− mice were partially protected from lung fibrosis compared to WT mice. Altogether, the results suggest TLRs 2 and 9 cooperate in neutrophil recruitment and subsequently disease severity and play an integral role in the outcome of HP.

Hypersensitivity Pneumonitis (HP) is an interstitial lung disease caused by repeated inhalation of a wide range of environmental antigens. It is characterized by alveolitis, granuloma formation, and fibrosis. Since HP is a T cell-mediated disease, it is important to determine the type of T cell response associated with granuloma formation and the factors that control this response. We hypothesized that HP is associated with a predominant Th17 cell response where both T-bet and TLRs 2 and 9 are controlling T cell response during HP. The results demonstrated a predominant Th17 response associated with granuloma formation in the lungs of C57BL/6J mice during granulomatous and chronic HP. We also found that T-bet KO mice exposed to Saccharopolyspora rectivirgula (SR) were characterized by exacerbated Th17 cell response accompanied by an increase in granuloma formation and collagen production in the lungs compared to WT exposed mice. In an attempt to find other factors regulating Th17 development, previous studies in our lab showed that TLR2/9 double knockout (DKO) exposed mice were characterized by a decrease in the percentage of Th17 cells in their lungs compared to WT mice. Consequently, we hypothesized that the decrease in Th17 response is attributed to defect in phagocytosis, or cytokines production, or antigen presentation ability of TLR2/9 DKO antigen presenting cells (APCs). In vitro, we found no significant difference in the phagocytosis ability of both WT and TLR2/9 DKO BMDMs (Bone Marrow Derived Macrophages) or BMDCs (Bone Marrow Derived Dendritic Cells). In vivo, there was no significant difference in the phagocytosis ability of both WT and TLR2/9 DKO alveolar macrophages. Although we found that both TLRs 2 and 9 were regulating cytokine production e.g. IL-6, IL-10, and TNFα from BMDMs, we did not find a defect in antigen presentation ability of both WT and TLR2/9 DKO splenic APCs. In conclusion, these findings suggested the following: (1) Both granulomatous and chronic HP is associated with a predominant Th17 response and granuloma formation; (2) T-bet plays a role in controlling disease severity and Th17 development during HP; (3) TLR2 and 9 do not affect APCs’ ability to phagocytose SR but they affect cytokine production; and (4) Both WT and TLR2/9 DKO APCs are equally efficient in presenting SR antigen to T cells.