Elizabeth A. Fitzpatrick, PhD

Our research focus is on the mechanisms of disease pathogenesis in Hypersensitivity Pneumonitis (HP).  HP is an interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease is characterized by alveolitis and granuloma formation that in some patients leads to chronic fibrotic lung disease.  There is a lack of successful therapeutic strategies to treat the disease underscoring the need for research into disease pathogenesis. 

Our laboratory has been studying the role of IFNg in the development of HP using the Saccharopolyspora rectivirgula (SR) animal model.  SR is a gram-positive thermophile that is commonly found in moldy hay and repeated inhalation leads to a form of HP known as Farmer’s lung.  C57BL/6 mice intranasally inoculated with SR for 3 days/week for 3 weeks develop an alveolitis that is initially neutrophilic but becomes more lymphocytic upon subsequent exposures. By the 3rd week of exposures mice develop granulomas, composed of monocytes and T lymphocytes surrounded by interstitial fibroblasts.  Studies using this animal model have suggested that IFNg plays an important role in the immunopathology.  IFNg is required for the development of granulomas in the lungs of mice following repeated exposure to SR.  One function of IFNg production is the expression of T cell chemokines that recruit T cells into the lung following SR exposure; in the absence of IFNg, T cell recruitment and subsequently granuloma formation are reduced.  These results have lead to two areas of research in the lab.  The first is to identify the cells and signaling pathways that regulate IFNg production following exposure to SR.  The second area of research is to identify the pattern recognition receptors (PRRs) that are involved in recruiting IFNg - producing immune cells into the lung. 

The first project in the lab focuses on identifying the cells that produce and/or regulate IFNg production following exposure to SR.  Our studies have revealed that neutrophils are a source of IFNg during the early stages of HP.  Intracellular cytokine staining performed on lung cells isolated from mice exposed to SR or saline revealed that approximately 30% of neutrophils expressed intracellular IFNg without additional in vitro stimulation with SR.  In addition, purified neutrophils expressed mRNA for IFNg as measured by RT-PCR and secreted IFNg protein into the culture supernatant following a 12 hr culture period.  Depletion of neutrophils in mice prior to exposure to SR resulted in significantly less IFNg mRNA expression than control mice exposed to SR.  Stimulation of IFNg production by neutrophils in vitro required IL-18 in combination with either IL-12 or IL-15.  Mice deficient in IL-18 signaling expressed less IFNg mRNA during the innate response to SR compared to WT mice suggesting that IL-18 plays a key role in induction of IFNg during HP.  Taken together these results suggest that neutrophils are a source of IFNg in the lung during the development of HP.  IFNg production in response to SR is dependent upon the transcription factor T-bet which also plays a role in Th1 cell development.  Mice deficient in T-bet develop a more severe form of HP as determined by increased collagen production and increased migration of Th17 cells into the lung.  Future studies are planned to determine the signaling pathways that lead to induction of T-bet and the mechanism by which T-bet inhibits disease severity.

The second project in the lab focuses on identifying the PRRs that are involved in recruiting neutrophils into the lung.  The production of pro-inflammatory cytokines and chemokines is dependent upon activation of the innate immune system by microbial products via PRRs.  These receptors make up a family of signaling receptors that recognize pathogen associated molecular patterns (PAMPs), which are conserved structures found almost exclusively on microbes.  The best-known family of PRRs is the Toll-Like-Receptors (TLRs); binding of PAMPs to TLRs leads to the recruitment of adaptor proteins to the receptor complex and induction of a signaling cascade that result in the activation of numerous pro-inflammatory genes.  MyD88 is the most commonly used adaptor protein; only TLRs 3 and 4 are not completely dependent upon it.  Stimulation of the MyD88 pathway leads to activation of the mitogen-activated protein kinase (MAPK) and NF-kB signaling pathways and is essential for pro-inflammatory cytokine and chemokine production such as TNF, IL-1, IL-12, IL-6, and IL-8.  Our research has determined that MyD88 is required for neutrophil recruitment following SR exposure. MyD88 KO mice have a significant decrease in neutrophils and myeloperoxidase (MPO) activity in the lung following SR exposure compared to WT mice.  Additionally, adherent cells isolated from MyD88 KO mice did not upregulate cytokine and chemokine mRNA in response to stimulation with SR compared to WT mice.  These results suggest that MyD88 is required for neutrophil recruitment into the lung following SR exposure and TLRs that use the MyD88 signaling pathway may play a role in the disease.  Two of the TLRs that use the MyD88 signaling pathway are TLR2 and TLR9.  TLR2 binds to a variety of ligands such as peptidoglycan, lipotechoic acids and lipoproteins from gram-positive bacteria and mycobacteria as well as yeast zymosan.  TLR9 is an intracellular receptor that binds to CpG DNA present in bacteria.  Our studies have demonstrated that mice genetically deficient in both TLR2 and TLR9 have reduced alveolitis following SR exposure compared to WT mice.  Long-term exposure of TLR2/9-/- mice to SR resulted in a less severe form of HP as measured by cytokine production and granuloma formation in the lungs in comparison to the WT mice and single TLR2-/- and TLR9-/- mice.  These results suggest that TLRs 2 and 9 cooperate in disease pathogenesis; however, since these double knockout mice are not completely deficient in neutrophil recruitment and cytokine production, these results suggest that additional receptors upstream of MyD88 also play a role.  Future studies will determine the individual roles of TLR2 and TLR9 as well as that of other MyD88-dependent TLRs that contribute to disease pathogenesis. 

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