Date of Award

12-2013

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

Elizabeth A. Fitzpatrick, Ph.D.

Committee

Maureen A. McGargill, Ph.D. Edward F. Rosloniec, Ph.D. Christopher M. Waters, Ph.D. Ae-Kyung Yi, Ph.D.

Keywords

Chronic hypersensitivity pneumonitis, Hypersensitivity pneumonitis, TLR2, TLR2/9, TLR9, Toll-like receptors.

Abstract

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.

DOI

10.21007/etd.cghs.2013.0015

Comments

Two year embargo expired December 2015

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