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"TOLL-LIKE RECEPTOR 2 REGULATION OF HOST IMMUNITY TO Mycobacterium tuberculosis"

by
Amanda McBride
MD/PhD Program
B.S. 2001, Rutgers University



Thesis Advisor: Padmini Salgame, Ph.D.
Professor
Department of Medicine

Thursday, April 21, 2011
MSB C-600, 12:00 P.M.


Abstract

Disease caused by the pulmonary pathogen Mycobacterium tuberculosis (Mtb) remains a major global health threat. Genetic studies have correlated single nucleotide polymorphisms within the Toll-like receptor (TLR) 2 gene with susceptibility to tuberculosis disease, suggesting an important role for TLR2 in host defense against Mtb. However, while there is substantial in vitro evidence that Mtb-TLR2 interactions have profound effects on antigen presenting cell (APC) functions, in vivo studies have thus far not been able to clarify the role of TLR2 in the host response to Mtb. Therefore, the overall focus of this study is to investigate mechanisms downstream of TLR2 in vivo which modulate immune responses during Mtb infection.
To clarify the function of TLR2 in the context of innate and adaptive immunity to Mtb, the goal of the first part of this study was to address the effects of TLR2 engagement in vivo on APC functions, as well as subsequent contributions to secondary (memory) responses to Mtb. The response of TLR2-deficient (TLR2 knockout [TLR2KO]) mice to primary and secondary aerosol challenges with the Erdman strain of Mtb was compared to that of wild-type (WT) mice based on assessment of Mtb-specific T helper 1 (Th1) cell development, bacterial burden, recruited T cell phenotype, and granulomatous response. Following both primary and secondary Mtb challenge, TLR2KO mice displayed similar kinetics and magnitudes of Mtb-specific interferon-gamma (IFN-) producing T cell development in the draining mediastinal lymph nodes (MLNs) and lungs. Upon re-challenge with Mtb, both WT and TLR2KO immune mice displayed similarly enhanced resistance to infection compared to their na´ve counterparts. The phenotypes of recruited T cells and the granulomatous responses in the lungs were also similar between WT and TLR2KO immune mice. Further, at early time points following primary challenge with Mtb, TLR2KO and WT mice displayed similar expression levels of interleukin-10 (IL-10) and the p40 subunit of interleukin-12 (IL-12p40) in the MLNs and in sorted lung APC populations. Together, these findings conclusively indicate that TLR2 signals do not modulate APC functions during the early host response to Mtb and do not influence memory immunity to Mtb.
Previous studies of TLR-deficient mice have yielded variable reports on their relative resistance to Mtb infection. Therefore, the goal of the second part of this study was to address the role of TLR2 during control of chronic Mtb infection. Following aerosol challenge with Mtb, TLR2KO mice were compromised in their ability to control bacterial burden during later stages of infection and prevent reactivation of chronic disease. While compact granulomas were observed in WT lungs, enhanced pulmonary inflammation and disorganized granuloma architecture were evident in TLR2KO lungs as infection progressed. The escalating lung pathology in TLR2KO mice was associated with increased overall cellular infiltration and a paradoxical reduction in the accumulation of regulatory T cells (Tregs) in the lungs as compared to WT. Examination of Rag2-deficient mice reconstituted with WT or TLR2KO T cells demonstrated that direct TLR2 signals in Tregs does not influence their expansion or accumulation in the lungs during Mtb infection. However, gene expression and protein analysis of TLR2KO lungs revealed decreased levels of CCL22, a chemokine crucial for mediating Treg recruitment to sites of inflammation. Together, these findings indicate a novel role for TLR2 in limiting inflammation during the control of chronic Mtb infection by mediating Treg recruitment to the Mtb granuloma.
Given the observation that TLR2KO mice display disorganized granuloma architecture during chronic Mtb infection, the goal of the third part of this study was to identify potential pathways downstream of TLR2 that may be involved in granuloma maintenance. Through genome-wide microarray analysis, we identified genes that were differentially regulated in TLR2KO lungs as compared to WT during Mtb infection. Most significantly, differentially regulated genes were found to be involved in cellular movement and immune cell trafficking and included genes encoding matrix metalloproteinases (MMPs). MMP-12, in particular, was found to be up-regulated during Mtb infection in a TLR2-dependent fashion. Examination of MMP-12-deficient (MMP-12 knockout [MMP12KO]) mice following Mtb aerosol challenge demonstrated overall decreased lung cell numbers as compared to WT, with no adverse effects on control of bacterial burden. These findings indicate a novel role for TLR2 in regulating production of MMPs during host responses to Mtb and identify a contribution of TLR2-induced MMP-12 in cellular recruitment to the lungs during Mtb infection.
Although TLR2 has been shown to induce opposing effects on APC functions, whether it preferentially signals in the context of heterodimers with TLR1 or TLR6 to regulate different functions is unknown. Further, it is unknown if signaling through TLR2/1 dimers modulates inflammatory responses differently than TLR2/6 dimers in vivo. Therefore, the final goal of this study was to determine the relative contributions of the TLR2 co-receptors TLR1 and TLR6 to TLR2-mediated control of host responses to Mtb. Analysis of cytokine responses to Mtb by APCs in vitro indicated an equivalent contribution of TLR1 and TLR6 to TLR2-mediated APC responses. To determine the in vivo contributions of these co-receptors, TLR1KO and TLR6KO mice were challenged with Mtb via aerosol along with control WT and TLR2KO mice. Comparison of lung bacterial burden demonstrated equivalent control of Mtb in the absence of either TLR as compared to WT. Further, while the granulomatous response was disorganized in TLR2KO lungs during chronic infection, both TLR1KO and TLR6KO mice exhibited focal, organized granulomas. These results indicate that TLR1 and TLR6 play redundant roles in TLR2-mediated host responses to Mtb.
Overall, the results of this study demonstrate that TLR2 responses to Mtb are necessary for generating appropriate control of Mtb infection by the host. The findings indicate that TLR2 signals are not required for early innate immune responses and initiation of primary and secondary adaptive immune responses. However, this study has uncovered a critical role for TLR2 in limiting excessive inflammation and maintaining granulomatous organization.


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