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"Novel Insights into Host Immunity
Against Mycobacterium Tuberculosis"

Jill Konowich
MD/PhD Program
B.S. 2006, Massachusetts Institute of Technology, Cambridge, MA

Thesis Advisor: Padmini Salgame, PhD
Professor, Department of Medicine

Friday, May 15, 2015
2:30 P.M., MSB C-600


Arguably the most successful pathogen of all time, Mycobacterium tuberculosis (Mtb) has persisted as a major global health threat due both to the high adaptability of Mtb as well as the emergence of drug resistant strains. Incidence of this highly communicable disease begins with inhalation of Mtb laden respiratory droplets. Mtb initially encounters alveolar macrophages expressing membrane-bound Toll-like Receptors (TLRs), including TLR2. TLR2 warrants particular attention because it recognizes a multitude of Mtb ligands. Moreover, polymorphisms within the TLR2 gene are associated with increased susceptibility to developing tuberculosis and reduced TLR2 expression. Evidence validates the considerable role of TLR2 in the host response to Mtb. Mtb-ligand driven activation of TLR2 modulates antigen presenting cell (APC) functionality and regulates the production of both pro- and anti-inflammatory cytokines essential to eliciting an adaptive immune response and eliminating the bacilli. Notwithstanding published data, the role of TLR2 in host immunity against Mtb is far from elucidated. My thesis projects, therefore, explore the contribution of TLR2 signaling in modulating immune responses to Mtb through: (I.) determining the role of TLR2 on nonhematopoietic- and hematopoietic- derived cells; (II.) understand TLR2s role in the granulomatous response; and (III.) investigate how TLR2 activate kinases which modulate macrophage antimycobacterial functions.
We have demonstrated that TLR2 is critical for host resistance against Mtb infection. However, which cell types are key players in this response remain unknown. TLR2 is expressed on leukocytes and stromal cells, including alveolar epithelium. Accumulating evidence has illustrated that alveolar epithelial cells have critical functions in host defense against TB. The contribution of TLR2 on stromal cells has yet to be addressed. To decipher TLR2s contribution on nonhematopoietic and hematopoietic cells over the course of Mtb infection, we transplanted congenically labeled bone marrow into lethally irradiated mice. Chimeric mice lacking TLR2 on either the hematopoietic (H-KO), nonhematopoietic (NH-KO), or both (TLR2KO) compartments were infected with Mtb Erdman. A WT chimeric mouse with TLR2 on both compartments was also generated as a control. All groups were evaluated based on bacterial burden, dissemination, cellular recruitment, gene expression, and granulomatous response. The H-KO mice exhibit increased bacterial burden and inflammation, decreased accumulation of Tregs, and disruption of the granuloma architecture. Thus, TLR2 signaling on the hematopoietic compartment is crucial for control of chronic murine Mtb infection. In contrast, the NH-KO mice displayed noticeably reduced granulomatous inflammation, reduced extrapulmonary dissemination, and decreased cellular recruitment to the lung. Thus, TLR2 signaling on the nonhematopoietic compartment controls inflammation and recruitment of the innate immune response.
These observations illuminate a novel role for TLR2 on nonhematopoietic cells and demonstrate a paradox within TLR2 signaling. TLR2 on hematopoietic cells plays a protective role by controlling infection and mitigating inflammation, while its role on nonhematopoietic cells is deleterious by promoting inflammation induced immunopathology and dissemination. Preservation of this delicate balanced response is essential to maintaining host control of TB and could lead to the development of cell-specific TLR2 inhibitors.
Chronic Mtb infection in the absence of TLR2 results in pneumonitis and disorganized granuloma architecture. The characteristic accumulation of Foxp3+ T regulatory cells (Tregs) in the tubercule granulomas was significantly reduced in TLR2KO mice and independent of TLR2 expression on Tregs. Given that myeloid cells release chemokines that attract Tregs to areas of inflammation, we investigated this potential role of TLR2 on myeloid cells. Prior to infection, TLR2KO mice were adoptively transferred with either TLR2 expressing WT BMDMs (WT_TLR2KO mice) or TLR2KO BMDMs (TLR2KO_TLR2KO mice). At 7 weeks post-infection, WT_TLR2KO mice displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages (TLR2KO_TLR2KO mice). However, the pulmonary bacterial burden between the two groups was not remarkable indicating that TLR2s role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. These findings reveal a novel role for TLR2 in mediating the recruitment of Foxp3+ Tregs to the lungs to control inflammation-induced pathology and help maintain a stable granuloma.
However, the mechanism behind Treg accumulation in Mtb infection remains uncertain. CCR4 is essential for T cell migration to sites of inflammation, including the lungs, and is highly expressed on Tregs. Considering that TLR2KO mice adoptively transferred with WT macrophages had significantly increased accumulation of Tregs coupled with increased levels of CCL22, we examined if loss of CCL22 or CCR4 prevent Treg accumulation during Mtb infection. Loss of CCL22 alone was not sufficient to prevent Treg accumulation. Potentially, CCR4 independent of a CCL22 gradient could control the recruitment of Tregs in response to Mtb infection. To investigate this, WT and CCR4KO mice were infected with Mtb, revealing that the absence of CCR4 resulted in improved control of Mtb infection and altered the kinetics of cellular recruitment. CCR4 appears to not only control T cell and Treg accumulation but also control the trafficking of leukocytes to the lungs. Loss of CCR4 also influences both the magnitude and timing of the TH1 adaptive immune response, making CCR4 a promising target for both small molecule inhibitors and vaccine development.
Although a vast array of mycobacterial moieties recognized by TLR2 induces a predominantly pro-inflammatory and TH1 polarized response, TLR2 signaling can lead to suppression of APCs functions. In the absence of TLR2, our group has demonstrated that the production of both anti-inflammatory cytokine interleukin 10 (IL-10) and pro-inflammatory cytokine interleukin 12 (IL-12) is ablated in Mtb-infected murine macrophages. TLR2 governs a delicate cytokine balance between IL-12, which promotes combating the pathogen, and IL-10, which prevents destruction of host tissues. This duality in TLR2 function extends beyond the macrophage, as evidenced by our TLR2 chimerism studies. While these opposing forces provide an indispensable self-regulation of the immune system, successful pathogens such as Mtb have evolved ways to override the immune system and promote their survival. Advantageously utilizing this duality, Mtb secretes virulence factors able to exploit this counterbalance in favor of immunosuppression. Extracellular-regulated kinase (ERK)1/2 is triggered downstream of TLR2 by Mtb infection and has been shown to control the differential production of IL-10 and IL-12. Furthermore, IL-10 induction is ERK1/2-dependent while blocking ERK1/2 enhances IL-12 production from Mtb-infected macrophages. Based on these findings, we surmised that inhibiting ERK1/2 could adventitiously shift TLR2 signaling in favor of IL-12 production and enhance macrophage mediated dispatching of Mtb. We blocked phosphorylation of ERK1/2 by using the inhibitor PD98059 (Pd) and performed intracellular Mtb growth assays in various types of macrophages, including murine bone marrow derived macrophages (BMDMs), murine peritoneal macrophages, and human monocytes. Inhibiting ERK1/2 in all of the above populations resulted in significantly improved control of Mtb growth. Inhibition of ERK1/2 in macrophages also resulted in a concomitant increase in IL12p40 expression and a decrease in IL-10 expression. Furthermore, ERK1KO macrophages infected with Mtb also exhibited improved control of bacteria. A mixed bone marrow chimera was generated with a 50:50 mix of ERK1KO and WT hematopoietic cells in a WT recipient mouse to evaluate in vivo differences in bacterial burden within the macrophage populations. Preliminary results suggest an early reduction in Mtb in ERK1KO macrophages. However, a more targeted model resulting in loss of just ERK1KO is necessary to full explore these differences. Therefore, ERK1/2 regulation may prove essential to boosting host immunity and improving clearance of Mtb infection.
Overall, the results of this study demonstrate the essential and multi-faceted role that TLR2 plays in host defense against Mtb. Our findings indicate that TLR2 on hematopoietic cells is crucial for control of bacterial burden, accumulation of Tregs, and maintenance of the dynamic granulomatous response. Macrophages play the most contributory role as instillation of TLR2+ macrophages into a TLR2-deficient host resulted in restoration of the WT phenotype. Furthermore, our in vitro studies of Mtb infected macrophages support that inhibition of downstream TLR2 signaling results in improved control of Mtb. We have also discovered that TLR2 on nonhematopoietic cells modifies the innate response and contributes towards inflammation. Thus, this dichotomous role in TLR2 signaling protects the host from Mtb infection but also allows Mtb to establish a foothold within the lungs. Future studies deciphering these pathways are necessary to understand TB pathogenesis and to develop more

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