Investigation Of Molecular Mechanism Of Mycobacterium Tuberculosis ManLAM Inducing IL-10 Producing B Cells And Regulating Th2 Immunity

Background:Tuberculosis (TB) remains a serious threat to public health, it ranks alongside HIV/AIDS as a leading cause of death worldwide. Recently, emerging evidences suggest that B cells could orchestrate the immune response against Mycobacterium tuberculosis (M. tb) by interacting with other immune cells in M. fb-infected mouse, nonhuman primates and humans. Therefore, the roles of B cells involved in anti-M. tb immunity need to be further clarified. A subset of B cells, IL-10-producing B cells (B10 cells), has been shown to negatively regulate cellular immune responses in infectious diseases caused by intracellular pathogens, including hepatitis B virus, HIV-1 and Listeria. However, the B10 cell roles in TB remains elusive. In addition, mannose-capped lipoarabinomanan (ManLAM), the main cell wall component and immunosuppressive epitope of M. tb, could induce IL-10 secretion of DCs, macrophages, as well as CD4+T cells. And more importantly, Toll-like receptor 2 (TLR2) has been incriminated as major signaling receptor that binds to ManLAM. Some studies also demonstrate that induction of B10 cells critically depends on intrinsic Toll-like receptors (TLRs) signaling.Objective:In this study, we want to explore whether M. tb and ManLAM could stimulate B cells to secrete IL-10, and demonstrate the detailed molecular mechanisms. Then, we want to further evaluate the regulatory effects of ManLAM-induced B10 cells on CD4+ T cell polarization in M.tb-immunized mice model, and further revalidate in different disease models, including autoimmune diseases and cancer.Method:In the present study, B10 cells was firstly analyzed by flow cytometry (FCM) in paripherial boold samples of TB patients. With the use of FCM, ELISA and qRT-PCR, ManLAM-induced B10 cells were detected in in vitro and in vivo mouse models. Molecular mechanisms involved in ManLAM-induced IL-10 production in B cells was analyzed by gene interference, western blotting, qRT-PCR, ELISA, FCM etc. The regulatory effects of ManLAM-induced B10 cells on CD4+T cell polarization in M. ^-immunized mice model, inflammatory bowel disease (IBD) mice model and hepatocellular carcinoma (HCC)-bearing mice model were analyzed by Transwell, HE stain, FCM etc.Results:The frequency of B10 cells in peripheral blood was significantly elevated in TB patients compared with healthy donors. Heat inactivated M. tb strains (H37Rv, BCG) and their ManLAM could induce IL-10 production of mouse B cells both in vitro and in vivo with the use of ManLAM-specific aptamer ZXL1 and ManLAM-deficient BCG strain (BCGA2196). Low concentration of ManLAM (0-10 ng/ml) mainly depended on TLR2 to induce IL-10 production of B cells, while depended on mannose receptor (MR) at relative higher concentration (10 ng/ml-10 ?g/ml). Molecular mechanisms analysis revealed that ManLAM increased ubiquitin specific protease 40 (USP40) expression, activated PI3K/AKT pathway by binding to TLR2 of B cells, and thus promoted downstream API and NF-?B signaling pathway to enhance IL-10 production.ManLAM-induced B10 cells inhibit Thl polarization of mouse CD4+T cells but promote Th2 polarization in vitro, and hindered the Thl immune response upon vaccination with inactive M. tb in in vivo mouse model. In mouse inflammatory bowel disease (IBD) and HCC-bearing mouse models, ManLAM-treated B cells significantly decreased IFN-y production of CD4+T cells in draining lymph nodes and enhance IL-4 production in an IL-10 dependent manner. The attenuation of the inflammatory leision in colitis and promotion of tumor growth may be attributed to suppression of pathogenic or protective Thl response by IL-10.Conclusions:Collectively, in this study, we have shown that ManLAM from M. tb induces B10 cells both in vitro and in vivo mouse model. Molecular mechanism analysis revealed that ManLAM increases ubiquitin specific protease 40 (USP40) expression, activates PI3K/AKT pathway by binding to TLR2 of B cells, and thus promotes downstream API and NF-?B signaling pathway to enhance IL-10 production. ManLAM-induced B10 cells skew CD4+T cells toward a Th2 subtype in inactivated M. tb immunization, IBD and tumor-bearing mice models. Here, our finding provides a novel mechanism in which M. tb ManLAM exploits B10 cells to negatively regulate anti-TB immunity.