Kallikrein 12 regulates innate resistance of murine macrophages against Mycobacterium bovis infection by modulating autophagy and apoptosis

Aims and objectives: Tuberculosis (TB) is a leading infectious cause of mortality worldwide and millions of people continue to develop active TB each year. Mycobacterium bovis (M. bovis) is a member of Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis and imposing high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases and their role is established in various physiological and pathological processes. Since the KLKs expression has been linked to various cancerous, degenerative and infectious conditions, it is likely that KLKs expression may mediate host immune response against M. bovis infection. Methods: In the current study, we investigated the expression of KLK12 in M. bovis infection in vivo and in vitro by using C57BL/6 mice and two types of murine macrophages, RAW264.7 cells and bone marrow derived macrophages (BMDMs). In this study, we used two strains of M. bovis, M. bovis C68004 strain and M. bovis N strain. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown BMDMs by using siRNA transfection. We investigated the expression of various proteins through QRT-PCR, Immunoblot and ELISA. Apoptotic cells were detected by flow cytometry. Furthermore, cell viability and phagocytic ability of macrophages was also determined after transfection and infection. CFU assay was performed to know the intracellular survival of M. bovis. Results: Our results exhibited an increase in the expression of KLK12 in the lung and spleen of M. bovis infected mice. A dose (MOI) and time dependent up-regulation was also noticed in RAW264.7 and BMDMs. Then, we evaluated the role of KLK12 in innate immune response regulation against M. bovis. Interestingly, knockdown of KLK12 resulted into significant down regulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were also significantly down regulated in KLK12 knockdown macrophages but difference in IL-10 and IFN-β expression was non-significant. Our finding also depicted that knockdown of KLK12 enhances the intracellular survival of M. bovis. Conclusion: Taken together, these findings suggest that up-regulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine and human TB.