TLR-2 mediated cytosolic-Ca²⁺ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-α production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages.

Abstract

The implications of TLR-2 mediated alterations in cytosolic-Ca²⁺((Ca²⁺)_c) levels in M. smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus, we observed TLR-2 signalling is required in the phagocytosis of M. smegmatis. M. smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca²⁺)_c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca²⁺)_c-surge triggered downstream ER-stress and superoxide (O₂^-) generation. The cross-talk between ER-stress and O₂^- amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O₂^--NO axis imperative in M. smegmatis-induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca²⁺)_c-surge and ensuing cross-talk between ER-stress and O₂^- potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.