Mycobacterium tuberculosis heat-shock protein 16.3 activates macrophages probably through TLR4/MyD88 pathway

Tuberculosis is a chronic disease caused by Mycobacterium tuberculosis (Mtb), and many previous studies indicated that Hsp16.3 may play an important role in long-term survival of Mtb within macrophages. However, the underlying mechanism of Hsp16.3 in protecting of Mtb is not clear. In this study, we constructed a prokaryotic expression vector, pET28a-Mtb Hsp16.3, which induced the expression of Mtb Hsp16.3 and purified the fusion protein through nickel-affinity chromatography. Subsequently, qPCR and EIA assays were used to assess the expression of TNF-α and IL-10 in RAW264.7 cells after treated with Mtb Hsp16.3 in vitro. Finally, we evaluated the impact of small interference RNA-mediated TLR4 silencing on Mtb Hsp16.3-induced macrophage activation, and further confirmed its molecular mechanism. We found that Mtb Hsp16.3 was successfully purified and could activate macrophages by enhancing the levels of TNF-α and IL-10 in vitro. In addition, knockdown of TLR4 via using siTLR4 could obviously reduce TNF-α and IL-10 expression and significantly increase the levels of IL-12 and IL-4. Moreover, Mtb Hsp16.3 could enhance the activation of MYD88-NF-κB signaling in RAW264.7 cells in a concentration dependent manner. Our data suggests that Mtb Hsp16.3 could activate macrophage through TLR4-NF-κB signaling pathway, providing a basis for further studies on the biological activity of Mtb Hsp16.3 and its role in the pathogenesis of TB.