The Mtb9.8 antigenic protein of Mycobacterium bovis/Mycobacterium tuberculosis has been identified as a target of the T-cell response. However, the interaction of Mtb9.8 with Toll-like receptors (TLRs) and the relevant signaling pathways have not been fully clarified. In this study, recombinant Mtb9.8 (rMtb9.8) derived from M. bovis-stimulated RAW264.7 cells initiated the secretion of TNF-α and IL-1β in a dose-dependent manner. Blocking assays show that TLR2-neutralizing antibody decreases the production of TNF-α and IL-1β. Moreover, NF-κB activation is associated with TNF-α and IL-1β production by rMtb9.8 stimulation, and rMtb9.8 stimulation also induces the phosphorylation of NF-κB p65 at Ser536 and its rapid nuclear translocation in RAW264.7 cells. Furthermore, NF-κB luciferase activity is rapidly activated in response to rMtb9.8 in RAW264.7 cells and is also significantly increased in rMtb9.8-induced HEK293-TLR2. However, these activations were abrogated in cells with a dominant-negative mutation of NF-κB p65 and by treatment with anti-TLR2 antibody. We also find that rMtb9.8 induces the activation of IRF-1. These findings indicate that M. bovis-derived rMtb9.8 activates the NF-κB pathway via TLR2 in RAW264.7 cells. In particular, it phosphorylates NF-κB p65 at Ser536 and induces nuclear translocation, thereby leading to the production of TNF-α and IL-1β, which correlates with the induction of IRF-1.
The TB9.8 of Mycobacterium bovis can induce strong antigen-specific T-cell responses in proliferation assays and IFN-γ assays. However, whether and how TB9.8 activates innate immune cells remain unclear. Therefore, recombinant protein TB9.8 (rTB9.8)-induced proinflammatory cytokine profile by RAW264.7 cells was investigated and the related signaling pathway was studied. Stimulation with rTB9.8 triggered RAW264.7 cells to produce IL-6 and IL-12 p40. In addition, rTB9.8 activated the mitogen-activated protein kinase (MAPK) cascade in RAW264.7 cells by inducing the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase (p38) and also promoted nuclear translocation of phosphorylated p38 and ERK1/2. Furthermore, rTB9.8 activated nuclear factor κB (NF-κB) signaling pathway by inducing p65 translocation into the nucleus and the phosphorylation of IκBα in the cytosol. Blocking assays showed that specific inhibitors of ERK1/2, p38, and IκBα can significantly reduce the expression of IL-6 and IL-12 p40, which demonstrated that rTB9.8-mediated cytokine production is dependent on the activation of these kinases. Thus, this study demonstrates that rTB9.8 can activate RAW264.7 and trigger IL-6 and IL-12 p40 production via the ERK, p38, and NF-κB signaling pathways.
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