Abstract
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.
Highlights
Mycobacterium bovis is a member of the Mycobacterium tuberculosis complex (MTBC), which causes a significant zoonosis constituting a public health problem[1]
Endotoxin contamination of prepared rMtb9.8 was removed by exposure of the material to Triton X-114, we investigated whether its pro-inflammatory activity was due to LPS contamination by measuring the sensitivity of the rMtb9.8 effect to polymyxin B (PB). rMtb9.8 was pre-treated with 10 μg/ml PB to inhibit any possible LPS-induced increase in TNF-α and IL-1β production
IL-1β production is significantly increased in tuberculosis pleurisy, which usually presents as a self-resolving type of primary tuberculosis[38]
Summary
Mycobacterium bovis is a member of the Mycobacterium tuberculosis complex (MTBC), which causes a significant zoonosis constituting a public health problem[1]. During the early stages of M. tuberculosis infection, macrophages serve as the main effector cells that are involved in the activation of cytokine production in a complex process of cross-regulation that limits bacterial survival and proliferation[5]. This cytokine network plays a crucial role in the inflammatory response and in the outcome of mycobacterial infections[6]. The results of this study indicate that rMtb9.8 is able to induce the production of TNF-α and IL-1β in RAW264.7 cells and that it activates the NF-κB pathway through TLR2-mediated signaling, thereby contributing to the induction of IRF-1
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