RTB10.4of Mycobacterium Bovis Mediated By TLR2Activating RAW264,7Pathway And Its Expression Of Cytokine

Macrophages play a significant role in the host defense mechanism of Mycobacterium bovis,Toll-like receptors (TLR) are key molecules involved in activating macrophages, Several proteinkinases, such as mitogen-activated protein kinases (MAPKs) and the nuclear translocation of theNuclear Factor-Kappa B (NF-κB) are also activated via the TLR signaling pathway，stimulating thetranscription and production of IL-1, IL-6and TNF-, activating the innate immune response. Toevaluate whether rTB10.4interact with Toll-like receptors (TLRs) and the signaling pathways for activeinflammation, this study has carried out mainly works as the following aspects:Firstly, we tested the activation role of M. bovis-derived recombinant TB10.4protein (rTB10.4) onthe expression of TNF-, IL-6, and IL-12p40in RAW264.7cells by ELISA. The result showed thatRAW264.7cells, exposed to rTB10.4, produced significantly higher levels of TNF-, IL-6, and IL-12p40in a dose-dependent manner than control groups. Furthermore, the expressions of TNF-, IL-6, andIL-12p40were inhibited in RAW264.7cells when TLR2signals were blockaded by treating withanti-TLR2. However, TLR4had no such effects after treating with anti-TLR4. These data suggested thatthe activation role of rTB10.4on expression of cytokines was mediated by TLR2signals.We examined whether rTB10.4could activate the MAPK pathway and then effect the expressionsof cytokines by the methods of Western Blot and Image Stream100. Our results showed that rTB10.4could activate the p38kinase (p38) and extracellular-regulated kinase (ERK) signals and induce thenuclear translocation of p38and ERK. However, rTB10.4had no effects on phosphorylation of JNKpathway. After treated with anti-TLR-2, the phosphorylations of p38and ERK1/2were significantlyreduced in RAW264.7cells. The inhibition of p38and ERK activity with inhibitor of SB203580(p38)or U0126(ERK1/2) had a significant effect on rTB10.4-induced secretion of TNF-, IL-6, and IL-12p40.To investigate whether TLR2could contribute to rTB10.4induces the activation of NF-κB byNF-κB luciferase activity detection. RAW264.7cells transfected with a luciferase reporter generevealed that rTB10.4stimulation significantly activated the NF-κB luciferase activity. Then wemeasured the rTB10.4-induced phosphorylation and nuclear translocation of NF-κB p65by Westernblot analysis，these data suggest that rTB10.4mediates the activation of NF-κB p65by triggering thedegradation of IκB in the cytoplasm in time-dependent manner. Then, we detected the ranscriptionalcompetence of nuclear NF-κB p65by rTB10.4stimulation using the ImageStream analysis, the resultsshowed that translocation of NF-κB p65was observely induced by rTB10.4. Furthmore, we pre-treatedRAW264.7cells with a specific NF-κB inhibitor BAY-117082. The production of TNF-or IL-12p40dramatically blocked by BAY-117082. These observations suggested that TB10.4induced theproduction of TNF-, IL-12p40, and IL-6via the NF-κBsignalling pathway.In summary, our findings provide evidence that rTB10.4promotes the production of TNF-, IL-6,and IL-12p40in RAW264.7cells via TLR2, and that the p38MAPK, ERK1/2and NF-κB pathways were essential to this process. Thus, our studies provide a better understanding of the molecularmechanisms influencing host/pathogen interactions necessary to prevent Mycobacterium bovisinfection.