The Role Of TLR4 In Mouse Macrophages Infected By M.vaccae

Nontuberculous mycobacterium(NTM) is a kind of environmental pathogens and widely exists in natural environment, which consists of Mycobacterium except MTC and M. leprae. In recent years, the bacterium has received attention with the increasing isolation rate of NTM in animals and humans infected. However, little attention is paied to the pathogenic mechanism of NTM, whereas most studies focus on the epidemiological investigation, drug resistance analysis and differential diagnosis. Macrophage plays a significant role in the host defense mechanism of Mycobacterium tuberculosis. Interaction of specific mycobacterial ligands with Toll-like receptor(TLR) leading to macrophage activation which triggers signaling pathways, such as mitogen-activated protein kinase(MAPK). It mediates translocation of the nuclear transcription factor Nuclear Factor-Kappa B(NF-κB) into the nucleus. NF-κB promotes transcription of the genes of host innate immune defense, subsequent expression of proinflammatory cytokines and chemokines. Secretion of cytokines regulates the innate immune response as well as initiates the adaptive immune response to MTB. In order to investigate whether Mycobacterium vaccae ligands with TLR and leads to macrophage activation which triggers signaling pathways, this study has been carried out as follows:Firstly, we had recultivated and identified the Mycobacterium vaccae which was isolated from the mesenteric lymph nodes of livestock, and then established the model of murine macrophage infected with the bacterium. We tested the activation role of M.vaccae on the expression of TLR4, TNF-α and IFN-γ in RAW264.7 cells by qRT-PCR and ELISA. The result showed that RAW264.7 cells exposed to M.vaccae produced significantly higher levels of TLR4,TNF-α and IFN-γ in a close-dependent manner than control groups. These data suggested the activation role of M.vaccae on the expression of cytokines was liganded by TLR4 signaling pathways in murine macrophage.We also tested the activation role of M.vaccae on the expression of IL-4, IL-10, IL-12,IL-1β, TNF-α and IFN-γ in RAW264.7 cells by ELISA. The result showed that RAW264.7 cells exposed to M.vaccae produced significantly higher levels of IL-4, IL-10, IL-12, IL-1β, TNF-αand IFN-γ in a close-dependent manner than control groups. Furthermore, the expression of IL-4and IL-10 were inhibited, and IL-1β was significantly increased in RAW264.7 cells when TLR4 signals were blocked by treating with TLR4 inhibitor. These data showed the activation role ofM.vaccae on the expression of IL-4 and IL-10 were liganded by TLR4 signaling pathways in murine macrophage. In addition, the expression of IL-1β was inhibited with the activation of TLR4. We examined whether M.vaccae could trigger the MAPK and NF-κB signaling pathways which liganded with TLR4 by qRT-PCR and Western Blot. Our results showed that M.vaccae could activate the expression of MyD88, TRIF and TRAF6 mRNA in RAW264.7 cells.Moreover, the expressions of MyD88 and TRAF6 mRNA were significantly inhibited when TLR4 signals were blocked by treating with TLR4 inhibitor in RAW264.7 cells. M.vaccae could activate the p38 kinase(p38), extracellular-regulated kinase(ERK) and c-Jun N-terminal kinase(JNK) pathway. After treating with TLR4 inhibitor, the phosphorylation of p38 and ERK1/2were significantly reduced in RAW264.7 cell. M.vaccae could also activate the NF-κB signals.The expression of NF-κB was increased after treating with TLR4 in RAW264.7 cell. These data showed that the activation role of M.vaccae on the expression p38 and ERK1/2 were liganded by TLR4 signaling pathways in murine macrophage. In addition, M.vaccae could inhibit the production of NF-κB by activating TLR4.In summary, our study firstly described the mechaisms between NTM and macrophage,which was induced by M.vaccae interacting with macrophages. TLR4 played a key role in RAW264.7 cells against M.vaccae infection, containing the expression of cytokines and transduction of signal pathways. Thus, this research provides scientific data in anti-NTM infection of hosts and a theoretical basis for the study of immune mechanism in NTM disease.