Recombinant TB9.8 of Mycobacterium bovis Triggers the Production of IL-12 p40 and IL-6 in RAW264.7 Macrophages via Activation of the p38, ERK, and NF-κB Signaling Pathways
- 400 Downloads
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.
KEY WORDSTB9.8 Mycobacterium bovis RAW264.7 cell MAPK NF-κB signaling pathways
This work was supported by the National Science Foundation of China (No. 31302130), the Special Fund for the Agricultural Science and Technology Innovation Program (ASTIP-IAS-11), and the National High Technology Research and Development Program of China (863 Program) (No. 2012AA101302). The authors are especially grateful to the imaging application scientist Miao Lin from Merck Chemicals (Shanghai) Co., Ltd., Beijing Branch.
Conflict of Interest
The authors have no financial conflicts of interest.
- 8.Magee, D.A., K.M. Conlon, N.C. Nalpas, J.A. Browne, C. Pirson, C. Healy, et al. 2014. Innate cytokine profiling of bovine alveolar macrophages reveals commonalities and divergence in the response to Mycobacterium bovis and Mycobacterium tuberculosis infection. Tuberculosis (Edinburgh, Scotland) 94: 441–450.CrossRefGoogle Scholar
- 14.Chan, E.D., K.R. Morris, J.T. Belisle, P. Hill, L.K. Remigio, P.J. Brennan, et al. 2001. Induction of inducible nitric oxide synthase-NO by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-κB signaling pathways. Infection and Immunity 69: 2001–2010.CrossRefPubMedCentralPubMedGoogle Scholar
- 18.Kang S.R., Han D.Y, Park K.I., Park H.S., Cho Y.B., Lee H.J., et al. 2011. Suppressive effect on lipopolysaccharide-induced proinflammatory mediators by Citrus aurantium L. in macrophage RAW 264.7 cells via NF-kappaB signal pathway. Evidence-based complementary and alternative medicine: eCAM 2011Google Scholar
- 19.Oh, Y.C., Y.H. Jeong, J.H. Ha, W.K. Cho, and J.Y. Ma. 2014. Oryeongsan inhibits LPS-induced production of inflammatory mediators via blockade of the NF-kappaB, MAPK pathways and leads to HO-1 induction in macrophage cells. BMC Complementary and Alternative Medicine 14: 242.CrossRefPubMedCentralPubMedGoogle Scholar
- 20.Kumar, A., R. Murphy, P. Robinson, L. Wei, and A.M. Boriek. 2004. Cyclic mechanical strain inhibits skeletal myogenesis through activation of focal adhesion kinase, Rac-1 GTPase, and NF-kappaB transcription factor. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology 18: 1524–1535.CrossRefGoogle Scholar
- 21.Li, W., Q. Zhao, W. Deng, T. Chen, M. Liu, and J. Xie. 2014. Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling. PLoS ONE 9: e94418.CrossRefPubMedCentralPubMedGoogle Scholar
- 23.Ilghari, D., K.L. Lightbody, V. Veverka, L.C. Waters, F.W. Muskett, P.S. Renshaw, et al. 2011. Solution structure of the Mycobacterium tuberculosis EsxG⋅EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes. Journal of Biological Chemistry 286: 29993–30002.CrossRefPubMedCentralPubMedGoogle Scholar
- 24.Billeskov, R., C. Vingsbo-Lundberg, P. Andersen, and J. Dietrich. 2007. Induction of CD8 T cells against a novel epitope in TB10.4: correlation with mycobacterial virulence and the presence of a functional region of difference-1. Molecular Immunology 179: 3973–3981.Google Scholar
- 25.Skjot, R.L., I. Brock, S.M. Arend, M.E. Munk, M. Theisen, T.H. Ottenhoff, et al. 2002. Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family. Infection and Immunity 70: 5446–5453.CrossRefPubMedCentralPubMedGoogle Scholar
- 28.George, T.C., S.L. Fanning, P. Fitzgerald-Bocarsly, R.B. Medeiros, S. Highfill, Y. Shimizu, et al. 2006. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. Journal of Immunological Methods 311: 117–129.CrossRefPubMedGoogle Scholar
- 30.Jung, S.-B., C.-S. Yang, J.-S. Lee, A.-R. Shin, S.-S. Jung, J.W. Son, et al. 2006. The mycobacterial 38-kDa glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes. Infection and Immunity 74: 2686–2696.CrossRefPubMedCentralPubMedGoogle Scholar
- 34.Prins, J.M., E.J. Kuijper, M. Mevissen, P. Speelman, and S. Van Deventer. 1995. Release of tumor necrosis factor alpha and interleukin 6 during antibiotic killing of Escherichia coli in whole blood: influence of antibiotic class, antibiotic concentration, and presence of septic serum. Infection and Immunity 63: 2236–2242.PubMedCentralPubMedGoogle Scholar
- 39.Fulton, S., J. Cross, Z. Toossi, and W. Boom. 1998. Regulation of interleukin-12 by interleukin-10, transforming growth factor-β, tumor necrosis factor-α, and interferon-γ in human monocytes infected with Mycobacterium tuberculosis H37Ra. Journal of Infectious Diseases 178: 1105–1114.CrossRefPubMedGoogle Scholar
- 45.Alemán, M., P. Schierloh, S. Silvia, R.M. Musella, M.A. Saab, M. Baldini, et al. 2004. Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients. Infection and Immunity 72: 5150–5158.CrossRefPubMedCentralPubMedGoogle Scholar
- 46.Lee, J.S., J. Son, S.B. Jung, Y.M. Kwon, C.S. Yang, J.H. Oh, et al. 2006. Ex vivo responses for interferon-gamma and proinflammatory cytokine secretion to low-molecular-weight antigen MTB12 of Mycobacterium tuberculosis during human tuberculosis. Scandinavian Journal of Immunology 64: 145–154.CrossRefPubMedGoogle Scholar
- 48.Valentinis, B., A. Bianchi, D. Zhou, A. Cipponi, F. Catalanotti, V. Russo, et al. 2005. Direct effects of polymyxin B on human dendritic cells maturation. The role of IkappaB-alpha/NF-kappaB and ERK1/2 pathways and adhesion. The Journal of Biological Chemistry 280: 14264–14271.CrossRefPubMedGoogle Scholar
- 50.Deng, W., W. Li, J. Zeng, Q. Zhao, C. Li, Y. Zhao, et al. 2014. Mycobacterium tuberculosis PPE family protein Rv1808 manipulates cytokines profile via co-activation of MAPK and NF-kappaB signaling pathways. Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 33: 273–288.CrossRefGoogle Scholar