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Mycoplasma hyopneumoniae induces pro-inflammatory cytokine and nitric oxide production through NFκB and MAPK pathways in RAW264.7 cells

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Abstract

In the present study, we confirmed the ability of M. hyopneumoniae to induce the secretion of large amount of proinflammatory cytokine and nitric oxide (NO) in murine macrophage RAW 264.7 cells. Moreover, M. hyopneumoniae-induced activation of the MAPK and NF-кB pathways by phosphorylation of ERK1/2, p38 and JNK/SAPK and by dissociation of IκB from NF-κB. Translocation of transcription factor NF-κB and its binding was confirmed through western blot and electromobility shift assay. From these results, we further hypothesized that these signal proteins were involved in M. hyopneumoniae-induced proinflammatory cytokines and NO productions in macrophages. Hence, we utilized specific blockers of MAPK and NF-κB to investigate the signaling pathway involvement in cytokine and NO production through pharmacological approaches. The results demonstrated significant inhibition of TNF-α, IL-1β, IL-6 and NO by MAPK inhibitors. NF-κB inhibitor PDTC significantly inhibited IL-1β and NO production. These findings contribute to the understanding of the mechanisms of immune reactivity and may ultimately prove useful in the development of new therapeutic strategies. In summary, we found critical evidence for the involvement of NF-κB and MAPK signaling pathways in the upregulation of proinflammatory cytokine and NO induced by M. hyopneumoniae.

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References

  • Amass SF, Clark LK, van Alstine WG, Bowersock TL, Murphy DA, Knox KE, Albregts SR (1994) Interaction of Mycoplasma hyopneumoniae and Pasteurella multocida infections in swine. J Am Vet Med Assoc 204:102–107

    CAS  PubMed  Google Scholar 

  • Asai T, Okada M, Ono M, Irisawa T, Mori Y, Yokomizo Y, Sato S (1993) Increased levels of tumor necrosis factor and interleukin 1 in bronchoalveolar lavage fluids from pigs infected with Mycoplasma hyopneumoniae. Vet Immunol Immunopathol 38:253–260

    Article  CAS  PubMed  Google Scholar 

  • Asai T, Okada M, Ono M, Mori Y, Yokomizo Y, Sato S (1994) Detection of interleukin-6 and prostaglandin E2 in bronchoalveolar lavage fluids of pigs experimentally infected with Mycoplasma hyponeumoniae. Vet Immunol Immunopathol 44:97–102

    Article  CAS  PubMed  Google Scholar 

  • Baeuerle PA, Henkel T (1994) Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12:141–179

    CAS  PubMed  Google Scholar 

  • Choi C, Kwon D, Jung K, Ha Y, Lee YH, Kim O, Park HK, Kim SH, Hwang KK, Chae C (2006) Expression of inflammatory cytokines in pigs experimentally infected with Mycoplasma hyopneumoniae. J Comp Pathol 134:40–46

    Article  CAS  PubMed  Google Scholar 

  • Collart MA, Baeuerle P, Vassalli P (1990) Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Mol Cell Biol 10:1498–1506

    CAS  PubMed  Google Scholar 

  • DeBey MC, Ross RF (1994) Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun 62:5312–5318

    CAS  PubMed  Google Scholar 

  • Digicaylioglu M, Lipton S (2001) Erthropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades. Nature 412:641–647

    Article  CAS  PubMed  Google Scholar 

  • Feng SH, Lo SC (1999) Lipid extract of Mycoplasma penetrans proteinase K-digested lipid-associated membrane proteins rapidly activates NF-kappaB and activator protein 1. Infect Immun 67:2951–1256

    CAS  PubMed  Google Scholar 

  • Friis NF, Szancer J (1994) Sensitivity of certain porcine and bovine mycoplasmas to antimicrobial agents in a liquid medium test compared to a disc assay. Acta Vet Scand 35:389–394

    CAS  PubMed  Google Scholar 

  • Garcia J, Lemercier B, Roman-Roman S, Rawadi G (1998) A Mycoplasma fermentans-derived synthetic lipopeptide induces AP-1 and NF-kappaB activity and cytokine secretion in macrophages via the activation of mitogen-activated protein kinase pathways. J Biol Chem 273:34391–34398

    Article  CAS  PubMed  Google Scholar 

  • Grimm S, Baeuerle PA (1993) The inducible transcription factor NF-kappa B: structure-function relationship of its protein subunits. Biochem J 290(Part 2):297–308

    CAS  PubMed  Google Scholar 

  • Hwang MH, Lim JH, Yun HI, Kim JC, Jung BY, Hsu WH, Park SC (2006) The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells. Vet J 172:556–560

    Article  CAS  PubMed  Google Scholar 

  • Hwang MH, Chang ZQ, Kang EH, Lim JH, Yun HI, Rhee MH, Jeong KS, Park SC (2008) Surfactin C inhibits Mycoplasma hyopneumoniae-induced transcription of proinflammatory cytokines and nitric oxide production in murine RAW 264.7 cells. Biotechnol Lett 30:229–233

    Article  CAS  PubMed  Google Scholar 

  • Jongeneel CV (1994) Regulation of the TNF alpha gene. Prog Clin Biol Res 388:367–381

    CAS  PubMed  Google Scholar 

  • Lim Y, Seok H (2002) Studies on the mycoplasmal pneumoniae in slaughter pigs. 1. Seasonal detection by gross finding of lung lesion and dot-ELISA technique. Korean J Vet Res 42:219–224

    Google Scholar 

  • Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ (1993) Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci USA 90:9730–9734

    Article  CAS  PubMed  Google Scholar 

  • Maes D, Segales J, Meyns T, Sibila M, Pieters M, Haesebrouck F (2008) Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol 126:297–309

    Article  CAS  PubMed  Google Scholar 

  • Muller JM, Ziegler-Heitbrock HW, Baeuerle PA (1993) Nuclear factor kappa B, a mediator of lipopolysaccharide effects. Immunobiology 187:233–256

    CAS  PubMed  Google Scholar 

  • Muneta Y, Uenishi H, Kikuma R, Yoshihara K, Shimoji Y, Yamamoto R, Hamashima N, Yokomizo Y, Mori Y (2003) Porcine TLR2 and TLR6: identification and Their Involvement in Mycoplasma hyopneumoniae Infection. J Interferon Cytokine Res 23:583–590

    Article  CAS  PubMed  Google Scholar 

  • Rawadi G, Ramez V, Lemercier B, Roman-Roman S (1998) Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis. J Immunol 160:1330–1339

    CAS  PubMed  Google Scholar 

  • Rawadi G, Garcia J, Lemercier B, Roman-Roman S (1999) Signal transduction pathways involved in the activation of NF-kappa B, AP-1, and c-fos by Mycoplasma fermentans membrane lipoproteins in macrophages. J Immunol 162:2193–2203

    CAS  PubMed  Google Scholar 

  • Sacht G, Marten A, Deiters U, Sussmuth R, Jung G, Wingender E, Muhlradt PF (1998) Activation of nuclear factor-kappaB in macrophages by mycoplasmal lipopeptides. Eur J Immunol 28:4207–4212

    Article  CAS  PubMed  Google Scholar 

  • Sanghera JS, Weinstein SL, Aluwalia M, Girn J, Pelech SL (1996) Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. J Immunol 156:4457–4465

    CAS  PubMed  Google Scholar 

  • Sohn MH, Lee KE, Choi SY, Kwon BC, Chang MW, Kim KE (2005) Effect of Mycoplasma pneumoniae lysate on interleukin-8 gene expression in human respiratory epithelial cells. Chest 128:322–326

    Article  CAS  PubMed  Google Scholar 

  • Stancovski I, Baltimore D (1997) NF-kappaB activation: the I kappaB kinase revealed? Cell 91:299–302

    Article  CAS  PubMed  Google Scholar 

  • Su B, Karin M (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 8:402–411

    Article  CAS  PubMed  Google Scholar 

  • Thacker EL, Thacker BJ, Kuhn M, Hawkins PA, Waters WR (2000) Evaluation of local and systemic immune responses induced by intramuscular injection of a Mycoplasma hyopneumoniae bacterin to pigs. Am J Vet Res 61:1384–1389

    Article  CAS  PubMed  Google Scholar 

  • Thanawongnuwech R, Young TF, Thacker BJ, Thacker EL (2001) Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model. Vet Immunol Immunopathol 79:115–127

    Article  CAS  PubMed  Google Scholar 

  • Thanawongnuwech R, Thacker B, Halbur P, Thacker EL (2004) Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Clin Diagn Lab Immunol 11:901–908

    CAS  PubMed  Google Scholar 

  • Whiteside ST, Israel A (1997) I kappa B proteins: structure, function and regulation. Semin Cancer Biol 8:75–82

    Article  CAS  PubMed  Google Scholar 

  • Yang SH, Sharrocks AD, Whitmarsh AJ (2003) Transcriptional regulation by the MAP kinase signaling cascades. Gene 320:13–21

    Article  Google Scholar 

  • You X, Yimou Wu, Zeng Y, Deng Z, Qiu H, Minjun Yu (2008) Mycoplasma genitalium-derived lipid-associated membrane proteins induce activation of MAPKs, NF-кB andAP-1 in THP-1cells. FEMS Immunol Med Microbiol 52:228–236

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by Korea Research Foundation (KRF) grant funded by the Korea government (MEST) (No. 2009-0071490) and Application Center Program, Ministry of Science and Technology and in part by Ministry of Commerce, Industry Energy (MOCIE) through the Center for Traditional Microorganism Resources (TMR) at Keimyung University.

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Correspondence to Seung-Chun Park.

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Mi-Hyun Hwang and Dereje Damte contributed equally to this work.

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Hwang, MH., Damte, D., Lee, JS. et al. Mycoplasma hyopneumoniae induces pro-inflammatory cytokine and nitric oxide production through NFκB and MAPK pathways in RAW264.7 cells. Vet Res Commun 35, 21–34 (2011). https://doi.org/10.1007/s11259-010-9447-5

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