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Inflammation

, Volume 37, Issue 3, pp 880–892 | Cite as

The Involvement of NADPH Oxidase-Mediated ROS in Cytokine Secretion from Macrophages Induced by Mycobacterium tuberculosis ESAT-6

  • Weiwei Liu
  • Yuan Peng
  • Yanlin Yin
  • Zhihui Zhou
  • Wanding Zhou
  • Yalei DaiEmail author
Article

Abstract

The 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. To understand the role of ESAT-6 in macrophage response against M. tuberculosis, the effects of ESAT-6 on macrophage generation of reactive oxygen species (ROS) and production of cytokines were studied. ESAT-6-induced macrophage secretion of monocyte chemoattractant protein-1 and TNF-α was found in a time- and dose-dependent manner. Signaling inhibition experiments indicate that NF-κB activation mediated by p38/JNK mitogen-activated protein kinase (MAPK) was involved in ESAT-6-triggered cytokine production. Moreover, TLR2 was engaged in ESAT-6-stimulated macrophage activation via rapidly induced ROS production and regulated activation of JNK/p38 MAPKs and NF-κB. More importantly, NADPH oxidase-mediated ROS generation is required during this process. Our study has identified a novel signal transduction pathway involving NADPH-ROS-JNK/p38-NF-κB in ESAT-6-induced cytokine production from macrophages. These findings provide an important evidence to understand the pathogenesis of M. tuberculosis infection in the modulation of the immune response.

KEY WORDS

macrophage ESAT-6 TLR2 ROS NADPH oxidase 

Notes

Acknowledgments

This study was supported by a grant from the National Basic Research program of China (973 Program, no. 2012CB518700).

Conflict of Interest

The authors declare that they have no conflicts of interest.

References

  1. 1.
    Yew, W.W., and C.C. Leung. 2008. Update in tuberculosis 2007. American Journal of Respiratory and Critical Care Medicine 177: 479–485.PubMedCrossRefGoogle Scholar
  2. 2.
    Warner, D.F., and V. Mizrahi. 2006. Tuberculosis chemotherapy: the influence of bacillary stress and damage response pathways on drug efficacy. Clinical Microbiology Reviews 19: 558–570.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Pieters, J. 2008. Mycobacterium tuberculosis and the macrophage: maintaining a balance. Cell Host & Microbe 3: 399–407.CrossRefGoogle Scholar
  4. 4.
    Zuñiga, J., D. Torres-García, T. Santos-Mendoza, T.S. Rodriguez-Reyna, J. Granados, and E.J. Yunis. 2012. Cellular and humoral mechanisms involved in the control of tuberculosis. Clinical and Developmental Immunology 2012: 1–18.CrossRefGoogle Scholar
  5. 5.
    Kumar, A., A. Farhana, L. Guidry, V. Saini, M. Hondalus, and A.J. Steyn. 2011. Redox homeostasis in mycobacteria: the key to tuberculosis control? Expert Reviews in Molecular Medicine e39: 1–25.Google Scholar
  6. 6.
    Segal, B.H., M.J. Grimm, A.N. Khan, W. Han, and T.S. Blackwell. 2012. Regulation of innate immunity by NADPH oxidase. Free Radical Biology and Medicine 53: 72–80.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Bafica, A., C.A. Scanga, C.G. Feng, C. Leifer, A. Cheever, and A. Sher. 2005. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. Journal of Experimental Medicine 202: 1715–1724.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Chow, J.C., D.W. Young, D.T. Golenbock, W.J. Christ, and F. Gusovsky. 1999. Toll-like receptor-4 mediates lipopolysaccharide induced signal transduction. The Journal of Biological Chemistry 274: 10689–10692.PubMedCrossRefGoogle Scholar
  9. 9.
    Chen, Y.C., C.C. Hsiao, C.J. Chen, C.H. Chin, S.F. Liu, C.C. Wu, H.L. Eng, T.Y. Chao, C.C. Tsen, Y.H. Wang, and M.C. Lin. 2010. Toll-like receptor 2 gene polymorphisms, pulmonary tuberculosis, and natural killer cell counts. BMC Medical Genetics 11: 1–10.Google Scholar
  10. 10.
    Kleinnijenhuis, J., M. Oosting, L.A.B. Joosten, M.G. Netea, and R. van Crevel. 2011. Innate immune recognition of Mycobacterium tuberculosis. Clinical and Developmental Immunology 2011: 1–12.CrossRefGoogle Scholar
  11. 11.
    Bloom, B.R., J. Flynn, K. McDonough, Y. Kress, and J. Chan. 1994. Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection. Immunobiology 191: 526–536.PubMedCrossRefGoogle Scholar
  12. 12.
    Kaufmann, S.H.E. 2001. How can immunology contribute to the control of tuberculosis? Nature Reviews Immunology 1: 20–30.PubMedCrossRefGoogle Scholar
  13. 13.
    Schaible, U.E., S. Sturgill-Koszycki, P.H. Schlesinger, and D.G. Russell. 1998. Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. Journal of Immunology 160: 1290–1296.Google Scholar
  14. 14.
    Underhill, D.M., A. Ozinsky, K.D. Smith, and A. Aderem. 1999. Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proceedings of the National Academy of Sciences of the United States of America 96: 14459–14463.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Drennan, M.B., D. Nicolle, V.J. Quesniaux, M. Jacobs, N. Allie, J. Mpagi, C. Fremond, H. Wagner, C. Kirschning, and B. Ryffel. 2004. Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection. The American Journal of Pathology 164: 49–57.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Reiling, N., C. Holscher, A. Fehrenbach, S. Kroger, C.J. Kirschning, S. Goyert, and S. Ehlers. 2002. Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis. The Journal of Immunology 169: 3480–3484.PubMedCrossRefGoogle Scholar
  17. 17.
    Bulut, Y., K.S. Michelsen, L. Hayrapetian, Y. Naiki, R. Spallek, M. Singh, and M. Arditi. 2005. Mycobacterium tuberculosis heat shock proteins use diverse toll like receptor pathways to activate pro-inflammatory signals. Journal of Biological Chemistry 280: 20961–20967.PubMedCrossRefGoogle Scholar
  18. 18.
    Abel, B., N. Thieblemont, V.J.F. Quesniaux, N. Brown, J. Mpagi, K. Miyake, F. Bihl, and B. Ryffel. 2002. Toll like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. Journal of Immunology 169: 3155–3162.CrossRefGoogle Scholar
  19. 19.
    Algood, H.M.S., J. Chan, and J.L. Flynn. 2003. Chemokines and tuberculosis. Cytokine and Growth Factor Reviews 14: 467–477.PubMedCrossRefGoogle Scholar
  20. 20.
    Court, N., V. Vasseur, R. Vacher, C. Frémond, Y. Shebzukhov, V.V. Yeremeev, I. Maillet, S.A. Nedospasov, S. Gordon, P.G. Fallon, H. Suzuki, B. Ryffel, and V.F. Quesniaux. 2010. Partial redundancy of the pattern recognition receptors, scavenger receptors, and C-type lectins for the long-term control of Mycobacterium tuberculosis infection. Journal of Immunology 184: 7057–7070.CrossRefGoogle Scholar
  21. 21.
    Carvalho, N.B., F.S. Oliveira, F.V. Dur˜aes, L.A. de Almeida, M. Flórido, L.O. Prata, M.V. Caliari, R. Appelberg, and S.C. Oliveira. 2011. Toll-like receptor 9 is required for full host resistance to Mycobacterium avium infection but plays no role in induction of Th1 responses. Infection and Immunity 79: 1638–1646.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Fietta, A.M., M. Morosini, F. Meloni, A.M. Bianco, and E. Pozzi. 2002. Pharmacological analysis of signal transduction pathways required for Mycobacterium tuberculosis-induced IL-8 and MCP-1 production in human peripheral monocytes. Cytokine 19: 242–249.PubMedCrossRefGoogle Scholar
  23. 23.
    Hasan, Z., J.M. Cliff, H.M. Dockrell, B. Jamil, M. Irfan, M. Ashraf, and R. Hussain. 2009. CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis. PLoS One e8459: 1–10.Google Scholar
  24. 24.
    Siveke, J.T., and A. Hamann. 1998. T helper 1 and T helper 2 cells respond differentially to chemokines. Journal of Immunology 160: 550–554.Google Scholar
  25. 25.
    Mendez, A., R. Hernandez-Pando, S. Contreras, D. Aguilar, and G.A.W. Rook. 2011. CCL2, CCL18 and sIL-4R in renal, meningeal and pulmonary TB; a 2 year study of patients and contacts. Tuberculosis 91: 140–145.PubMedCrossRefGoogle Scholar
  26. 26.
    Hussain, R., A. Ansari, N. Talat, Z. Hasan, and G. Dawood. 2011. CCL2/MCP-1 genotype-phenotype relationship in latent tuberculosis infection. PLoS One 6: 1–7.Google Scholar
  27. 27.
    Triebold, K.J., K. Pfeffer, C.J. Lowenstein, R. Schreiber, T.W. Mak, and B.R. Bloom. 1995. Tumor necrosis factor-α is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2: 561–572.PubMedCrossRefGoogle Scholar
  28. 28.
    Roca, F.J., and L. Ramakrishnan. 2013. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell 153: 521–534.PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Roach, D.R., A.G.D. Bean, C. Demangel, M.P. France, H. Briscoe, and W.J. Britton. 2002. TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. Journal of Immunology 168: 4620–4627.CrossRefGoogle Scholar
  30. 30.
    Pallen, M.J. 2002. The ESAT-6/WXG100 superfamily—and a new Gram-positive secretion system? Trends in Microbiology 10: 209–212.PubMedCrossRefGoogle Scholar
  31. 31.
    Renshaw, P.S., P. Panagiotidou, A. Whelan, S.V. Gordon, R.G. Hewinson, R.A. Williamson, and M.D. Carr. 2002. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex. Implications for pathogenesis and virulence. Journal of Biological Chemistry 277: 21598–21603.PubMedCrossRefGoogle Scholar
  32. 32.
    Guinn, K.M., M.J. Hickey, S.K. Mathur, K.L. Zakel, J.E. Grotzke, D.M. Lewinsohn, S. Smith, and D.R. Sherman. 2004. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Molecular Microbiology 51: 359–370.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Derrick, S.C., and S.L. Morris. 2007. The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression. Cellular Microbiology 9: 1547–1555.PubMedCrossRefGoogle Scholar
  34. 34.
    Hsu, T., S.M. Hingley-Wilson, B. Chen, M. Chen, A.Z. Dai, P.M. Morin, C.B. Marks, J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R.G. Russell, S.C. Derrick, F.M. Collins, S.L. Morris, C.H. King, and W.R. Jacobs Jr. 2003. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proceedings of the National Academy of Sciences 100: 12420–12425.CrossRefGoogle Scholar
  35. 35.
    Meher, A.K., N.C. Bal, K.V.R. Chary, and A. Arora. 2006. Mycobacterium tuberculosis H37Rv ESAT-6-CFP-10 complex formation confers thermodynamic and biochemical stability. The FEBS Journal 273: 1445–1462.PubMedCrossRefGoogle Scholar
  36. 36.
    Brodin, P., L. Majlessi, L. Marsollier, M.I. de Jonge, D. Bottai, C. Demangel, J. Hinds, O. Neyrolles, P.D. Butcher, C. Leclerc, S.T. Cole, and R. Brosch. 2006. Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infection and Immunity 74: 88–98.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Pang, X., B. Samten, G. Cao, X. Wang, A.R. Tvinnereim, X.L. Chen, and S.T. Howard. 2013. MprAB regulates the espA operon in Mycobacterium tuberculosis and modulates ESX-1 function and host cytokine response. Journal of Bacteriology 195: 66–75.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Chatterjee, S., V.P. Dwivedi, Y. Singh, I. Siddiqui, P. Sharma, L. Van Kaer, D. Chattopadhyay, and G. Das. 2011. Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner. PLoS Pathogens e1002378: 1–12.Google Scholar
  39. 39.
    Wang, X., P.F. Barnes, K.M. Dobos-Elder, J.C. Townsend, Y.T. Chung, H. Shams, S.E. Weis, and B. Samten. 2009. ESAT-6 inhibits production of IFN-gamma by Mycobacterium tuberculosis-responsive human T cells. Journal of Immunology 182: 3668–3677.CrossRefGoogle Scholar
  40. 40.
    Zhang, L., H. Zhang, Y. Zhao, F. Mao, J. Wu, B. Bai, Z. Xu, Y. Jiang, and C. Shi. 2012. Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages. DNA and Cell Biology 31: 171–179.PubMedCrossRefGoogle Scholar
  41. 41.
    Mishra, B.B., P. Moura-Alves, A. Sonawane, N. Hacohen, G. Griffiths, L.F. Moita, and E. Anes. 2010. Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome. Cellular Microbiology 12: 1046–1063.PubMedCrossRefGoogle Scholar
  42. 42.
    Pathak, S.K., S. Basu, K.K. Basu, A. Banerjee, S. Pathak, A. Bhattacharyya, T. Kaisho, M. Kundu, and J. Basu. 2007. Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nature Immunology 8: 610–618.PubMedCrossRefGoogle Scholar
  43. 43.
    Yin, Y., W. Liu, G. Ji, and Y. Dai. 2013. The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis. European Journal of Cell Biology 92: 150–159.PubMedCrossRefGoogle Scholar
  44. 44.
    Brodin, P., I. Rosenkrands, P. Andersen, S.T. Cole, and R. Brosch. 2004. ESAT-6 proteins: protective antigens and virulence factors? Trends in Microbiology 12: 500–508.PubMedCrossRefGoogle Scholar
  45. 45.
    Guinn, K.M., M.J. Hickey, S.K. Mathur, K.L. Zakel, J.E. Grotzke, D.M. Lewinsohn, S. Smith, and D.R. Sherman. 2004. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Molecular Microbiology 51: 359–370.PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Song, C.H., J.S. Lee, S.H. Lee, K. Lim, H.J. Kim, J.K. Park, T.H. Paik, and E.K. Jo. 2003. Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes. Journal of Clinical Immunology 23: 194–201.PubMedCrossRefGoogle Scholar
  47. 47.
    Schorey, J.S., and A.M. Cooper. 2003. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way. Cellular Microbiology 5: 133–142.PubMedCrossRefGoogle Scholar
  48. 48.
    A, S.K., K. Bansal, S. Holla, S. Verma-Kumar, P. Sharma, and K.N. Balaji. 2012. ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling. Molecular Immunology 49: 655–663.PubMedCrossRefGoogle Scholar
  49. 49.
    Ganguly, N., P.H. Giang, S.K. Basu, F. Mir, I. Siddiqui, and P. Sharma. 2007. Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2. BMC Immunology 8: 1–12.CrossRefGoogle Scholar
  50. 50.
    Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annual Review of Immunology 18: 621–663.PubMedCrossRefGoogle Scholar
  51. 51.
    Fang, F.C. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nature Reviews Microbiology 2: 820–832.PubMedCrossRefGoogle Scholar
  52. 52.
    Li, Q., and J.F. Engelhardt. 2006. Interleukin-1beta induction of NF kappaB is partially regulated by H2O2-mediated activation of NF kappaB-inducing kinase. Journal of Biological Chemistry 281: 1495–1505.PubMedCrossRefGoogle Scholar
  53. 53.
    Lu, Y., and L.M. Wahl. 2005. Oxidative stress augments the production of matrix metalloproteinase-1, cyclooxygenase-2 and prostaglandin E2 through enhancement of NF-kappa B activity in lipopolysaccharide-activated human primary monocytes. Journal of Immunology 175: 5423–5429.CrossRefGoogle Scholar
  54. 54.
    Segal, B.H., M.J. Grimm, A.N. Khan, W. Han, and T.S. Blackwell. 2012. Regulation of innate immunity by NADPH oxidase. Free Radical Biology and Medicine 53: 72–80.PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Cave, A. 2009. Selective targeting of NADPH oxidase for cardiovascular protection. Current Opinion in Pharmacology 9: 208–213.PubMedCrossRefGoogle Scholar
  56. 56.
    Brodin, P., I. Rosenkrands, P. Andersen, S.T. Cole, and R. Brosch. 2004. ESAT-6 proteins: protective antigens and virulence factors? Trends in Microbiology 12: 500–508.PubMedCrossRefGoogle Scholar
  57. 57.
    Blumenthal, A., S. Ehlers, M. Ernst, H.D. Flad, and N. Reiling. 2002. Control of mycobacterial replication in human macrophages: roles of extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinase pathways. Infection and Immunity 70: 4961–4967.PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Green, J.A., P.T. Elkington, C.J. Pennington, F. Roncaroli, S. Dholakia, R.C. Moores, A. Bullen, J.C. Porter, D. Agranoff, D.R. Edwards, and J.S. Friedland. 2010. Mycobacterium tuberculosis upregulates microglial matrix metalloproteinase-1 and -3 expression and secretion via NF-kappaB- and activator protein-1-dependent monocyte networks. Journal of Immunology 184: 6492–6503.CrossRefGoogle Scholar
  59. 59.
    Feng, Y., X. Yang, Z. Liu, Y. Liu, B. Su, Y. Ding, L. Qin, H. Yang, R. Zheng, and Z. Hu. 2008. Continuous treatment with recombinant Mycobacterium tuberculosis CFP-10-ESAT-6 protein activated human monocyte while deactivated LPS-stimulated macrophage. Biochemical and Biophysical Research Communications 365: 534–540.PubMedCrossRefGoogle Scholar
  60. 60.
    Bishai Jr., W.R., S. Chatterjee, V.P. Dwivedi, Y. Singh, I. Siddiqui, P. Sharma, L. Van Kaer, D. Chattopadhyay, and G. Das. 2011. Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner. PLoS Pathogens e1002378: 1–12.Google Scholar
  61. 61.
    Yang, C.S., D.M. Shin, H.M. Lee, J.W. Son, S.J. Lee, S. Akira, M.A. Gougerot-Pocidalo, J. El-Benna, H. Ichijo, and E.K. Jo. 2008. ASK1-p38 MAPK-p47phox activation is essential for inflammatory responses during tuberculosis via TLR2-ROS signalling. Cellular Microbiology 10: 741–754.PubMedCrossRefGoogle Scholar
  62. 62.
    Martindale, J.L., and N.J. Holbrook. 2002. Cellular response to oxidative stress: signaling for suicide and survival. Journal of Cellular Physiology 192: 1–15.PubMedCrossRefGoogle Scholar
  63. 63.
    Mossman, B.T., K.M. Lounsbury, and S.P. Reddy. 2006. Oxidants and signaling by mitogen-activated protein kinases in lung epithelium. American Journal of Respiratory Cell and Molecular Biology 34: 666–669.PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Thannickal, V.J., and B.L. Fanburg. 2000. Reactive oxygen species in cell signaling. American Journal of Physiology - Lung Cellular and Molecular Physiology 279: L1005–L1028.PubMedGoogle Scholar
  65. 65.
    Leto, T.L., S. Morand, D. Hurt, and T. Ueyama. 2009. Targeting and regulation of reactive oxygen species generation by Nox family NADPH oxidases. Antioxidants and Redox Signaling 11: 2607–2619.PubMedCentralPubMedCrossRefGoogle Scholar
  66. 66.
    Yang, C.S., D.M. Shin, K.H. Kim, Z.W. Lee, C.H. Lee, S.G. Park, Y.S. Bae, and E.K. Jo. 2009. NADPH oxidase 2 interaction with TLR2 is required for efficient innate immune responses to mycobacteria via cathelicidin expression. Journal of Immunology 182: 3696–3705.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Weiwei Liu
    • 1
  • Yuan Peng
    • 1
  • Yanlin Yin
    • 1
  • Zhihui Zhou
    • 1
  • Wanding Zhou
    • 1
  • Yalei Dai
    • 1
    Email author
  1. 1.Department of ImmunologyTongji University School of MedicineShanghaiChina

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