Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.
References
[1]
Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2: 569–579.
[2]
Denzer K, Kleijmeer MJ, Heijnen HF, Stoorvogel W, Geuze HJ (2000) Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci 113 Pt 19: 3365–3374.
[3]
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9: 581–593.
[4]
Thery C, Duban L, Segura E, Veron P, Lantz O, et al. (2002) Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nat Immunol 3: 1156–1162.
[5]
Kim SH, Lechman ER, Bianco N, Menon R, Keravala A, et al. (2005) Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis. J Immunol 174: 6440–6448.
[6]
Kim SH, Bianco NR, Shufesky WJ, Morelli AE, Robbins PD (2007) Effective treatment of inflammatory disease models with exosomes derived from dendritic cells genetically modified to express IL-4. J Immunol 179: 2242–2249.
[7]
Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, et al. (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 7: 297–303.
[8]
Yu S, Liu C, Su K, Wang J, Liu Y, et al. (2007) Tumor exosomes inhibit differentiation of bone marrow dendritic cells. J Immunol 178: 6867–6875.
[9]
Liu C, Yu S, Zinn K, Wang J, Zhang L, et al. (2006) Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function. J Immunol 176: 1375–1385.
[10]
Clayton A, Mitchell JP, Court J, Linnane S, Mason MD, et al. (2008) Human tumor-derived exosomes down-modulate NKG2D expression. J Immunol 180: 7249–7258.
[11]
Taylor DD, Gercel-Taylor C, Lyons KS, Stanson J, Whiteside TL (2003) T-cell apoptosis and suppression of T-cell receptor/CD3-zeta by Fas ligand-containing membrane vesicles shed from ovarian tumors. Clin Cancer Res 9: 5113–5119.
[12]
Taylor DD, Gercel-Taylor C (2005) Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects. Br J Cancer 92: 305–311.
[13]
Valenti R, Huber V, Filipazzi P, Pilla L, Sovena G, et al. (2006) Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer Res 66: 9290–9298.
[14]
Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z (2007) Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res 67: 7458–7466.
[15]
Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS (2007) Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood 110: 3234–3244.
[16]
Bhatnagar S, Schorey JS (2007) Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory. J Biol Chem 282: 25779–25789.
[17]
Quah BJ, O'Neill HC (2007) Mycoplasma contaminants present in exosome preparations induce polyclonal B cell responses. J Leukoc Biol 82: 1070–1082.
[18]
Cassell GH, Cole BC (1981) Mycoplasmas as agents of human disease. N Engl J Med 304: 80–89.
[19]
Blanchard A, Montagnier L (1994) AIDS-associated mycoplasmas. Annu Rev Microbiol 48: 687–712.
[20]
Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62: 1094–1156.
[21]
Tsai S, Wear DJ, Shih JW, Lo SC (1995) Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation. Proc Natl Acad Sci U S A 92: 10197–10201.
[22]
Macpherson I, Russell W (1966) Transformations in hamster cells mediated by mycoplasmas. Nature 210: 1343–1345.
[23]
Namiki K, Goodison S, Porvasnik S, Allan RW, Iczkowski KA, et al. (2009) Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS One 4: e6872.
[24]
Zhang S, Tsai S, Lo SC (2006) Alteration of gene expression profiles during mycoplasma-induced malignant cell transformation. BMC Cancer 6: 116.
[25]
Paton GR, Jacobs JP, Perkins FT (1965) Chromosome changes in human diploid-cell cultures infected with Mycoplasma. Nature 207: 43–45.
[26]
Feng SH, Tsai S, Rodriguez J, Lo SC (1999) Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells. Mol Cell Biol 19: 7995–8002.
[27]
Zhang B, Shih JW, Wear DJ, Tsai S, Lo SC (1997) High-level expression of H-ras and c-myc oncogenes in mycoplasma-mediated malignant cell transformation. Proc Soc Exp Biol Med 214: 359–366.
[28]
Flanagan BF, Newton DJ, Johnson PM (1998) Mycoplasma origin of tumor cell protein. Nat Med 4: 133.
[29]
Schmidhauser C, Dudler R, Schmidt T, Parish RW (1990) A mycoplasmal protein influences tumour cell invasiveness and contact inhibition in vitro. J Cell Sci 95(Pt 3): 499–506.
[30]
Rawadi G, Roman-Roman S, Castedo M, Dutilleul V, Susin S, et al. (1996) Effects of Mycoplasma fermentans on the myelomonocytic lineage. Different molecular entities with cytokine-inducing and cytocidal potential. J Immunol 156: 670–678.
[31]
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.
[32]
Kostyal DA, Butler GH, Beezhold DH (1995) Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes. Infect Immun 63: 3858–3863.
[33]
Sugimura K, Ohno T, Kimura Y, Kimura T, Azuma I (1992) Arginine deiminase gene of an AIDS-associated mycoplasma, Mycoplasma incognitus. Microbiol Immunol 36: 667–670.
[34]
Pietsch K, Ehlers S, Jacobs E (1994) Cytokine gene expression in the lungs of BALB/c mice during primary and secondary intranasal infection with Mycoplasma pneumoniae. Microbiology 140(Pt 8): 2043–2048.
[35]
Zurita-Salinas CS, Palacios-Boix A, Yanez A, Gonzalez F, Alcocer-Varela J (1996) Contamination with Mycoplasma spp. induces interleukin-13 expression by human skin fibroblasts in culture. FEMS Immunol Med Microbiol 15: 123–128.
[36]
Bennett RH, Jasper DE (1977) Immunosuppression of humoral and cell-mediated responses in calves associated wtih inoculation of Mycoplasma bovis. Am J Vet Res 38: 1731–1738.
[37]
Bergquist LM, Lau BH, Winter CE (1974) Mycoplasma-associated immunosuppression: effect on hemagglutinin response to common antigens in rabbits. Infect Immun 9: 410–415.
[38]
Cole BC, Wells DJ (1990) Immunosuppressive properties of the Mycoplasma arthritidis T-cell mitogen in vivo: inhibition of proliferative responses to T-cell mitogens. Infect Immun 58: 228–236.
[39]
Chan PJ, Seraj IM, Kalugdan TH, King A (1996) Prevalence of mycoplasma conserved DNA in malignant ovarian cancer detected using sensitive PCR-ELISA. Gynecol Oncol 63: 258–260.
[40]
Huang S, Li JY, Wu J, Meng L, Shou CC (2001) Mycoplasma infections and different human carcinomas. World J Gastroenterol 7: 266–269.
Beatty WL, Ullrich HJ, Russell DG (2001) Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event. Eur J Cell Biol 80: 31–40.
[43]
Tarshis M, Yavlovich A, Katzenell A, Ginsburg I, Rottem S (2004) Intracellular location and survival of Mycoplasma penetrans within HeLa cells. Curr Microbiol 49: 136–140.
[44]
Brenner C, Wroblewski H, Le Henaff M, Montagnier L, Blanchard A (1997) Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines. Infect Immun 65: 4322–4329.
[45]
Herbelin A, Ruuth E, Delorme D, Michel-Herbelin C, Praz F (1994) Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes. Infect Immun 62: 4690–4694.
[46]
Kostyal DA, Butler GH, Beezhold DH (1994) A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes. Infect Immun 62: 3793–3800.
[47]
Rieger A, Bar-Or A (2008) B-cell-derived interleukin-10 in autoimmune disease: regulating the regulators. Nat Rev Immunol 8: 486–487.
[48]
Burdin N, Rousset F, Banchereau J (1997) B-cell-derived IL-10: production and function. Methods 11: 98–111.
[49]
Madan R, Demircik F, Surianarayanan S, Allen JL, Divanovic S, et al. (2009) Nonredundant roles for B cell-derived IL-10 in immune counter-regulation. J Immunol 183: 2312–2320.
[50]
Gong M, Meng L, Jiang B, Zhang J, Yang H, et al. (2008) p37 from Mycoplasma hyorhinis promotes cancer cell invasiveness and metastasis through activation of MMP-2 and followed by phosphorylation of EGFR. Mol Cancer Ther 7: 530–537.
