Abstract
Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.
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Change history
26 May 2016
In the version of this article initially published, Mathias Chamaillard was inadvertently omitted from the list of authors and from the Author Contributions section. The error has been corrected in the HTML and PDF versions of the article.
References
Freytag, L.C. & Clements, J.D. Mucosal adjuvants. Vaccine 23, 1804–1813 (2005).
Elson, C.O. & Ealding, W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J. Immunol. 132, 2736–2741 (1984).
Porgador, A., Staats, H.F., Itoh, Y. & Kelsall, B.L. Intranasal immunization with cytotoxic T lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal-mucosa-associated lymphoid tissue. Infect. Immun. 66, 5876–5881 (1998).
Sunahara, R.K., Dessauer, C.W., Whisnant, R.E., Kleuss, C. & Gilman, A.G. Interaction of Gsα with the cytosolic domains of mammalian adenylyl cyclase. J. Biol. Chem. 272, 22265–22271 (1997).
Lycke, N., Tsuji, T. & Holmgren, J. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur. J. Immunol. 22, 2277–2281 (1992).
Williamson, E., Westrich, G.M. & Viney, J.L. Modulating dendritic cells to optimize mucosal immunization protocols. J. Immunol. 163, 3668–3675 (1999).
Datta, S.K. et al. Mucosal adjuvant activity of cholera toxin requires TH17 cells and protects against inhalation anthrax. Proc. Natl. Acad. Sci. USA 107, 10638–10643 (2010).
Braun, M.C., He, J., Wu, C.Y. & Kelsall, B.L. Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor β1 and β2 chain expression. J. Exp. Med. 189, 541–552 (1999).
Xu-Amano, J. et al. Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces TH2 cells in mucosa-associated tissues. J. Exp. Med. 178, 1309–1320 (1993).
Yamamoto, S. et al. A nontoxic mutant of cholera toxin elicits TH2-type responses for enhanced mucosal immunity. Proc. Natl. Acad. Sci. USA 94, 5267–5272 (1997).
la Sala, A. et al. Cholera toxin inhibits IL-12 production and CD8-α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function. J. Exp. Med. 206, 1227–1235 (2009).
Li, X. et al. Divergent requirement for Gαs and cAMP in the differentiation and inflammatory profile of distinct mouse TH subsets. J. Clin. Invest. 122, 963–973 (2012).
Mattsson, J. et al. Cholera toxin adjuvant promotes a balanced TH1–TH2–TH17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b+ DCs. Mucosal Immunol. 8, 815–827 (2015).
Shaw, M.H., Reimer, T., Kim, Y.G. & Nuñez, G. NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Curr. Opin. Immunol. 20, 377–382 (2008).
Inohara, N. et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J. Biol. Chem. 278, 5509–5512 (2003).
Girardin, S.E. et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872 (2003).
Park, J.H. et al. RICK (RIP2) mediates innate immune responses induced through Nod1 and Nod2 but not TLRs. J. Immunol. 178, 2380–2386 (2007).
Hasegawa, M. et al. A critical role of RICK (RIP2) polyubiquitination in Nod-induced NF-κB activation. EMBO J. 27, 373–383 (2008).
Pavot, V. et al. Cutting edge: new chimeric NOD2–TLR2 adjuvant drastically increases vaccine immunogenicity. J. Immunol. 193, 5781–5785 (2014).
Magalhaes, J.G. et al. Nod2-dependent TH2 polarization of antigen-specific immunity. J. Immunol. 181, 7925–7935 (2008).
Fritz, J.H. et al. Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 26, 445–459 (2007).
Allen, E.K. et al. Characterization of the nasopharyngeal microbiota in health and during rhinovirus challenge. Microbiome 2, 22 (2014).
Warner, N. & Núñez, G. MyD88: a critical adaptor protein in innate immunity signal transduction. J. Immunol. 190, 3–4 (2013).
Kawai, T. & Akira, S. Toll-like receptors and their cross-talk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011).
Reinhardt, R.L., Liang, H.E. & Locksley, R.M. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat. Immunol. 10, 385–393 (2009).
Eto, D. et al. IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (TFH) differentiation. PLoS One 6, e17739 (2011).
Karnowski, A. et al. B and T cells collaborate in antiviral responses via IL-6, IL-21, and transcriptional activator and coactivator, Oct2 and OBF-1. J. Exp. Med. 209, 2049–2064 (2012).
de Rooij, J. et al. Epac is a Rap1 guanine-nucleotide exchange factor directly activated by cyclic AMP. Nature 396, 474–477 (1998).
Warner, N. et al. A genome-wide siRNA screen reveals positive and negative regulators of the NOD2 and NF-κB signaling pathways. Sci. Signal. 6, rs3 (2013).
Ivanov, I.I. et al. Specific microbiota direct the differentiation of IL-17-producing T helper cells in the mucosa of the small intestine. Cell Host Microbe 4, 337–349 (2008).
Shulzhenko, N. et al. Cross-talk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut. Nat. Med. 17, 1585–1593 (2011).
Abt, M.C. et al. Commensal bacteria calibrate the activation threshold of innate antiviral immunity. Immunity 37, 158–170 (2012).
Mazmanian, S.K., Round, J.L. & Kasper, D.L. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625 (2008).
Ellouz, F., Adam, A., Ciorbaru, R. & Lederer, E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 59, 1317–1325 (1974).
Magalhaes, J.G. et al. Nucleotide-oligomerization-domain-containing proteins instruct T cell helper type 2 immunity through stromal activation. Proc. Natl. Acad. Sci. USA 108, 14896–14901 (2011).
Duan, W. et al. Innate signals from Nod2 block respiratory tolerance and program TH2-driven allergic inflammation. J. Allergy Clin. Immunol. 126, 1284–1293.e10 (2010).
Ma, C.S. et al. Early commitment of naive human CD4+ T cells to the T follicular helper (TFH) cell lineage is induced by IL-12. Immunol. Cell Biol. 87, 590–600 (2009).
Schmitt, N. et al. Human dendritic cells induce the differentiation of interleukin-21-producing T follicular helper-like cells through interleukin-12. Immunity 31, 158–169 (2009).
Jiang, V., Jiang, B., Tate, J., Parashar, U.D. & Patel, M.M. Performance of rotavirus vaccines in developed and developing countries. Hum. Vaccin. 6, 532–542 (2010).
Levine, M.M. Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine. BMC Biol. 8, 129 (2010).
Lopman, B.A. et al. Understanding reduced rotavirus vaccine efficacy in low socio-economic settings. PLoS One 7, e41720 (2012).
Grassly, N.C. et al. Mucosal immunity after vaccination with monovalent and trivalent oral poliovirus vaccine in India. J. Infect. Dis. 200, 794–801 (2009).
Johnson, R.A. et al. Comparison of immune responses to the O-specific polysaccharide and lipopolysaccharide of Vibrio cholerae O1 in Bangladeshi adult patients with cholera. Clin. Vaccine Immunol. 19, 1712–1721 (2012).
Leung, D.T. et al. Comparison of memory B cell, antibody-secreting cell and plasma antibody responses in young children, older children and adults with infection caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh. Clin. Vaccine Immunol. 18, 1317–1325 (2011).
Yatsunenko, T. et al. Human gut microbiome viewed across age and geography. Nature 486, 222–227 (2012).
Korpe, P.S. & Petri, W.A. Jr. Environmental enteropathy: critical implications of a poorly understood condition. Trends Mol. Med. 18, 328–336 (2012).
Chattha, K.S., Vlasova, A.N., Kandasamy, S., Rajashekara, G. & Saif, L.J. Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model. J. Immunol. 191, 2446–2456 (2013).
Oh, J.Z. et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity 41, 478–492 (2014).
Kim, D. et al. Suppression of allergic diarrhea in murine ovalbumin-induced allergic diarrhea model by PG102, a water-soluble extract prepared from Actinidia arguta. Int. Arch. Allergy Immunol. 150, 164–171 (2009).
Wu, H.Y., Nguyen, H.H. & Russell, M.W. Nasal lymphoid tissue (NALT) as a mucosal immune-inductive site. Scand. J. Immunol. 46, 506–513 (1997).
Franchi, L., Eigenbrod, T. & Núñez, G. Cutting edge: TNF-α mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation. J. Immunol. 183, 792–796 (2009).
Hasegawa, M. et al. Transitions in oral and intestinal microflora composition and innate-immune-receptor-dependent stimulation during mouse development. Infect. Immun. 78, 639–650 (2010).
Ubeda, C. et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. J. Clin. Invest. 120, 4332–4341 (2010).
Acknowledgements
This work was supported by US National Institutes of Health (NIH) grant R01 DK61707 (G.N.), the University of Michigan's Cancer Center Support grant 5 P30 CA46592 (G.N.), the DFG Cluster of Excellence 'Inflammation at Interfaces' (P.R.) and the BMBF grant TP5 (P.R.). We thank M. Zeng for critical review of the manuscript, L. Haynes for animal husbandry and the University of Michigan Germ-Free Animal Core Facility and Host Microbiome Initiative (G.N.) for support.
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D.K., Y.-G.K. and G.N. conceived the study. D.K. performed most of the experiments. Y.-G.K. performed several experiments. S.-U.S. and D.-J.K. helped with experiments. D.P., D.J.P. and P.R. generated and performed initial characterization of Itgax-Cre;Nod2fl/fl mice. M.C. provided critical materials. N.K., D.J.P., P.R. and N.I. helped in the design of several experiments and provided critical advice. D.K., Y.-G.K. and G.N. wrote the manuscript, with contributions from all of the authors.
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Kim, D., Kim, YG., Seo, SU. et al. Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin. Nat Med 22, 524–530 (2016). https://doi.org/10.1038/nm.4075
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DOI: https://doi.org/10.1038/nm.4075
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