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Unmethylated CpG motifs in Toxoplasma gondii DNA induce TLR9- and IFN-β-dependent expression of α-defensin-5 in intestinal epithelial cells

Published online by Cambridge University Press:  02 November 2015

MIGUEL H. SANTAMARIA ,
EUGENIA PEREZ CABALLERO  and
RICARDO S. CORRAL
MIGUEL H. SANTAMARIA
Affiliation:
Laboratorio de Biología Experimental, Centro de Estudios Metabólicos, Vega Lamera 8, C.P. 39005 Santander, Spain
EUGENIA PEREZ CABALLERO
Affiliation:
Laboratorio de Biología Experimental, Centro de Estudios Metabólicos, Vega Lamera 8, C.P. 39005 Santander, Spain
RICARDO S. CORRAL*
Affiliation:
Servicio de Parasitología-Chagas, Hospital de Niños “Ricardo Gutiérrez”, Gallo 1330, C1425 Buenos Aires, Argentina
*
*Corresponding author. Servicio de Parasitología-Chagas, Hospital de Niños “Ricardo Gutiérrez”, Gallo 1330, C1425 Buenos Aires, Argentina. E-mail: ricardocorral56@hotmail.com
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Summary

The gut epithelial barrier is a strategic place to prevent, or at least to limit, parasite dissemination upon oral infection with Toxoplasma gondii. Innate immunity to this pathogen results from delicate interactions involving different components of the infecting agent and the host. We herein aimed to examine the molecular mechanism by which protozoan DNA boosts the production of α-defensin-5 (DEFA-5), the main antimicrobial peptide at the target site of infection. The present study shows that DEFA-5 is rapidly upregulated in intestinal epithelial cells following intracellular Toll-like receptor 9 (TLR9) activation by unmethylated CpG motifs in DNA from T. gondii (CpG-DNA). Concomitantly, CpG-DNA purified from the pathogen markedly increased TLR9 mRNA expression levels in the Caco-2 cell line. We further verified that DEFA-5 production was dependent on interferon-β released from these cells upon treatment with CpG-DNA prepared from tachyzoites. Our results suggest that, in protozoan DNA-stimulated intestinal epithelial cells, the TLR9/interferon-β/DEFA-5 pathway may initiate an innate anti-T. gondii response without the need of parasite invasion. These findings highlight the key role of the gut epithelium in Toxoplasma recognition and amplification of local host defence against this microbe, thereby contributing to gain insight into immunoprotective mechanisms and to improve therapeutic strategies.

Keywords

Toxoplasma gondiiparasite DNAunmethylated CpGToll-like receptor 9α-defensin-5interferon-βintestinal epithelial cells
Type
Research Article
Information
Parasitology , Volume 143 , Issue 1 , January 2016 , pp. 60 - 68
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Copyright
Copyright © Cambridge University Press 2015 

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References

REFERENCES

Andrade, W. A., Souza, M. C., Ramos-Martinez, E., Nagpal, K., Dutra, M. S., Melo, M. B., Bartholomeu, D. C., Ghosh, S., Golenbock, D. T. and Gazzinelli, R. T. (2013). Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice. Cell Host and Microbe 13, 42–53.CrossRefGoogle ScholarPubMed
Beiting, D. P. (2014). Protozoan parasites and type I interferons: a cold case reopened. Trends in Parasitology 30, 491–498.CrossRefGoogle ScholarPubMed
Cardoso, L. S., Araujo, M. I., Góes, A. M., Pacífico, L. G., Oliveira, R. R. and Oliveira, S. C. (2007). Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microbial Cell Factories 6, 1.CrossRefGoogle ScholarPubMed
Cohen, S. B. and Denkers, E. Y. (2015). The gut mucosal immune response to Toxoplasma gondii . Parasite Immunology 37, 108–117.CrossRefGoogle ScholarPubMed
Colonne, P. M., Eremeeva, M. E. and Sahni, S. K. (2011). Beta interferon-mediated activation of signal transducer and activator of transcription protein 1 interferes with Rickettsia conorii replication in human endothelial cells. Infection and Immunity 79, 3733–3743.CrossRefGoogle ScholarPubMed
Corral, R. S., Iñiguez, M. A., Duque, J., López-Pérez, R. and Fresno, M. (2007). Bombesin induces cyclooxygenase-2 expression through the activation of the nuclear factor of activated T cells and enhances cell migration in Caco-2 colon carcinoma cells. Oncogene 26, 958–969.CrossRefGoogle ScholarPubMed
Diefenbach, A., Schindler, H., Donhauser, N., Lorenz, E., Laskay, T., MacMicking, J., Röllinghoff, M., Gresser, I. and Bogdan, C. (1998). Type 1 interferon (IFNalpha/beta) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8, 77–87.CrossRefGoogle ScholarPubMed
Ewaschuk, J. B., Backer, J. L., Churchill, T. A., Obermeier, F., Krause, D. O. and Madsen, K. L. (2007). Surface expression of Toll-like receptor 9 is upregulated on intestinal epithelial cells in response to pathogenic bacterial DNA. Infection and Immunity 75, 2572–2579.CrossRefGoogle ScholarPubMed
Foureau, D. M., Mielcarz, D. W., Menard, L. C., Schulthess, J., Werts, C., Vasseur, V., Ryffel, B., Kasper, L. H. and Buzoni-Gatel, D. (2010). TLR9-dependent induction of intestinal α-defensins by Toxoplasma gondii . Journal of Immunology 184, 7022–7029.CrossRefGoogle ScholarPubMed
Frye, M., Bargon, J., Lembcke, B., Wagner, T. O. and Gropp, R. (2000). Differential expression of human α- and β-defensins mRNA in gastrointestinal epithelia. European Journal of Clinical Investigation 30, 695–701.CrossRefGoogle ScholarPubMed
Garberi, J. C., Blanco, J. C., Angel, S. O., Pzsenny, V., Arakelian, M. C. and Pastini, A. (1990). Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites. Preliminary characterization of its genome. Memórias do Instituto Oswaldo Cruz 85, 429–434.CrossRefGoogle ScholarPubMed
Grigat, J., Soruri, A., Forssmann, U., Riggert, J. and Zwirner, J. (2007). Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family. Journal of Immunology 179, 3958–3965.CrossRefGoogle ScholarPubMed
Han, S. H., Kim, Y. E., Park, J. A., Park, J. B., Kim, Y. S., Lee, Y., Choi, I. G. and Kwon, H. J. (2009). Expression of human beta-defensin-2 gene induced by CpG-DNA in human B cells. Biochemical and Biophysical Research Communications 389, 443–448.CrossRefGoogle ScholarPubMed
Han, S. J., Melichar, H. J., Coombes, J. L., Chan, S. W., Koshy, A. A., Boothroyd, J. C., Barton, G. M. and Robey, E. A. (2014). Internalization and TLR-dependent type I interferon production by monocytes in response to Toxoplasma gondii . Immunology and Cell Biology 92, 872–881.CrossRefGoogle ScholarPubMed
Jarczak, J., Kościuczuk, E. M., Lisowski, P., Strzałkowska, N., Jóźwik, A., Horbańczuk, J., Krzyżewski, J., Zwierzchowski, L. and Bagnicka, E. (2013). Defensins: natural component of human innate immunity. Human Immunology 74, 1069–1079.CrossRefGoogle ScholarPubMed
Juarez, E., Nuñez, C., Sada, E., Ellner, J. J., Schwander, S. K. and Torres, M. (2010). Differential expression of Toll-like receptors on human alveolar macrophages and autologous peripheral monocytes. Respiratory Research 11, 2.CrossRefGoogle ScholarPubMed
Khan, A., Taylor, S., Su, C., Sibley, L. D., Paulsen, I. and Ajioka, J. W. (2007). Genetics and genome organization of Toxoplasma gondii . In Toxoplasma: Molecular and Cellular Biology (ed. Ajioka, J. W. and Doldati, D.), pp. 193–207. Horizon Scientific Press, Norfolk, UK.Google Scholar
Kingma, S. D., Li, N., Sun, F., Valladares, R. B., Neu, J. and Lorca, G. L. (2011). Lactobacillus johnsonii N6·2 stimulates the innate immune response through Toll-like receptor 9 in Caco-2 cells and increases intestinal crypt Paneth cell number in biobreeding diabetes-prone rats. Journal of Nutrition 141, 1023–1028.CrossRefGoogle ScholarPubMed
Knuefermann, P., Schwederski, M., Velten, M., Krings, P., Ehrentraut, H., Rüdiger, M., Boehm, O., Fink, K., Dreiner, U., Grohé, C., Hoeft, A., Baumgarten, G., Koch, A., Zacharowski, K. and Meyer, R. (2008). Bacterial DNA induces myocardial inflammation and reduces cardiomyocyte contractility: role of toll-like receptor 9. Cardiovascular Research 78, 26–35.CrossRefGoogle ScholarPubMed
Lazarovici, A., Zhou, T., Shafer, A., Dantas Machado, A. C., Riley, T. R., Sandstrom, R., Sabo, P. J., Lu, Y., Rohs, R., Stamatoyannopoulos, J. A. and Bussemaker, H. J. (2013). Probing DNA shape and methylation state on a genomic scale with DNase I. Proceedings of the National Academy of Sciences of the United States of America 110, 6376–6381.CrossRefGoogle ScholarPubMed
Lu, W. and de Leeuw, E. (2013). Pro-inflammatory and pro-apoptotic properties of human defensin 5. Biochemical and Biophysical Research Communications 436, 557–562.CrossRefGoogle ScholarPubMed
Melo, M. B., Nguyen, Q. P., Cordeiro, C., Hassan, M. A., Yang, N., McKell, R., Rosowski, E. E., Julien, L., Butty, V., Dardé, M. L., Ajzenberg, D., Fitzgerald, K., Young, L. H. and Saeij, J. P. (2013). Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways. PLoS Pathogens 9, e1003779.CrossRefGoogle ScholarPubMed
Mesbah, M. and Whitman, W. B. (1989). Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. Journal of Chromatography A 479, 297–306.CrossRefGoogle ScholarPubMed
Minns, L. A., Menard, L. C., Foureau, D. M., Darche, S., Ronet, C., Mielcarz, D. W., Buzoni-Gatel, D. and Kasper, L. H. (2006). TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii . Journal of Immunology 176, 7589–7597.CrossRefGoogle ScholarPubMed
Monroy, F. P. (2008). Toxoplasma gondii: effect of infection on expression of 14-3-3 proteins in human epithelial cells. Experimental Parasitology 118, 134–138.CrossRefGoogle ScholarPubMed
Mowat, A. (2011). Innate immunity in the intestine. Journal of Innate Immunity 3, 541–542.CrossRefGoogle ScholarPubMed
Orellana, M. A., Suzuki, Y., Araujo, F. and Remington, J. S. (1991). Role of beta interferon in resistance to Toxoplasma gondii infection. Infection and Immunity 59, 3287–3290.CrossRefGoogle ScholarPubMed
Pagnini, C., Corleto, V. D., Mangoni, M. L., Pilozzi, E., Torre, M. S., Marchese, R., Carnuccio, A., Giulio, E. D. and Delle Fave, G. (2011). Alteration of local microflora and α-defensins hyper-production in colonic adenoma mucosa. Journal of Clinical Gastroenterology 45, 602–610.CrossRefGoogle ScholarPubMed
Parker, D., Martin, F. J., Soong, G., Harfenist, B. S., Aguilar, J. L., Ratner, A. J., Fitzgerald, K. A., Schindler, C. and Prince, A. (2011). Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract. MBio 2, e00016–11.CrossRefGoogle ScholarPubMed
Peterson, L. W. and Artis, D. (2014). Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nature Reviews Immunology 14, 141–153.CrossRefGoogle ScholarPubMed
Platz, J., Beisswenger, C., Dalpke, A., Koczulla, R., Pinkenburg, O., Vogelmeier, C. and Bals, R. (2004). Microbial DNA induces a host defense reaction of human respiratory epithelial cells. Journal of Immunology 173, 1219–1223.CrossRefGoogle ScholarPubMed
Raetz, M., Hwang, S. H., Wilhelm, C. L., Kirkland, D., Benson, A., Sturge, C. R., Mirpuri, J., Vaishnava, S., Hou, B., Defranco, A. L., Gilpin, C. J., Hooper, L. V. and Yarovinsky, F. (2013). Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells. Nature Immunology 14, 136–142.CrossRefGoogle ScholarPubMed
Raj, P. A. and Dentino, A. R. (2002). Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiology Letters 206, 9–18.CrossRefGoogle ScholarPubMed
Ronet, C., Darche, S., Leite de Moraes, M., Miyake, S., Yamamura, T., Louis, J. A., Kasper, L. H. and Buzoni-Gatel, D. (2005). NKT cells are critical for the initiation of an inflammatory bowel response against Toxoplasma gondii . Journal of Immunology 175, 899–908.CrossRefGoogle ScholarPubMed
Rumio, C., Besusso, D., Palazzo, M., Selleri, S., Sfondrini, L., Dubini, F., Ménard, S. and Balsari, A. (2004). Degranulation of Paneth cells via toll-like receptor 9. American Journal of Pathology 165, 373–381.CrossRefGoogle ScholarPubMed
Salzman, N. H., Ghosh, D., Huttner, K. M., Paterson, Y. and Bevins, C. L. (2003). Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature 422, 522–526.CrossRefGoogle ScholarPubMed
Salzman, N. H., Hung, K., Haribhai, D., Chu, H., Karlsson-Sjöberg, J., Amir, E., Teggatz, P., Barman, M., Hayward, M., Eastwood, D., Stoel, M., Zhou, Y., Sodergren, E., Weinstock, G. M., Bevins, C. L., Williams, C. B. and Bos, N. A. (2010). Enteric defensins are essential regulators of intestinal microbial ecology. Nature Immunology 11, 76–83.CrossRefGoogle ScholarPubMed
Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A. and Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology 21, 1–26.CrossRefGoogle Scholar
Schulthess, J., Fourreau, D., Darche, S., Meresse, B., Kasper, L., Cerf-Bensussan, N. and Buzoni-Gatel, D. (2008). Mucosal immunity in Toxoplasma gondii infection. Parasite 15, 389–395.CrossRefGoogle ScholarPubMed
Shoda, L. K. M., Kegerreis, K. A., Suarez, C. E., Roditi, I., Corral, R. S., Bertot, G. M., Norimine, J. and Brown, W. C. (2001). DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide. Infection and Immunity 69, 2162–2171.CrossRefGoogle Scholar
Sonaimuthu, P., Fong, M. Y., Kalyanasundaram, R., Mahmud, R. and Lau, Y. L. (2014). Sero-diagnostic evaluation of Toxoplasma gondii recombinant Rhoptry antigen 8 expressed in E. coli . Parasites & Vectors 7, 297.CrossRefGoogle ScholarPubMed
Takeda, S., Miyazaki, D., Sasaki, S., Yamamoto, Y., Terasaka, Y., Yakura, K., Yamagami, S., Ebihara, N. and Inoue, Y. (2011). Roles played by toll-like receptor-9 in corneal endothelial cells after herpex simplex virus type 1 infection. Investigative Ophthalmology and Visual Science 52, 6729–6736.CrossRefGoogle Scholar
Tanaka, T., Rahman, M. M., Battur, B., Boldbaatar, D., Liao, M., Umemiya-Shirafuji, R., Xuan, X. and Fujisaki, K. (2010). Parasiticidal activity of human α-defensin-5 against Toxoplasma gondii . In Vitro Cellular and Developmental Biology Animal 46, 560–565.CrossRefGoogle ScholarPubMed
Vaishnava, S., Behrendt, C. L., Ismail, A. S., Eckmann, L. and Hooper, L. V. (2008). Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface. Proceedings of the National Academy of Sciences of the United States of America 105, 20858–20863.CrossRefGoogle ScholarPubMed
Watson, J. L. and McKay, D. M. (2006). The immunophysiological impact of bacterial CpG DNA on the gut. Clinica Chimica Acta 364, 1–11.CrossRefGoogle ScholarPubMed
Wattrang, E., Berg, M. and Magnusson, M. (2005). Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro . Veterinary Immunology and Immunopathology 107, 265–279.CrossRefGoogle ScholarPubMed
Wehkamp, J., Salzman, N. H., Porter, E., Nuding, S., Weichenthal, M., Petras, R. E., Shen, B., Schaeffeler, E., Schwab, M., Linzmeier, R., Feathers, R. W., Chu, H., Lima, H. Jr., Fellermann, K., Ganz, T., Stange, E. F. and Bevins, C. L. (2005). Reduced Paneth cell alpha-defensins in ileal Crohn's disease. Proceedings of the National Academy of Sciences of the United States of America 102, 18129–18134.CrossRefGoogle ScholarPubMed