Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 neg is online at: average * The Journal of Immunology published online 21 June 2010 Immune Cells in Spleen and Mucosa from submission to initial decision + 4 weeks from acceptance to publication TLR5 Signaling Stimulates the Innate Production of IL-17 and IL-22 by CD3 CD127 Laurye Van Maele, Christophe Carnoy, Delphine Cayet, Pascal Songhet, Laure Dumoutier, Isabel Ferrero, Laure Janot, François Erard, Julie Bertout, Hélène Leger, Florent Sebbane, Arndt Benecke, Jean-Christophe Renauld, Wolf-Dietrich Hardt, Bernhard Ryffel and Jean-Claude Sirard http://www.jimmunol.org/content/early/2010/06/21/jimmun ol.1000115 J Immunol Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. 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Published June 21, 2010, doi:10.4049/jimmunol.1000115 The Journal of Immunology TLR5 Signaling Stimulates the Innate Production of IL-17 and IL-22 by CD3negCD127+ Immune Cells in Spleen and Mucosa Laurye Van Maele,*,†,‡,x,{,1 Christophe Carnoy,*,†,‡,x,{,1 Delphine Cayet,*,†,‡,x,{,1 Pascal Songhet,‖ Laure Dumoutier,# Isabel Ferrero,** Laure Janot,††,‡‡ Franc¸ois Erard,††,‡‡ Julie Bertout,{ He´le`ne Leger,x,xx,{{ Florent Sebbane,*,†,‡,x,{ Arndt Benecke,x,xx,{{ Jean-Christophe Renauld,# Wolf-Dietrich Hardt,‖ Bernhard Ryffel,††,‡‡ and Jean-Claude Sirard*,†,‡,x,{ In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes Downloaded from associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3negCD127+ immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3negCD127+ cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ oll-like receptors are key players in innate immunity and RecentstudieshighlightedthecontributionofIL-17A,IL-17F,and are essential for sensing microbial components and trigger- IL-22 to defensive reactions within the mucosa (3–6). These cyto- T ing the host defense (1). At the luminal interface, the TLR kines help orchestrate innate immunity by stimulating epithelial response is mediated by the epithelium and mainly consists of neu- cells to produce defense molecules, matrix proteases, and tissue trophil recruitment and activation (2). After microbes cross the ep- repair molecules (7, 8). The source of IL-17A, IL-17F, and IL-22 ithelium, sensing occurs within the lamina propria. However, the varies. During an adaptive response, the lymphocytes that differen- nature of the TLR-mediated innate cells and defense factors that are tiate into Th17 cells are the main producers of cytokines (9). IL-17A triggered by microbial desequestration has yet to be defined. can rapidly be produced during innate responses to bacteria or mi- crobial molecular patterns by gd T lymphocytes in a TLR4- by guest on September 26, 2021 dependent manner, NKT cells activated with a-galactosylceramide, *Institut Pasteur de Lille, Centre d’Infection et d’Immunite´ de Lille; †Institut Na- or lymphoid tissue inducer (LTi)-like cells following stimulation tional de la Sante´ et de la Recherche Me´dicale Unite´ 1019; ‡Centre National de la with the TLR2/Dectin-1 agonist zymosan (10–12). NK-like and Recherche Scientifique Unite´ Mixte de Recherche 8204; xUniversite´ Lille Nord de France; {{Institut de Recherche Interdisciplinaire, Centre National de la Recherche LTi-like innate lymphocytes expressing IL-7Ra, NKp46, the Scientifique Unite´ de Service et de Recherche 3078; {Institut Fe´de´ratif de Recherche transcription factor RORgt, and eventually CCR6 are sources of xx †† 142, Lille; Institut des Hautes E´tudes Scientifiques, Bures-sur-Yvette; University of IL-22 and/or IL-17 in mucosa under steady-state conditions (13– Orle´ans and ‡‡Centre National de la Recherche Scientifique Molecular Immunology and Embryology Unite´ Mixte de Recherche 6218, Institut de Transgenose, Orle´ans, 18). Interestingly, microbial flora-colonizing mucosa are required to ‖ France; Institute of Microbiology, Eidgeno¨ssiche Technische Hochschule Zurich, switch on lasting IL-17 and IL-22 production (15, 17). In absence Zurich; **Ludwig Institute for Cancer Research, Lausanne Branch, University of of these innate lymphocytes, infectious colitis is exacerbated, sug- Lausanne, Epalinges, Switzerland; and #Ludwig Institute for Cancer Research, Brussels Branch, de Duve Institute, Universite´ catholique de Louvain, Brussels, gesting an operational role of IL-22 and IL-17 in the gut’s innate Belgium immunity (15, 17). However, the link between TLR-mediated sig- 1L.V.M., C.C., and D.C. contributed equally to this work. naling, Th17-related cytokine production by innate immune cells, Received for publication January 15, 2010. Accepted for publication May 11, 2010. and mucosal defenses has not been defined. This work was supported by the Institut National de la Sante´ et de la Recherche The ability of TLR5 signaling to induce mucosal production of Me´dicale (to. C.C., L.V.M., D.C., and J.-C.S.), the Institut Pasteur de Lille, the IL-17 and IL-22 and thereby promote antimicrobial defense has Universite´ Lille Nord de France, and the Re´gion Nord Pas de Calais (ARCir Europe). W.-D.H. and J.-C.S. are funded by the European Community (Grant INCO-CT-2006- never been investigated. TLR5 detects flagellins—the main pro- 032296). tein of bacterial flagella (19). Flagellins are expressed by bacteria, Microarray data were deposited in the publicly available database (http://mace.ihes. particularly pathogenic bacteria, in the gut and the lung and acti- fr) with accession number 2844328654. vate epithelial TLR5 signaling (19–21). Flagellin expression is Address correspondence and reprint requests to Dr. Jean-Claude Sirard, Center for switched off as soon as bacteria translocate into the lamina propria Infection and Immunity of Lille, Institut National de la Sante´ et de la Recherche (22). Detection of flagellin molecules represents therefore an Me´dicale Unite´ 1019, Centre National de la Recherche Scientifique Unite´ Mixte de Recherche 8204, Institut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille alarm signal for subepithelial invasion and/or disruption of the Cedex, France. E-mail address: [email protected] epithelial barrier function. TLR5 signaling is rapidly induced in The online version of this article contains supplemental material. the lamina propria dendritic cells (DCs) of the small intestine (23). Abbreviations used in this paper: Ct, cycle threshold; DC, dendritic cell; DTX, diph- In the current study, we show that flagellin activates (via DCs) the theria toxin; gc, common g-chain; LTi, lymphoid tissue inducer; ns, nonsignificant; splenic and mucosal production of IL-17 and IL-22 and the sub- WT, wild-type. sequent expression of target genes. This TLR5-mediated response Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 was associated with a unique population of immune cells express- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000115 2 TLR-MEDIATED INNATE IMMUNE CELL ACTIVATION ing CD127 but not CD3 that resembles LTi cells, LTi-like, or NK- Microarrays neg + like innate lymphocytes. Our findings suggest that CD3 CD127 Total RNA (2 mg) was processed on the Mouse Whole Genome Arrays innate immune cells may be instrumental to the host’s mucosal version 2.0 (Applied Biosystems) (27). Data were analyzed using the defense through the early production of Th17-related cytokines. NeONORM method, and heat maps were created as described previously (27, 28). Gene Ontology was analyzed using the Panther Protein Classi- Materials and Methods fication System (www.pantherdb.org). Mice Statistical analysis Specific pathogen-free mouse strains C57BL/6J, C57BL/6J-Ly5.1, BALB/ The Mann-Whitney U test and the Graphpad Prism software 5.0 were used 2 2 2 2 2 2 2 2 2 2 2 2 c, and Tcrb / , Tcrd / , Tcrb / Tcrd / , Tlr5 / (24); Myd88 / (25); in analyses. The Limma test with Benjamini-Hochberg false discovery rate transgenic animals for pre-TCRa, Cd11c-DTR-EGFP (Itagx-DTR/EGFP) (FDR) correction was used for high-throughput PCR with TaqMan Low 2 2 2 2 2 2 (26); Rag2 / Il2rg / backcrossed on C57BL/6J mice; Cd1d / Density Arrays.