ARTICLE Received 24 Jan 2015 | Accepted 13 May 2015 | Published 25 Jun 2015 DOI: 10.1038/ncomms8464 OPEN IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2 þ Vg6 þ gd T cells Aoi Akitsu1,2,3,4,5, Harumichi Ishigame1,w, Shigeru Kakuta1,w, Soo-hyun Chung1,5, Satoshi Ikeda1, Kenji Shimizu1,5, Sachiko Kubo1,5, Yang Liu1,w, Masayuki Umemura6, Goro Matsuzaki6, Yasunobu Yoshikai7, Shinobu Saijo1,w & Yoichiro Iwakura1,2,4,5 Interleukin-17 (IL-17)-producing gd T(gd17) cells have been implicated in inflammatory diseases, but the underlying pathogenic mechanisms remain unclear. Here, we show that both CD4 þ and gd17 cells are required for the development of autoimmune arthritis in IL-1 receptor antagonist (IL-1Ra)-deficient mice. Specifically, activated CD4 þ T cells direct gd T-cell infiltration by inducing CCL2 expression in joints. Furthermore, IL-17 reporter mice reveal that the Vg6 þ subset of CCR2 þ gd T cells preferentially produces IL-17 in inflamed joints. Importantly, because IL-1Ra normally suppresses IL-1R expression on gd T cells, IL-1Ra-deficient mice exhibit elevated IL-1R expression on Vg6 þ cells, which play a critical role in inducing them to produce IL-17. Our findings demonstrate a pathogenic mechanism in which adaptive and innate immunity induce an autoimmune disease in a coordinated manner. 1 Laboratory of Molecular Pathogenesis, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. 2 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan. 3 Research Fellow of the Japan Society for the Promotion of Science (JSPS), Tokyo 102-0083, Japan. 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama 332-0012, Japan. 5 Division of Experimental Animal Immunology, Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba 278-0022, Japan. 6 Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213, Japan. 7 Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. w Present addresses: RIKEN Center for Integrative Medical Sciences, Kanagawa 230-0045, Japan (H.I.); Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan (S.K.); Renji Hospital Clinical Stem Cell Research Center, Shanghai Jiao Tong University School of Medicine, Shang Hai 200127, China (Y.L.); Department of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba 263-8522, Japan (S.S.). Correspondence and requests for materials should be addressed to Y.I. (email: [email protected]). NATURE COMMUNICATIONS | 6:7464 | DOI: 10.1038/ncomms8464 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8464 nterleukin (IL)-17 plays important roles in the development of development of arthritis. Both Vg6 þ and Vg4 þ gd T cells autoimmune diseases, such as rheumatoid arthritis and were recruited to the joints, but only the Vg6 þ subset efficiently Ipsoriasis, by inducing expression of proinflammatory cyto- produced IL-17, because Il1rn À / À Vg6 þ cells intrinsically kines and chemokines, recruiting neutrophils and activating expressed high levels of IL-1R due to the loss of down- T cells and B cells1,2. Although helper CD4 þ T (Th17) cells regulation of IL-1R expression by IL-1Ra. These observations are well-known producers of IL-17 that contribute to the provide novel insights into the pathogenic mechanism underlying development of autoimmune diseases, recent studies showed the development of autoimmune arthritis in Il1rn À / À mice. that innate immune cells and innate-like cells are also important 3,4 sources of IL-17 in local inflammatory tissues . Mouse Results autoimmune disease models have revealed that IL-17-producing cd T cells mainly produce IL-17 in Il1rn À / À mouse joints.We gd T(gd17) cells are an important innate source of IL-17 analysed IL-17-producing cells in arthritic Il1rn À / À mice. Both (refs 5–13). In collagen-induced arthritis, experimental auto- gd17 cell and Th17 cell populations were elevated in draining immune encephalomyelitis and psoriasis-like skin inflammation, lymph nodes (LNs) of affected joints (Fig. 1a,b), whereas pro- the synergy between gd17 and ab T cells is important for disease þ þ þ þ 5,6,11,14 portions of IL-17 CD8 T cells and IL-17 DX5 T cells were development , but it remains unclear how gd17 cells induce unchanged (Supplementary Fig. 1). Notably, most joint-infiltrat- tissue-specific inflammation. ing IL-17-producing cells were gd T cells, whereas Th17 cells were gd17 cells share many characteristics with Th17 cells. However, rare (Fig. 1c). Moreover, immunofluorescence staining revealed in contrast to Th17 cells in which differentiation in the periphery that IL-17 in the joints was primarily expressed in gd T cells is required for IL-17 production, the functional potential of gd17 gd 15–17 (Fig. 1d). These observations suggest that 17 cells play a cells is already determined during intrathymic development . pathogenic role in the development of arthritis in Il1rn À / À mice. These gd thymocytes, which express the transcription factor 18 RORgt and the signature cytokine receptor IL-23R , leave the þ thymus as functionally committed cells19. Therefore, gd T cells cd and CD4 T cells are involved in arthritis development.To analyse the pathogenic roles of gd T cells and CD4 þ T cells, we produce IL-17 directly following stimulation with IL-1b and IL- À / À 23 without T cell receptor (TCR) stimulation in the periphery5,13. injected either anti-gd TCR or anti-CD4 mAb into Il1rn Although the expression of IL-23R on gd17 cells is constitutive5, mice before onset of disease. Both antibody treatments sig- expression of IL-1R in the periphery is tissue-type dependent20. nificantly suppressed the incidence of arthritis (Fig. 2a,b). Il1r1 À / À mice or anti-IL-1R monoclonal antibody (mAb) Although the severity scores, determined by the swelling and treatment abrogates IL-17 production in gd T cells11,20, ankylosing changes of the affected ankles of antibody-treated suggesting that IL-1R expression plays a critical role in IL-17 mice, were similar to those of untreated mice (Supplementary production. However, the regulatory mechanism of IL-1R Fig. 2a,b), the microscopic histological scores significantly expression remains unclear. In addition to RORgt, transcription decreased following treatment with anti-gd TCR mAb (Fig. 2c,d). factors such as Blk21, Hes-1 (ref. 22), NFkB23, SOX4 and SOX13 Anti-gd TCR mAb treatment depleted 70–90% of gd T cells at (ref. 24) are also required for gd17 cell development. early time points, but the population gradually recovered In mice, the TCRg locus consists of seven Vg (Vg1–Vg7) genes (Supplementary Fig. 2c), probably due to the development of that are closely correlated with effector functions, although in antibodies against this mAb. Nonetheless, the anti-gd TCR mAb most strains Vg3 is a pseudogene25. IL-17 is produced by Vg4 þ treatment greatly decreased the gd17 population (Fig. 2e; and Vg6 þ gd T cells26 (Heilig and Tonegawa’s nomenclature27). Supplementary Fig. 2d), suggesting that anti-gd TCR mAb effectively depleted gd17 cells, at least at earlier times. IL-17 Although overall gene expression patterns are similar between À these two subsets18, each subset has distinct features. Vg6 þ gd T production in the gd TCR population was not elevated in these mice (Supplementary Fig. 2d). Most populations of CD4 þ cells express the invariant Vg6/Vd1 TCR, develop only in the late- À / À embryonic thymus and preferentially localize in the uterus, vagina, T cells, including Th17 cells, remained depleted in Il1rn lung, dermis and peritoneal cavity28,29. On the other hand, Vg4 þ mice on day 11 after anti-CD4 mAb treatment (Fig. 2f; gd T cells have a more diverse TCR repertoire, and develop in Supplementary Fig. 2e). These results suggest that both gd T cells and CD4 þ T cells are involved in the development of arthritis both foetal and adult thymus. Subsequently, they circulate in À / À 30 in Il1rn mice. However, arthritis developed normally in blood and reside in the dermis and secondary lymphoid organs . À / À À / À þ Tcrd Il1rn mice (Supplementary Fig. 2f), and IL-17- However, the differences between the pathogenic roles of Vg6 À À À and Vg4 þ gd17 cells, particularly the contribution of Vg6 þ gd17 producing CD4 CD8 gdTCR T cells were increased in the cells to inflammatory diseases, remain unclear. LNs and joints of these mice (Supplementary Fig. 2g). The mechanism of gd17 cell migration into inflammatory sites is also poorly understood. Recent gene array analysis showed that Both cd17 and CD4 þ T cells collaborate to develop arthritis. the expression of chemokine receptors such as CCR6, CCR2 and Next, we directly examined the pathogenic role of gd T cells and CXCR6 is upregulated in gd17 thymocytes18. Moreover, CCR6 CD4 þ T cells by adoptive cell transfer. gd T cells derived from expression is often used as a marker of gd17 cells17. However, it Cd4 À / À Il1rn À / À mice and/or CD4 þ T cells derived from remains unknown whether these chemokine receptors function in Tcrd À / À Il1rn À / À mice were transferred into scid/scid mice. gd17 cell migration. We found that scid/scid mice that received transfer of whole- IL-1 receptor antagonist (IL-1Ra, gene symbol: Il1rn)isan Il1rn À / À T cells or a mixture of gd and CD4 þ T cells developed endogenous inhibitor of IL-1 activity that competes with IL-1a arthritis, whereas those that received transfer of gd or CD4 þ and IL-1b for IL-1R binding.