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• W2803433102 abstract "•T cell production of GM-CSF is dispensable for the initiation of arthritis•GM-CSF from stromal cells is crucial for the initiation of autoimmune arthritis•GM-CSF-producing synovial-resident ILCs augment autoimmune arthritis•ILC production of GM-CSF is stimulated by IL-2, IL-33, or TLR9 ligands Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention. Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention. Proinflammatory cytokines such as IL-1, IL-6, IL-17, IL-23, GM-CSF, and TNF-α are important in the development and maintenance of chronic inflammatory disorders such as autoimmune disease. Neutralization of these cytokines or blockade of their receptors is effective in hampering the progression of tissue inflammation and inducing long-term remission in autoimmune disorders (Cho and Feldman, 2015Cho J.H. Feldman M. Heterogeneity of autoimmune diseases: pathophysiologic insights from genetics and implications for new therapies.Nat. Med. 2015; 21: 730-738Crossref PubMed Scopus (154) Google Scholar). Recent genome-wide association studies of immune-mediated diseases have revealed common inflammatory pathways, involving the genes encoding these cytokines and receptors, in various autoimmune diseases (Parkes et al., 2013Parkes M. Cortes A. van Heel D.A. Brown M.A. Genetic insights into common pathways and complex relationships among immune-mediated diseases.Nat. Rev. Genet. 2013; 14: 661-673Crossref PubMed Scopus (386) Google Scholar). It remains to be determined, however, how key inflammatory cytokines control non-lymphoid as well as lymphoid target cells in autoimmune tissue inflammation, how environmental factors, as well as genetic factors, contribute to the inflammation, and how the cytokine-dependent inflammatory pathways can be targeted to treat or prevent autoimmune diseases. IL-17-producing T helper (Th17) cells play critical roles for host defense against infectious pathogens but can also mediate various autoimmune or inflammatory reactions. They express the lineage defining transcription factor Rorγt and require IL-6 and TGF-β for their differentiation and IL-1 and IL-23 for their terminal effector functions (Ivanov et al., 2006Ivanov I.I. McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. Cua D.J. Littman D.R. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3963) Google Scholar, McGeachy et al., 2009McGeachy M.J. Chen Y. Tato C.M. Laurence A. Joyce-Shaikh B. Blumenschein W.M. McClanahan T.K. O’Shea J.J. Cua D.J. The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo.Nat. Immunol. 2009; 10: 314-324Crossref PubMed Scopus (784) Google Scholar, Veldhoen et al., 2006Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (3008) Google Scholar). In rheumatoid arthritis (RA), for example, it has been shown that various inflammatory cytokines including TNF-α, IL-1, and IL-6 are involved in joint inflammation and that T cells interact with tissue-resident macrophage-like or fibroblast-like synoviocytes (FLSs) in destroying cartilage and bone in the joint (Bartok and Firestein, 2010Bartok B. Firestein G.S. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis.Immunol. Rev. 2010; 233: 233-255Crossref PubMed Scopus (1260) Google Scholar). It still remains unclear, however, how arthritogenic Th17 cells mediate chronic tissue inflammation in the joint via local cytokine and cellular networks. GM-CSF is a key proinflammatory cytokine for the activation of dendritic cells (DCs) and macrophages; for example, DCs respond to GM-CSF to secrete IL-6 and IL-23, which sustain pathogenic Th17 cells in vivo (Sonderegger et al., 2008Sonderegger I. Iezzi G. Maier R. Schmitz N. Kurrer M. Kopf M. GM-CSF mediates autoimmunity by enhancing IL-6-dependent Th17 cell development and survival.J. Exp. Med. 2008; 205: 2281-2294Crossref PubMed Scopus (199) Google Scholar). Moreover, IL-1 and IL-23 signaling drives Rorγt-expressing Th17 cells to secrete GM-CSF, perpetuating autoimmune inflammation, for example, in mouse experimental autoimmune encephalomyelitis (EAE) (Codarri et al., 2011Codarri L. Gyülvészi G. Tosevski V. Hesske L. Fontana A. Magnenat L. Suter T. Becher B. RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.Nat. Immunol. 2011; 12: 560-567Crossref PubMed Scopus (908) Google Scholar, El-Behi et al., 2011El-Behi M. Ciric B. Dai H. Yan Y. Cullimore M. Safavi F. Zhang G.X. Dittel B.N. Rostami A. The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.Nat. Immunol. 2011; 12: 568-575Crossref PubMed Scopus (814) Google Scholar). The antigen-presentation capacity of monocytes and synovial inflammatory macrophages can also be enhanced by stimulation with GM-CSF through upregulation of MHC class II expression (Alvaro-Gracia et al., 1989Alvaro-Gracia J.M. Zvaifler N.J. Firestein G.S. Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.J. Exp. Med. 1989; 170: 865-875Crossref PubMed Scopus (131) Google Scholar). In addition, GM-CSF signaling evokes an inflammatory signature in CCR2+Ly6Chi monocytes and drives them to induce tissue damage (Croxford et al., 2015Croxford A.L. Lanzinger M. Hartmann F.J. Schreiner B. Mair F. Pelczar P. Clausen B.E. Jung S. Greter M. Becher B. The cytokine GM-CSF drives the inflammatory signature of CCR2+ monocytes and licenses autoimmunity.Immunity. 2015; 43: 502-514Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). GM-CSF thus appears to possess pleiotropic effects on monocytes and/or DCs and Th17 cells, augmenting the activation of innate and adaptive immune cells and amplifying tissue inflammation. The SKG strain of mice, carrying a point mutation in the gene encoding the T cell receptor (TCR)-proximal signaling molecule ZAP-70, develops CD4+ T cell-mediated autoimmune arthritis, which clinically and immunologically resembles RA in humans (Hata et al., 2004Hata H. Sakaguchi N. Yoshitomi H. Iwakura Y. Sekikawa K. Azuma Y. Kanai C. Moriizumi E. Nomura T. Nakamura T. Sakaguchi S. Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice.J. Clin. Invest. 2004; 114: 582-588Crossref PubMed Scopus (243) Google Scholar, Sakaguchi et al., 2003Sakaguchi N. Takahashi T. Hata H. Nomura T. Tagami T. Yamazaki S. Sakihama T. Matsutani T. Negishi I. Nakatsuru S. Sakaguchi S. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice.Nature. 2003; 426: 454-460Crossref PubMed Scopus (656) Google Scholar). The mice spontaneously develop the disease in a microbially conventional environment but not under a specific-pathogen-free (SPF) condition. Yet the disease can be induced in SPF SKG mice by stimulation of innate immunity via Toll-like receptors (TLRs), the Dectin pathway, or complement activation pathways (Hashimoto et al., 2010Hashimoto M. Hirota K. Yoshitomi H. Maeda S. Teradaira S. Akizuki S. Prieto-Martin P. Nomura T. Sakaguchi N. Köhl J. et al.Complement drives Th17 cell differentiation and triggers autoimmune arthritis.J. Exp. Med. 2010; 207: 1135-1143Crossref PubMed Scopus (156) Google Scholar, Yoshitomi et al., 2005Yoshitomi H. Sakaguchi N. Kobayashi K. Brown G.D. Tagami T. Sakihama T. Hirota K. Tanaka S. Nomura T. Miki I. et al.A role for fungal beta-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice.J. Exp. Med. 2005; 201: 949-960Crossref PubMed Scopus (346) Google Scholar). We previously demonstrated, by using SKG mice, how self-reactive T cells are generated in the process of thymic-positive and -negative selection (Sakaguchi et al., 2003Sakaguchi N. Takahashi T. Hata H. Nomura T. Tagami T. Yamazaki S. Sakihama T. Matsutani T. Negishi I. Nakatsuru S. Sakaguchi S. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice.Nature. 2003; 426: 454-460Crossref PubMed Scopus (656) Google Scholar), become activated in the periphery by recognizing self-antigens, differentiate into arthritogenic Th17 cells upon stimulation of innate immunity (Hirota et al., 2007aHirota K. Hashimoto M. Yoshitomi H. Tanaka S. Nomura T. Yamaguchi T. Iwakura Y. Sakaguchi N. Sakaguchi S. T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis.J. Exp. Med. 2007; 204: 41-47Crossref PubMed Scopus (383) Google Scholar), migrate into the joints (Hirota et al., 2007bHirota K. Yoshitomi H. Hashimoto M. Maeda S. Teradaira S. Sugimoto N. Yamaguchi T. Nomura T. Ito H. Nakamura T. et al.Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model.J. Exp. Med. 2007; 204: 2803-2812Crossref PubMed Scopus (705) Google Scholar), and aggress self-antigens expressed by synoviocytes (Ito et al., 2014Ito Y. Hashimoto M. Hirota K. Ohkura N. Morikawa H. Nishikawa H. Tanaka A. Furu M. Ito H. Fujii T. et al.Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease.Science. 2014; 346: 363-368Crossref PubMed Scopus (65) Google Scholar). In addition, dysfunction of Foxp3+ regulatory T cells due to the ZAP-70 mutation facilitates autoimmune T cells to expand, become activated, and exert their effector functions, causing autoimmune diseases in a wide spectrum of organs or tissues (Tanaka et al., 2010Tanaka S. Maeda S. Hashimoto M. Fujimori C. Ito Y. Teradaira S. Hirota K. Yoshitomi H. Katakai T. Shimizu A. et al.Graded attenuation of TCR signaling elicits distinct autoimmune diseases by altering thymic T cell selection and regulatory T cell function.J. Immunol. 2010; 185: 2295-2305Crossref PubMed Scopus (77) Google Scholar). These features make this spontaneous model of autoimmune arthritis suitable for elucidating how Th17 cells mediate autoimmune diseases, especially RA, via interacting with other lymphoid and non-lymphoid cells at the inflammation site and controlling their production of inflammatory cytokines. In this report, we showed via the SKG model of autoimmune arthritis that arthritogenic Th17 cells orchestrated the progression of chronic joint inflammation by stimulating radio-resistant stromal cells including FLSs to secrete GM-CSF and subsequently by expanding GM-CSF-producing innate lymphoid cells (ILCs). Notably, GM-CSF secretion from ILCs was regulated by IL-2, the alarmin IL-33, and endogenous TLR-9 ligands released from damaged tissue-resident cells, in inflamed joints. The results demonstrate how antigen-specific self-reactive T cells stimulate the local cellular and cytokine networks that drive chronic tissue inflammation. A single injection of 20 mg mannan, an activator of the lectin pathway for complement activation, is able to synchronously evoke T cell-mediated autoimmune arthritis within 2–3 weeks in SPF SKG mice with an increase in Th17 cells in lymph nodes and joints (Hashimoto et al., 2010Hashimoto M. Hirota K. Yoshitomi H. Maeda S. Teradaira S. Akizuki S. Prieto-Martin P. Nomura T. Sakaguchi N. Köhl J. et al.Complement drives Th17 cell differentiation and triggers autoimmune arthritis.J. Exp. Med. 2010; 207: 1135-1143Crossref PubMed Scopus (156) Google Scholar). In the draining lymph nodes and inflamed joints of mannan-treated SKG mice, approximately 2% and 7%, respectively, of CD4+ T cells co-expressed IL-17 and GM-CSF, but not IFN-γ (Figures 1A and 1B ). In addition, GM-CSF (encoded by Csf2)-deficient (Csf2−/−) SKG mice were highly resistant to the induction of autoimmune arthritis by mannan as were Il17a−/− SKG mice, indicating a crucial role of GM-CSF for arthritis development in SKG mice (Figure 1C; Hirota et al., 2007aHirota K. Hashimoto M. Yoshitomi H. Tanaka S. Nomura T. Yamaguchi T. Iwakura Y. Sakaguchi N. Sakaguchi S. T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis.J. Exp. Med. 2007; 204: 41-47Crossref PubMed Scopus (383) Google Scholar). To determine the origin of such GM-CSF-producing cells in arthritic SKG mice, we generated IL-17-fate reporter SKG mice by crossing SKG mice with Il17aCre and R26ReYFP fate reporter strains (Hirota et al., 2011Hirota K. Duarte J.H. Veldhoen M. Hornsby E. Li Y. Cua D.J. Ahlfors H. Wilhelm C. Tolaini M. Menzel U. et al.Fate mapping of IL-17-producing T cells in inflammatory responses.Nat. Immunol. 2011; 12: 255-263Crossref PubMed Scopus (855) Google Scholar). Following mannan treatment, more than 30% of CD4+ T cells in inflamed joints were eYFP+, indicating that they were producing IL-17 or had once produced the cytokine (exTh17 cells) (Figure 1D). In addition, one-third of eYFP+ cells were producing GM-CSF, indicating that IL-17-producing CD4+ T cells produced GM-CSF in inflamed joints. Also, only ∼5% of eYFP+ cells were producing IFN-γ, suggesting that differentiation toward Th1-like cells was not the main cell fate of Th17 or exTh17 cells in this model, in contrast with EAE, in which the vast majority of exTh17 cells were producing IFN-γ (Hirota et al., 2011Hirota K. Duarte J.H. Veldhoen M. Hornsby E. Li Y. Cua D.J. Ahlfors H. Wilhelm C. Tolaini M. Menzel U. et al.Fate mapping of IL-17-producing T cells in inflammatory responses.Nat. Immunol. 2011; 12: 255-263Crossref PubMed Scopus (855) Google Scholar). Next, we adoptively transferred CD4+ T cells from Csf2−/− or wild-type (WT) SKG mice into Rag2−/− mice to determine the pathogenicity of GM-CSF-producing CD4+ T cells (Figure 1E). Csf2−/− SKG CD4+ T cells were able to induce autoimmune arthritis in all the recipient mice, although arthritis was significantly less severe than that induced by WT SKG CD4+ T cell transfer. Analysis of IL-17 and GM-CSF production by CD4+ T cells in the spleen and inflamed joints revealed that IL-17-producing CD4+ T cells, which were crucial for initiating SKG autoimmune arthritis (Hirota et al., 2007aHirota K. Hashimoto M. Yoshitomi H. Tanaka S. Nomura T. Yamaguchi T. Iwakura Y. Sakaguchi N. Sakaguchi S. T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis.J. Exp. Med. 2007; 204: 41-47Crossref PubMed Scopus (383) Google Scholar), had similarly differentiated from Csf2−/− or WT SKG CD4+ T cells (Figure 1F). These results demonstrated that both T cell-derived and non-T cell-derived GM-CSF contributed to joint inflammation in SKG mice and that GM-CSF from Th cells was not mandatory for this induction of autoimmune arthritis. To further assess the possible contribution of GM-CSF from non-CD4 T cells to arthritis development, we transferred Csf2−/− or WT SKG CD4+ T cells into Rag2−/− or Csf2−/−Rag2−/− mice (Figure 2A). Transfer of Csf2−/− SKG CD4+ T cells induced histologically evident arthritis in Rag2−/− mice although the arthritis was macroscopically less severe than WT SKG CD4+ T cell transfer (Figures 2B and 2C). In contrast, not only Csf2−/− SKG CD4+ T cells but also WT SKG CD4+ T cells completely failed to induce arthritis macroscopically and histologically in Csf2−/−Rag2−/− mice. Intracellular cytokine staining of transferred Csf2−/− or WT SKG CD4+ T cells revealed that both populations were activated and had differentiated into IL-17-producing CD4+ T cells regardless of whether the hosts produced GM-CSF or not (Figures 2D and 2E). Thus, the failure in arthritis development following transfer of CD4+ T cells into Csf2−/− hosts could be attributed not to impaired Th17 cell differentiation but to impaired GM-CSF production by certain host non-T cells stimulated by Th17 cells. We next attempted to determine the non-T cell source of GM-CSF in inflamed joints in SKG mice. FLSs are known to be key effector cells capable of secreting large amounts of pro-inflammatory mediators (e.g., tissue-degrading enzymes and cytokines such as TNF-α), which destroy the cartilage and bone in SKG arthritis as in human RA (Bartok and Firestein, 2010Bartok B. Firestein G.S. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis.Immunol. Rev. 2010; 233: 233-255Crossref PubMed Scopus (1260) Google Scholar, Hata et al., 2004Hata H. Sakaguchi N. Yoshitomi H. Iwakura Y. Sekikawa K. Azuma Y. Kanai C. Moriizumi E. Nomura T. Nakamura T. Sakaguchi S. Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice.J. Clin. Invest. 2004; 114: 582-588Crossref PubMed Scopus (243) Google Scholar). Gene expression analysis of FLSs that were freshly isolated from SKG arthritic joints and stimulated in vitro with recombinant IL-17 revealed quick upregulation within 1–3 hr of the expression of Csf2, Ccl20, Cxcl1, Cxcl5, Il6, Nfkbiz, Lif, and Zc3h12a (Figure 3A). Yet activated FLSs themselves neither expressed GM-CSF receptor-alpha nor responded to recombinant GM-CSF (Figure S1A and data not shown). The cells predominantly expressing GM-CSF receptor-alpha in inflamed joints were CD11b+Ly-6C+Ly-6G− inflammatory monocytes, as reported previously with EAE (Croxford et al., 2015Croxford A.L. Lanzinger M. Hartmann F.J. Schreiner B. Mair F. Pelczar P. Clausen B.E. Jung S. Greter M. Becher B. The cytokine GM-CSF drives the inflammatory signature of CCR2+ monocytes and licenses autoimmunity.Immunity. 2015; 43: 502-514Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), indicating that this population could be a main target of GM-CSF in the joint (Figure S1B). In addition, cell transfer of WT, but not Il17a−/−, SKG CD4+ T cells into Rag2−/− mice significantly induced Csf2, Ccl20, and Il6 transcription in CD45−podoplanin+ synoviocytes that were purified from the joints 4 weeks after cell transfer (Figure 3B). Thus, one of the cellular targets of arthritogenic Th17 cells is FLSs, which secrete GM-CSF upon IL-17 stimulation. Next, in order to search for a potential hematopoietic source of GM-CSF other than T cells in joint inflammation, we prepared single-cell suspensions from enzyme-digested inflamed joints in Rag2−/− mice that had received Csf2−/− or WT SKG CD4+ T cells. By intracellular GM-CSF staining, CD4+ and non-CD4+ T cell populations in CD45+ hematopoietic cells contained similar percentages of GM-CSF-producing cells in Rag2−/− mice transferred with WT SKG CD4+ T cells, whereas non-CD4 cells were the only source of GM-CSF in those transferred with Csf2−/− SKG CD4+ T cells (Figure 4A). To characterize the non-CD4 source of GM-CSF, various CD45+ cell populations were purified from SKG inflamed joints by the use of cell lineage markers and assessed for their cytokine expression by quantitative RT-PCR (Figure 4B). The vast majority of joint-infiltrating cells were CD11b+Ly-6G− inflammatory monocytes or macrophages and CD11b+Ly-6G+ neutrophils, a common feature of Th17 cell-mediated inflammation (Iwakura et al., 2011Iwakura Y. Ishigame H. Saijo S. Nakae S. Functional specialization of interleukin-17 family members.Immunity. 2011; 34: 149-162Abstract Full Text Full Text PDF PubMed Scopus (975) Google Scholar). However, these populations of neutrophils and inflammatory monocytes did not express Csf2 while they specifically expressed Il1b. In contrast, lymphoid populations including CD4+ T cells and lineage marker-negative ILCs expressed Csf2 but not Il1b. 2% to 4% of CD45+ lymphocytes in arthritic joints were lineage-negative CD45+ ILCs, which were equivalent in ratio between Rag2−/− mice transferred with Csf2−/− or WT SKG CD4+ T cells (Figures 4C, 4D, and S2A for gating strategy). We also found a fraction of synovial-resident ILCs in healthy joints of SKG mice (which are on the BALB/c background) and other mouse strains (Figures 4E, S3A, and S4). Expansion of these synovial ILCs expressing Ki-67 was detectable only in inflamed joints, but not in the draining LNs or bone marrow (BM), suggesting that synovial ILCs preferentially expanded in inflamed joints, but might scarcely migrate to the adjacent lymphoid organs (Figures 4E, 4F, and S3B). By flow cytometry, most of the joint-infiltrating ILCs expressed IL-7Ra, CD25, IL-33Ra, and CD44, while nearly a half of them expressed c-kit and/or MHC class II, and ∼10% of them expressed CCR6 (Figure 4G). Further analysis of transcription factors defining ILC subsets revealed that approximately 60% and 6% of the ILCs expressed Gata-3 or Rorγt, respectively, indicating predominant expansion of ILC2s in arthritic joints, as shown in intestinal infection (Figures 4H and 4I; Hoyler et al., 2012Hoyler T. Klose C.S. Souabni A. Turqueti-Neves A. Pfeifer D. Rawlins E.L. Voehringer D. Busslinger M. Diefenbach A. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells.Immunity. 2012; 37: 634-648Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). Indeed, GM-CSF-producing ILCs in healthy and inflamed joints expressed Gata-3 and/or IL-13, which are the signature transcription factor and cytokine, respectively, of ILC2s (Figures 4J and S3A). ILCs with a similar phenotype were also present in normal joints and expanded in collagen antibody-induced arthritis in C57/BL6 mice, indicating that the presence and expansion of GM-CSF-producing synovial ILCs was not dependent on the mouse genetic background or the mode of arthritis induction by autoimmune Th17 cells or autoantibody (Figures 4K and S4). In addition, inflamed joints contained Rorγt-expressing ILC3s (Figure 4H). As ILC3s were reportedly able to produce GM-CSF in the intestine to maintain gut homeostasis (Mortha et al., 2014Mortha A. Chudnovskiy A. Hashimoto D. Bogunovic M. Spencer S.P. Belkaid Y. Merad M. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis.Science. 2014; 343: 1249288Crossref PubMed Scopus (558) Google Scholar), we attempted with IL-17-fate reporter SKG mice to determine whether joint ILC3s, whose signature cytokine is IL-17, also produced GM-CSF. In correlation with the small percentage (∼6%) of Rorγt-expressing ILC3s shown in Figure 4H, joint ILCs contained a small fraction (∼4%) of IL-17-fate reporter-positive cells producing GM-CSF, suggesting that a part of GM-CSF-producing ILCs were derived from ILC3s in this model (Figure 4L). Synovial ILCs also highly expressed the transcription factor encoded by Bhlhe40, which reportedly controlled GM-CSF production by pathogenic Th cells (Figure 4M; Lin et al., 2014Lin C.C. Bradstreet T.R. Schwarzkopf E.A. Sim J. Carrero J.A. Chou C. Cook L.E. Egawa T. Taneja R. Murphy T.L. et al.Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation.Nat. Commun. 2014; 5: 3551Crossref PubMed Scopus (102) Google Scholar, Martínez-Llordella et al., 2013Martínez-Llordella M. Esensten J.H. Bailey-Bucktrout S.L. Lipsky R.H. Marini A. Chen J. Mughal M. Mattson M.P. Taub D.D. Bluestone J.A. CD28-inducible transcription factor DEC1 is required for efficient autoreactive CD4+ T cell response.J. Exp. Med. 2013; 210: 1603-1619Crossref PubMed Scopus (68) Google Scholar). To further determine the contribution of these ILCs to arthritis development, we attempted to selectively deplete ILCs by anti-Thy1.2 mAb in Thy1.2+ Rag2−/− mice that had been transferred with CD4+ T cells from Thy1.1+ congenic SKG mice (Figure 4N). Multiple injections of 500 μg anti-Thy1.2 mAb every week indeed reduced the severity of autoimmune arthritis significantly. Taken together, synovial ILCs selectively expanded in arthritic joints, secreting GM-CSF and contributing to the development of autoimmune arthritis. To assess the contribution of GM-CSF from either stromal cells or ILCs to autoimmune arthritis, we x-irradiated (6Gy) Rag2−/− or Csf2−/−Rag2−/− mice, reconstituted the mice with Rag2−/− or Csf2−/−Rag2−/− BM cells, transferred to them Csf2−/− SKG CD4+ T cells 6 weeks after BM reconstitution, and assessed arthritis score 12 weeks after CD4+ T cell transfer (Figure 5A). Rag2−/− BM-transferred x-irradiated Csf2−/− Rag2−/− mice and Csf2−/−Rag2−/− BM-transferred x-irradiated Rag2−/− mice, in which GM-CSF production was restricted to ILCs or radio-resistant stromal cells including FLSs, respectively, developed significantly less severe arthritis than Rag2−/− BM-transferred x-irradiated Rag2−/− mice, in which both ILCs and radio-resistant stromal cells produced GM-CSF (Figure 5B). The percentage of total synovial ILCs expanding in inflamed joints was comparable between Rag2−/− BM transfer and Csf2−/−Rag2−/− BM transfer, although the total number of synovial ILCs was smaller in the latter chimeras presumably because of less severe synovial inflammation (Figure 5C, data not shown). Also, the latter still possessed a small number of recipient-derived GM-CSF-producing ILCs (Figure 5D), indicating that reduction, if not complete abrogation, of GM-CSF production in ILCs was able to attenuate arthritis severity. In addition, by Csf2−/− BM transfer, the proportion of ILCs co-expressing GM-CSF and IL-13 decreased, with similar proportions of total IL-13+ ILCs, suggesting that joint inflammation had expanded Csf2−/− ILCs, which appeared to be hardly pathogenic (Figures 5D and 5E). To further assess the contribution of ILCs to arthritis development, we x-irradiated Thy1.1+ Rag2−/− mice, reconstituted them with BM cells from Thy1.1+ Rag2−/− mice and Thy1.2+ Csf2−/− SKG mice at a 1:1 ratio, and treated the mice 6 weeks later with a single injection of 20 mg mannan and subsequently with 500 μg anti-Thy1.1 mAb once a week. Anti-Thy-1.1 treatment selectively depleted synovial Thy1.1+ GM-CSF-producing ILCs, while control mAb treatment allowed similar expansion of synovial Thy1.1+ GM-CSF-producing ILCs and Thy1.2+ GM-CSF-nonproducing ILCs (Figure 5F). This selective depletion of synovial GM-CSF-producing ILCs significantly reduced the severity of arthritis (Figure 5G). These results collectively indicate that GM-CSF from both ILCs and radio-resistant stromal cells synergistically contributes to the development of severe autoimmune arthritis in SKG mice. Given the finding that IL-2, IL-7, and IL-33 control the effector function of ILC2s in inflamed tissues (Klose and Artis, 2016Klose C.S. Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis.Nat. Immunol. 2016; 17: 765-774Crossref PubMed Scopus (621) Google Scholar), we assessed their contribution to GM-CSF production by ILCs. Among various tissue homogenates prepared from normal mice, joint tissues substantially expressed IL-33, which significantly increased in arthritic joints at both the mRNA and protein levels compared with unaffected joints (Figures 6A and 6B ). This was consistent with the finding by others that IL-33 was constitutively expressed in the nucleus of some immune cells and stromal cells in inflamed joints (Fock et al., 2013Fock V. Mairhofer M. Otti G.R. Hiden U. Spittler A. Zeisler H. Fiala C. Knöfler M. Pollheimer J. Macrophage-derived IL-33 is a critical factor for placental growth.J. 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• W2803433102 date "2018-06-01" @default.
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• W2803433102 title "Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis" @default.
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• W2803433102 doi "https://doi.org/10.1016/j.immuni.2018.04.009" @default.
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