A crucial role for interleukin (IL)-1 in the induction of IL-17–producing T cells that mediate autoimmune encephalomyelitis

It was recently demonstrated that interleukin (IL)-23–driven IL-17–producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I–deficient (IL-1RI−/−) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI−/− compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI−/− mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI−/− mice, and IL-23–induced IL-17 production was substantially enhanced by IL-1α or IL-1β, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor κB, and novel protein kinase C isoforms in IL-1– and IL-23–mediated IL-17 production. Tumor necrosis factor α also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

T cells play a pathogenic role in many autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, and multiple sclerosis (MS). MS and the mouse model, experimental autoimmune encephalomyelitis (EAE), are chronic infl ammatory diseases of the central nervous system (1), which until recently were assumed to be mediated by Th1 cells (2). However, IFN-γ KO mice have an increased susceptibility to EAE (3), and disease is exacerbated in mice defi cient in the Th1-polarizing cytokine IL-12 (4). In contrast, induction of disease is blocked in mice defi cient in the novel IL-12 family member, IL-23 (4), which promotes the diff erentiation of IL-17-producing (ThIL-17) cells (5,6). The demonstration that adoptive transfer of autoantigen-specifi c ThIL-17 cells, but not Th1 cells, induced EAE (6), as well as studies in collagen-induced arthritis (7), has estab-lished a defi nitive pathogenic role for these cells in T cell-mediated autoimmune diseases.
Recent reports have demonstrated that ThIL-17 cells are a distinct subtype from Th1 and Th2 cells and do not share a precursor intermediate (8,9). Indeed, their diff erentiation process is inhibited by the signature Th1 and Th2 cytokines, IFN-γ and IL-4 (8). IL-23 can act on eff ector and memory CD4 + T cells to induce T cell proliferation and IL-17 secretion (5), but it can also promote the diff erentiation of naive T cells into ThIL-17 cells (8,9). ThIL-17 cells may function in autoimmunity by promoting the induction of IL-1β and TNF-α. It has been shown that IL-1β and TNF-α can mediate infl ammatory pathology in many autoimmune diseases, and antibodies or receptor antagonists of these infl ammatory cytokines are eff ective therapeutics. In rheumatoid arthritis, IL-17 is thought to play a major role upstream of IL-1 (10,11) in promoting autoimmune joint destruction. Furthermore, studies with IL-1RI −/− mice have established a role for IL-1 in It was recently demonstrated that interleukin (IL)-23-driven IL-17-producing (ThIL-17) T cells mediate infl ammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specifi c ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I-defi cient (IL-1RI −/− ) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was signifi cantly lower in IL-1RI −/− compared with wild-type mice, and this correlated with a failure to induce autoantigen-specifi c ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI −/− mice by adoptive transfer of autoantigen-specifi c cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI −/− mice, and IL-23induced IL-17 production was substantially enhanced by IL-1 or IL-1 , even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor B, and novel protein kinase C isoforms in IL-1-and IL-23mediated IL-17 production. Tumor necrosis factor also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our fi ndings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis
the induction of EAE (12). In addition, IL-1β is present in MS lesions and families with a high IL-1β to IL-1 receptor antagonist (IL-1Ra) production ratio are at a greater risk of having a relative with relapse-onset MS than those with a low ratio (13). However, the precise role of IL-1 in the pathogenesis of EAE is unclear.
We demonstrate that IL-23 alone is a poor stimulus for T cell IL-17 production and that IL-1 is essential for its induction. Immunization with foreign or self-antigens and a Tolllike receptor ligand or CFA demonstrated that IL-1 plays a vital and selective role in promoting antigen-specifi c ThIL-17 cells in vivo. Furthermore, a failure to induce EAE in IL-1RI −/− mice was associated with a failure to generate ThIL-17 cells. IL-1 acted directly on T cells in synergy with IL-23 to promote IL-17 secretion. TNF-α also synergized with IL-23 to induce IL-17, but this was dependant on IL-1. Our fi ndings demonstrate a central role for IL-1 upstream of IL-17 in promoting autoimmunity in EAE by driving the diff erentiation of pathogenic ThIL-17 cells.

RESULTS AND DISCUSSION IL-1RI is required for the induction of ThIL-17 cells
In a study on the role of IL-1RI in the adjuvant eff ect of the Toll-like receptor agonist LPS, we discovered that IL-1 is  were injected with MOG in CFA followed by PT as described above or with MOG-specifi c T cells (9 × 10 6 cells/mouse) generated from the spleens of C57BL/6 mice with EAE. Disease scores (n = 5) ± SD.

BRIEF DEFINITIVE REPORT
selectively required for induction of ThIL-17 cells. Parenteral immunization of C57BL/6 mice with KLH in the presence of LPS induced antigen-specifi c T cells that secreted IFN-γ, IL-10, and IL-17, whereas immunization with KLH in the absence of adjuvant induced little or no antigen-specifi c cytokine production ( Fig. 1 A). Immunization of IL-1RI −/− mice with KLH and LPS also induced antigen-specifi c IFN-γ and IL-10 production. However, antigen-specifi c IL-17 production was signifi cantly lower in IL-1RI −/− than in wild-type C57BL/6 mice. Antigenspecifi c proliferation was not signifi cantly diff erent between the groups (not depicted). IL-17 concentrations induced by PMA and anti-CD3 were also signifi cantly lower in cells from IL-1RI −/− mice ( Fig. 1 B). Thus, IL-1 is selectively required for antigen or anti-CD3 and PMA-induced IL-17 production.

IL-1RI-defi cient mice are resistant to EAE, and this correlates with reduced autoantigen-specifi c IL-17 production
We next examined the possibility that IL-1 may function in the pathology of EAE by promoting pathogenic autoantigenspecifi c ThIL-17 cells. Wild-type C57BL/6 mice developed severe EAE after immunization with myelin oligodendrocyte glycoprotein (MOG) and CFA. In contrast, the incidence of EAE (2/15 IL-1RI −/− vs. 13/15 in C57BL/6) was considerably lower in IL-1RI −/− mice ( Fig. 2 A). This is consistent with studies indicating an important role for IL-1 in EAE (12,14). However, we demonstrate that the resistance of IL-1RI −/− mice to the development of EAE correlates with a specifi c defect in activating autoantigen-specifi c ThIL-17 cells (Fig. 2 B). MOG-specifi c IL-17 production was selectively reduced in IL-1RI −/− mice in comparison with wildtype mice (Fig. 2 B). MOG-specifi c proliferation and secretion of IL-6, IL-10, and TNF-α were comparable in IL-1RI −/− and wild-type mice. Production of IFN-γ was reduced, but to a modest extent (twofold reduction compared with a 22-fold reduction in IL-17 production).
We examined the ability of MOG-specifi c T cells from wild-type mice to induce EAE in IL-1RI −/− mice and compared this with standard EAE induction. Transfer of a MOGspecifi c T cell line from C57BL/6 mice with advanced EAE into IL-1RI −/− mice induced EAE in fi ve out of fi ve mice, whereas mice injected directly with MOG and CFA did not develop disease (Fig. 2 C), demonstrating that IL-1RI is dispensable in the development of EAE after induction of the autoantigen-specifi c ThIL-17 T cells. Our data support fi ndings that neuroantigen-specifi c ThIL-17 cells can transfer EAE (6) and suggest that the major role of IL-1 in the development and IFN-γ was performed. Percentages refer to IL-17 production by gated CD3 + T cells. Splenic CD4 + (C) and CD8 + (D) T cells (5 × 10 5 cells/ml) from C57BL/6 mice were incubated with 10 U/ml IL-2 and 1 μg/ml anti-CD3 antibody alone or together with 0.1-1 ng/ml IL-23 and/or 1 ng/ml IL-1α or IL-1β. IL-17 concentrations were measured by ELISA in supernatants collected after 3 d. IL-23 plus IL-1 versus IL-23 only: *, P < 0.05; **, P < 0.01; ***, P < 0.001. of EAE is in promoting pathogenic ThIL-17 cells. The clinical symptoms in EAE are ameliorated by blocking IL-17 (9,15), and injection of anti-IL-17 delays the onset of disease and prevents the characteristic mononuclear cell infi ltrate in spinal cord white matter (9). Our data place IL-1 upstream of IL-17 and suggest that the effi cacy of blocking IL-1 as a therapy for EAE may refl ect a failure to activate ThIL-17 cells.
IL-1 synergizes with IL-23 to promote T cell IL-17 production Because our data pointed to a selective eff ect of IL-1 in promoting ThIL-17 cells, we determined whether IL-1 directly induced IL-17 secretion by T cells in vitro. Stimulation with rIL-23 induced IL-17 production by spleen cells from C57BL/6 but not from IL-1RI −/− mice (Fig. 3 A). However, production of IL-17 in response to IL-23 alone was relatively low. Furthermore, IL-1 alone did not induce IL-17 secretion by spleen cells but signifi cantly augmented IL-23-induced IL-17 production. Intracellular cytokine staining revealed that IL-17 was produced by CD3 + T cells (Fig. 3 B). Furthermore, the addition of anti-IFN-γ increased the frequency of IL-17-secreting T cells after stimulation with IL-1 and IL-23, consistent with a negative eff ect of IFN-γ on IL-17 production (9).

BRIEF DEFINITIVE REPORT
To determine whether the synergistic eff ect of IL-1 on IL-23-induced IL-17 production was mediated directly on T cells or required accessory cells, we assessed the eff ects of IL-1 and IL-23 on purifi ed T cells. CD4 + and CD8 + T cells from C57BL/6 mice were stimulated with IL-23 in the presence or absence of IL-1. IL-23 at concentrations of up to 1 ng/ml did not induce signifi cant IL-17 production (Fig. 3 C). However, the addition of IL-1α or IL-1β resulted in a dramatic synergistic eff ect with IL-23 in promoting IL-17 secretion. Both CD4 + and CD8 + T cells have been implicated in the pathology of MS (16) and EAE, and ThIL-17 T cells have been shown to induce EAE (6). Collectively with our data, these fi ndings suggest that the requirement for IL-1RI to promote ThIL-17 cells and EAE refl ects the need for synergy between IL-1 and IL-23 to induce IL-17 production.
To address whether the eff ects of IL-23 and IL-1 involved amplifi cation of a TCR-activated pathway, we examined the eff ects of IL-1 and IL-23 in the presence or absence of anti-CD3. IL-1-and IL-23-induced IL-17 production was retained in the absence of anti-CD3 (Fig. 4 A). Recent studies have indicated that IL-23 acts by promoting the survival and expansion of ThIL-17 T cells (17). Analysis of IL-17 mRNA by real-time PCR revealed that IL-23 or IL-1 and IL-23, but not IL-1 alone, induced potent IL-17 gene transcription (Fig. 4 B). Therefore, our data suggest that IL-23 is vital for induction of IL-17 mRNA expression. The recently documented ability of IL-6 and TGF-β to promote the diff erentiation of ThIL-17 cells (17)(18)(19) may be dependant on IL-1. We have found that IL-6 can synergize with IL-23 to promote IL-17 production, but this eff ect was abrogated in cells from IL-1RI −/− mice (not depicted). Alternatively, IL-1 and IL-23 may act independently of IL-6 and TGF-β to promote IL-17 production from T cells. However, the relative roles of IL-23, IL-6, TGF-β, and IL-1 in the diff erentiation and survival of ThIL-17 cells require further investigation.
Our data suggest that in addition to roles for IL-23, as well as CD28 and ICOS costimulation (9), in promoting the diff erentiation of naive T cells into ThIL-17 cells, there is an additional and central requirement for IL-1.

Signaling pathways involved in IL-1-and IL-23-induced IL-17 production
Using specifi c inhibitors of intracellular signaling pathways, we found that novel isoforms of protein kinase C (PKC), NF-κB, and PI3K are essential for IL-1 and IL-23 to promote IL-17 production by T cells (Fig. 4 C). These inhibitors completely blocked IL-1-and IL-23-induced IL-17 production, which was observed in the presence or absence of TCR stimulation, indicating that the cytokines can activate signaling pathways that directly promote IL-17 production.
PKCθ-defi cient mice are resistant to EAE and this correlates with reduced IL-17 in the central nervous systems of these mice (20). Our data support an important role for PKCθ (21) in IL-1-and IL-23-mediated induction of T cell IL-17 production. We also demonstrate that PI3K and NF-κB are important in the induction of IL-17 (22) and that phosphorylation of Akt (a readout for PI3K activation) and IκB-α were induced independently by IL-1 and IL-23 (Fig. 4 D). It was reported that MAP kinase pathways are involved in IL-17 transcriptional regulation in human T cells (23). We demonstrate that in murine cells, inhibitors of ERK (UO126) and p38 (SB203580) did not inhibit IL-17 and, notably, ERK inhibition enhanced IL-17 production (Fig. 4 C). However, in the absence of TCR stimulation, inhibition of p38 reduced IL-17 production in response to IL-1 and IL-23.
Because there are complex cytokine networks operating at sites of infl ammation, several cytokines, including IL-23, IL-1, and TNF-α, may contribute to amplify IL-17 production. For example, IL-17 can synergize with TNF-α and IL-1 to promote infl ammatory gene expression (28). Our demonstration that IL-1 acts upstream of IL-17 in EAE, combined with fi ndings that IL-17 can promote IL-1, indicates that there may be a "vicious circle" with IL-1 and IL-17 promoting each other and amplifying the autoimmune response.
Our fi ndings provide further evidence for a role of innate immune responses in directing adaptive immunity. Specifically, in addition to the established proinfl ammatory role of IL-1, our fi ndings reveal a hitherto unrecognized regulatory role for IL-1α and IL-1β in selectively promoting pathogenic ThIL-17 cells that mediate autoimmunity.

MATERIALS AND METHODS
Mice. C57BL/6 and IL-1RI −/− mice were obtained from Harlan Ltd. and bred at Trinity College Dublin under specifi c pathogen-free conditions. Mice were maintained according to the regulations of the European Union and the Irish Department of Health.
Immunization experiments. Mice were immunized s.c. in the fl ank with KLH (10 μg per mouse; endotoxin-free; Calbiochem) alone or together with LPS (10 μg per mouse; Escherichia coli, serotype R515; Qbiogene), or with PBS. Mice were killed after 7 d and inguinal lymph nodes were removed.
T cell purifi cation. Mouse T cell CD4 and CD8 subset column kits (R&D Systems) were used to purify CD4 + and CD8 + cells from the spleens of C57BL/6 mice. T cell purity was routinely >95%. T cells and spleen cells were incubated with IL-1α, IL-1β, TNF-α, and IL-23 (endotoxin content was <1 EU/μg protein in all cases; R&D Systems) alone or in combination. In the studies on intracellular signaling pathways, all inhibitors were obtained from Calbiochem. For experiments on purifi ed T cells, IL-2 (Hoff man-La Roche Inc.) and anti-CD3 were also included except where stated. Intracellular cytokine staining. Spleen cells from C57BL/6 mice were incubated for 72 h with medium or cytokines in the presence of 10 μg/ml brefeldin A (for the last 4 h) before extracellular staining with anti-CD3. Cells were washed, blocked with 1 μg/ml of Fcγ blocker (BD Biosciences), fi xed, permeabilized (Fix and Perm cell permeabilization kit; Caltag), and stained with anti-IL-17, anti-IFN-γ antibody, or the appropriate isotype control. Flow cytometric analysis was performed using a FACSCalibur fl ow cytometer (Becton Dickinson).
Western blotting. CD3 + cells (10 6 cells/ml) were purifi ed from the spleens of C57BL/6 mice and incubated with 10 ng/ml IL-1, 10 ng/ml IL-23, or IL-1 and IL-23 in the presence or absence of 1 μg/ml anti-CD3. Cell lysates were prepared and resolved on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and blocked. Membranes were incubated with antibodies specifi c for phospho-Akt and phospho-IκBα (Cell Signaling Technology) and β-actin (Sigma-Aldrich). After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature and developed by chemiluminescence (Lite-Ablot; Euroclone).
Real-time PCR. CD3 + T cells (3.5 × 10 6 cells/ml) were stimulated with 1 μg/ml anti-CD3 and 10 ng/ml IL-1, 10 ng/ml IL-23, or IL-1 and IL-23. RNA was harvested from the cells 48 h later using an RNEasy isolation kit (QIAGEN) followed by reverse transcription using a QuantiTect Reverse transcription kit (QIAGEN). Real-time PCR for the detection of IL-17 mRNA was performed using sense and antisense primers for IL-17A (Mm00439619_m1; Applied Biosystems) and a FAM-labeled MGB Taqman probe. 18S ribosomal RNA was used as an endogenous control. Samples were assayed on an Applied Biosystems 7300 PCR machine.
Statistical analysis. Cytokine levels were compared by one-way ANOVA. Where signifi cant diff erences were found, the Tukey-Kramer multiple comparisons test was used to identify diff erences between individual groups. Signifi cance of the results from real-time PCR was determined using Newman-Keul's test.
We would like to thank Dr. Thomas Connor, Department of Physiology, Trinity College Dublin, for help with real-time PCR. This work was supported by a Science Foundation Ireland Principal Investigator Award (no. 00/PI.I/B045 to K.H.G. Mills).
K.H.G. Mills is a founder, director, and shareholder in Opsona Therapeutics, a University campus company involved in the development of antiinfl ammatory therapeutics. The authors declare no other potential confl icts of interest.