It has been suggested that IL-17RC forms a complex with IL-17RA to mediate the functions of IL-17A and IL-17F homodimers as well as IL-17A/F heterodimers. It is still unclear whether IL-17RC is absolutely required for the signaling of IL-17 cytokines in vivo. By using Il-17rc–deficient mice, we show that IL-17RC is essential for the signaling of IL-17A, IL-17F, and IL-17A/F both in vitro and in vivo. IL-17RC does not preassociate with IL-17RA on the cell surface; rather IL-17A can induce the formation of an IL-17RC and IL-17RA complex. This process is not dependent on the intracellular similar expression to fibroblast growth factor genes and IL-17Rs (SEFIR) domain of IL-17RC, but the SEFIR is essential in IL-17A signal transduction. Finally, Il-17rc−/− mice develop much milder disease in an experimental autoimmune encephalomyelitis model, supporting an essential role for IL-17RC in mediating immune-mediated CNS inflammation.
Interleukin-17 family cytokines consist of six structurally related members, namely, IL-17A through IL-17F. IL-17A, also called CTLA-8 (1) or IL-17 (2), is the founding member of this family of cytokines. Yao et al. (2) first identified IL-17A as a novel cytokine that shares 57% identity with Herpesvirus saimiri gene 13. IL-17F was independently cloned by two groups based on its homology to IL-17A (3, 4). Among all of the IL-17 family cytokines, IL-17F shares the highest homology, ~50%, with IL-17A. In fact, IL-17A and IL-17F can form not only homodimers but also IL-17A/F heterodimers (5, 6). These three IL-17 ligands share largely overlapping biological functions, with their potencies on most of the target cells ranked as IL-17A > IL-17A/F > IL-17F (5–8). On the basis of the study of IL-17F crystal structure, it has been suggested that all of the IL-17 family cytokines may adopt a cysteine knot structure also found in the nerve growth factor and platelet-derived growth factor families of cytokines (9). Many leukocyte subsets, including CD4 T cells, CD8 T cells, γδ T cells, NKT cells, and lymphoid tissue inducer cells express IL-17A and IL-17F (10–12). Importantly, IL-17A and IL-17F are signature cytokines for a newly defined T helper subset, termed Th17 cells (13, 14).
IL-17RA encodes a type I transmembrane protein and defines a novel cytokine receptor family due to its lack of major homology to any other known cytokine receptors (2). Thus far, four additional IL-17R family members, designated IL-17RB, IL-17RC, IL-17RD, and IL-17RE, have been categorized into this family based on structural similarity (15, 16). IL-17RA is also an essential receptor component for IL-17F homodimer and IL-17A/F heterodimer (7, 8). It was noted from the original study that the binding potency of IL-17A to IL-17RA is somewhat weaker than the biological activity of IL-17A on its target cells (17). It was speculated that there might be an additional receptor chain expressed on the cell surface and required for the biological functions of IL-17A (17). Consistent with this premise, human IL-17A and IL-17F fail to elicit biological responses from Il-17ra–deficient mouse fibroblasts overexpressing human IL-17RA (18). Interestingly, responses are restored when these cells are cotransfected with both human IL-17RA and IL-17RC, suggesting that IL-17RC is the requisite second chain of the IL-17R complex (18). Both IL-17RA and IL-17RC are broadly expressed, particularly on epithelial cells as well as on fibroblasts and other stromal cells (2, 17, 19). The expression patterns of IL-17RA and IL-17RC, however, are not completely overlapping. For example, IL-17RA, but not IL-17RC, is found on various immune cell types (19). It is, therefore, speculated that IL-17RA may be sufficient for mediating IL-17A signaling in cells that lack IL-17RC (20, 21). Recent mechanistic studies, nonetheless, further support that both IL-17RA and IL-17RC may be required for the signaling of IL-17A and IL-17F. First, IL-17RC has high binding affinities to both IL-17A and IL-17F. In contrast, IL-17RA has a high binding affinity to IL-17A, with much weaker affinity to IL-17F (21). These differences in binding affinity may partially explain the weaker biological potencies of IL-17F compared with those of IL-17A on many cell types. Second, the structural complex of IL-17F and IL-17RA, recently solved by Ely et al. (22), suggests that there is a preference in forming the heterodimeric receptor complex. Finally, our initial characterization of Il-17rc–deficient mice further supports that IL-17RC is indeed required for the functions of IL-17A and IL-17F (23).
IL-17RA forms a preassembled complex without ligand binding (24). It is unclear, however, whether IL-17RA and IL-17RC are preassembled on the cell surface without binding to their ligands. It has shown that when both IL-17RA and IL-17RC are overexpressed in HEK293 cells they may form a preassociated complex in the absence of ligand, as demonstrated by the ability of one receptor chain to coimmunoprecipitate with the other receptor chain (18). Endogenous IL-17RA or IL-17RC, however, cannot be precipitated if the other chain is overexpressed in the cells (18), suggesting that there is no or a very low degree of preassociation of IL-17RA and IL-17RC chains on the cell surface. One of the key features of IL-17R family members is the presence of the similar expression to fibroblast growth factor genes and IL-17Rs (SEFIR) domain, which is similar to the TIR domain in the IL-1/TLR family (25). Deletions or mutations in the SEFIR domain of IL-17RA lead to the abolishment of the downstream signaling induced by IL-17A (26). A SEFIR domain-containing adaptor protein, Act1, has been shown to directly bind to IL-17RA through SEFIR domain-dependent interactions (27, 28). Act1 is essential for the functions of IL-17A and IL-17F, because Act1-deficient cells are compromised in most IL-17–induced signaling (28). TRAF6, which has a SEFIR domain, has also been reported to mediate the signaling of IL-17A (29). In addition, IL-17A can activate NF-κB, MAPK, and C/EBP pathways, all of which are important for the transcription of IL-17–induced genes (16).
IL-17A and IL-17F elicit broad proinflammatory responses from the various tissues and cell types known to express the IL-17R complex. IL-17A and IL-17F induce the expression of many genes involved in tissue-mediated innate immunity. These genes can be categorized as proinflammatory chemokines, cytokines, antimicrobial peptides, and proteins involved in tissue remodeling and acute phase response (14). In addition, IL-17A and IL-17F are very potent activators of neutrophils. Both cytokines promote neutrophil expansion through the induction of G-CSF. Furthermore, they also potentiate the recruitment of neutrophils through the augmentation of the expression of chemokines (14). Another key feature of the IL-17 pathway is its ability to synergize with other cytokines, such as TNF-α, IL-1, and IFN-γ, in the induction proinflammatory responses from various cell types (30, 31).
The cellular functions of IL-17A and IL-17F suggest that these cytokines may have important roles in host defense against infections. Both Il-17a– and Il-17ra–deficient mice show increased susceptibility to a variety of extracellular pathogens, including both Gram-positive and Gram-negative bacteria, such as Staphyloccus aureus and Klebsiella pneumoniae, the yeast Candida albicans, and the protozoal parasite Toxoplasma gondii (20, 32–34). In addition, the IL-17 pathway also participates in both local and systemic immune responses. For example, the delayed-type hypersensitivity response and Ab responses are reduced in Il-17a−/− mice (32–35). Similarly, Il-17ra−/− mice are protected in preclinical arthritis, inflammatory bowel disease models, supporting the critical role of IL-17RA in mediating the biological functions of IL-17 in vivo (36–38). Finally, elevated IL-17A and IL-17F have been detected in many human autoimmune diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (39–42), suggesting that the IL-17 pathway may play a potential pathogenic role in human diseases. Blocking the IL-17 pathway ameliorates disease in many preclinical animal models, including collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) (43–46).
In summary, studies with both Il-17a−/− and Il-17ra−/− mice have established the important immunological functions of the IL-17 pathway. However, despite rapid advancement in the field of IL-17 biology, many questions still remain, particularly the specific role of IL-17RC in the IL-17 signaling pathway and in the development of autoimmune diseases. In this study, we for the first time fully characterize Il-17rc–deficient mice. We show that IL-17RC is essential in mediating the functions of IL-17A and IL-17F, both in vitro and in vivo, and demonstrate a critical role for IL-17RC in mediating the pathogenesis of EAE.
Materials and Methods
Il-17rc knockout (KO) mice were generated by Lexicon Pharmaceuticals (The Woodlands, TX) as previously described (23). Il-17rb KO mice were also generated by Lexicon Pharmaceuticals and described in Supplemental Material. Il-17rb KO mice were bred for five generations onto BALB/c background. Il-17rc KO mice used were those bred for three generations onto C57BL/6 background (for in vitro studies and the myelin oligodendrocyte glycoprotein [MOG] EAE study) or those on congenic C57BL/6 background (for MOG-EAE study). Results of the MOG-EAE study obtained from cohorts of mice on the mixed background or congenic background were comparable. All of the animal experiments were approved by the Genentech Institutional Animal Care and Use Committee.
Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and Il-17rc–deficient embryos at embryonic day 14.5 and maintained in DMEM supplemented with 10% FBS (Hyclone, Logan, UT), nonessential amino acids (0.1 mM; Invitrogen, Carlsbad, CA), 2-ME (55 nM; Invitrogen), penicillin G (100 μg/ml), and streptomycin (100 μg/ml). Lung fibroblasts were isolated from 6-wk-old WT and Il-17rc–deficient mice and maintained in DMEM supplemented with 10% FBS (Hyclone), glutamine (2 mM), penicillin G (100 μg/ml), and streptomycin (100 μg/ml).
Cytokine stimulation and ELISA
All of the recombinant cytokines, including murine IL-17A, IL-17F, IL-17A/F, IL-17E, and human IL-17A were purchased from R&D Systems (Minneapolis, MN). All cytokine stimulation lasts 24 h, unless otherwise noted. All ELISAs were performed with DuoSet ELISA kits from R&D Systems, unless otherwise noted. For IL-17E stimulation, splenocytes, at 5× 106 cells per 250 μl per 96-well plate, from Il-17rb– or Il-17rc–deficient mice and WT littermate controls were cultured for 72 h in DMEM supplemented with 10% FBS (Hyclone), glutamine (2 mM), penicillin G (100 μg/ml), and streptomycin (100 μg/ml) in the presence or absence of 10 ng/ml murine IL-17E. The supernatant was harvested and assayed for IL-5 level by ELISA (BD Biosciences, San Jose, CA).
Small interfering RNA knockdown
Neonatal human foreskin fibroblasts (HFFs) were purchased from Cascade Biologics and cultured in medium 106 supplemented with Low Serum Growth Supplement (Cascade Biologics, Portland, OR). Small interfering RNA (siRNA) oligonucleotides for human ACT1, IL-17RA, and IL-17RC, as well as a nontargeting siRNA pool (as a negative control), were obtained from Dharmacon (Lafayette, CO) (ON-TARGETplus SMARTpool). At the time of plating (into a 24-well plate), 75,000 HFFs were transfected with siRNA oligonucleotides (to a final concentration of 100 nM) using TransIT-TKO transfection reagent (Mirus Bio, Madison, WI). Human IL-17A was added to the cells (to a final concentration of 25 ng/ml) 32 h after transfection/plating. Cells were harvested at 48 h for RNA isolation (RNeasy Mini Kit; Qiagen, Valencia, CA). Cells in duplicate wells were harvested at 72 h for flow cytometry analysis using biotinylated anti-human IL-17RA and IL-17RC (both raised in goat; R&D Systems). Conditioned media were harvested at 72 h for human G-CSF ELISA (R&D Systems)
RNA isolation and real-time RT-PCR
RNA was isolated using the RNeasy Mini Kit (Qiagen). For the MOG-EAE study, RNA from the brain at day 15 was isolated using RNeasy Lipid Tissue Mini Kit. A 5-mm fragment (~100 mg) near the optic tracts was selected to enrich for lesions. We conducted real-time RT-PCR on an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) with primers, probes, and TaqMan One-Step RT-PCR Master Mix (Applied Biosystems). Samples were normalized to the control housekeeping gene RPL-19 (mouse) or HPRT1 (human) and reported according to the ΔΔCT method: ΔΔCT = ΔCT sample − ΔCT reference. Primers and probes for mouse C/EBPδ and IκBζ and human ACT1, IL-17RA, IL-17RC, and HPRT1 were purchased from Applied Biosystems as TaqMan Gene Expression Assays. Mouse ribosomal housekeeping gene RPL-19, forward, 5′-GCA TCC TCA TGG AGC ACA T-3′, reverse, 5′-CTG GTC AGC CAG GAG CTT-3′, and probe, 5′-CTT GCG GGC CTT GTC TGC CTT-3′ (FAM, TAMRA). Mouse CXCL1, forward, 5′-CCG AAG TCA TAG CCA CAC TC-3′, reverse, 5′-TTT CTG AAC CAA GGG AGC TT-3′, and probe, 5′-AAG GCA AGC CTC GCG ACC AT-3′ (FAM, TAMRA). Mouse CXCL2, forward, 5′-ACA TCC AGA GCT TGA GTG TGA-3′, reverse, 5′-GCC CTT GAG AGT GGC TAT G-3′, and probe, 5′-CCC ACT GCG CCC AGA CAG AA-3′ (FAM, TAMRA). Mouse CCL5, forward, 5′-TGC TCC AAT CTT GCA GTC GT-3′, reverse, 5′-CTT CTT CTC TGG GTT GGC ACA-3′, and probe, 5′-TTT GTC ACT CGA AGG AAC CGC CAA GT-3′ (FAM, TAMRA). Mouse CCL20, forward, 5′-AAT GCT ATC ATC TTT CAC ACG AAG A-3′, reverse, 5′-CAG CCC TTT TCA CCC AGT TC-3′, and probe, 5′-AAG AAA ATC TGT GTG CGC TGA TCC AAA GC-3′ (FAM, TAMRA).
Transfection, coimmunoprecipitation, and Western blot
Mouse cDNA expressing FLAG-tagged full-length IL-17RC (GenBank protein database, accession no. CAD23360) and FLAG-tagged IL-17RC deletion mutant (IL-17RCΔ, lacking aas 497–647) were generated by PCR and cloned into a pRK-5 vector (BD Biosciences). They were then subcloned into a retroviral bicistronic vector that expresses GFP (pMSCV-IRES-GFP) and used to transfect Phoenix Ecotropic cells (ATCC, Manassas, VA). Viral supernatant suspensions were used to infect Il-17rc–deficient MEFs to restore murine IL-17RC expression. GFP+ MEFs were purified by FACS and passaged until they spontaneously immortalized. To examine the expression level of IL-17RC in WT, Il-17rc–deficient, FLAG-tagged reconstituted MEFs, flow cytometry analysis was done using biotinylated anti-FLAG Ab (Sigma-Aldrich, St. Louis, MO). For coimmunoprecipitation, Il-17rc–deficient MEFs and MEFs that stably express FLAG-tagged IL-17RC or IL-17RCΔ were stimulated with recombinant mouse IL-17A (50 ng/ml) for various length of time. Cell extracts were incubated with FLAG M2 resin (Sigma-Aldrich) overnight. Beads were then washed three times with lysis buffer and eluted with 3× FLAG solution (Sigma-Aldrich). Eluted proteins were analyzed on Novex Bis-Tris Gel (Invitrogen) followed by immunoblot with anti-mouse IL-17RA (R&D Systems).
For the cell signaling study in IL-17RC WT and KO MEFs, cells were stimulated with IL-17A (50 ng/ml) for various length of time and lysed with radioimmunoprecipitation assay buffer. Lysates were analyzed on Novex Bis-Tris Gel followed by immunoblot with anti-mouse IκB-α (Cell Signaling Technology, Danvers, MA) and anti-mouse actin (Sigma-Aldrich).
In vitro osteoclastogenesis
Bone marrow cells (BMCs) were isolated from tibiae of 7- to 9-wk-old BALB/c mice. Osteoblasts were isolated from calvaria of E19 IL-17RC KO and WT mice using standard methods (47). For coculture, calvaria-derived osteoblasts were seeded on day 0 at 0.5 × 105 cells per well in a 48-well plate in complete media (a-MEM supplemented with 10 nM 1α,25(OH)2D3, 1 μM PGE2, and10% FBS) with indicated reagents (IL-17A, IL-17F, or osteoprotegerin, all at 50 ng/ml). On day 1, 1 × 106 freshly isolated BMCs were added to the osteoblast culture. On day 5, 50% of the medium was refreshed. On day 10, osteoclastogenesis was assessed using tartrate-resistant acid phosphatase (TRAP) staining according to the manufacturer’s directions (Sigma-Aldrich).
T cell isolation and differentiation
Naive T cells (CD4+CD25−CD45RBhi) were purified from splenocytes, prelabeled with anti-CD4, anti-CD25, and anti-CD45RB (all from BD Pharmingen, San Diego, CA), of 9-wk-old WT and IL-17RC KO littermates by FACS-based sorting. A total of 106 naive T cells per milliliter were activated by plate-bound anti-CD3 (2C11, 5 μg/ml) and soluble anti-CD28 (37.5.1, 1 μg/ml) under Th0, in the presence of 10 μg/ml anti–IL-4 and 10 μg/ml anti–IFN-γ, or Th1 conditions with 5 ng/ml IL-12 and 10 μg/ml anti-IL-4. Anti-mouse IL-4 and anti-mouse IFN-γ Abs are from BD Phamingen. Recombinant mouse IL-12 is from R&D Systems. In addition, IL-17 (20 ng/ml; R&D Systems) was added to assess the affect of IL-17 on IFN-γ production. On day 3, supernatant was harvested for IFN-γ measurement, and T cells from both Th0 and Th1 conditions were expended in 4 ml fresh media. On day 7, resting Th cells were collected and counted. A total of 106 fully differentiated Th cells were restimulated with plate-bound anti-CD3 (2C11, 1 μg /ml) and soluble anti-CD28 (37.5.1, 1 μg /ml) without addition of cytokines. Forty-eight hours later, supernatant from these reactivated T cells was harvested for IFN-γ measurement by ELISA (BD Biosciences).
Induction of EAE
For EAE induction, 8-wk-old IL-17RC KO and WT littermates were immunized s.c. with 200 μg MOG35–55 peptide emulsified in 800 μg CFA supplemented with Mycobacterium tuberculosis at the back near the base of the tail. On day 0 and day 2 after immunization, 200 ng pertussis toxin was administered i.p. Clinical disease was scored three times per week starting at day 7 as follows: 0, no clinical disease; 1, limp tail or hind limb weakness but not both; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, moribund.
MOG recall assay and lymph node intracellular FACS analysis
Mice immunized with MOG35–55 were perfused with heparin–saline and sacrificed 21 d after immunization. Spleens were removed, and splenocytes were cultured in DMEM supplemented with 10% FBS (Hyclone), glutamine (2 mM), penicillin G (100 μg/ml), and streptomycin (100 μg/ml) in the presence of MOG35–55 at concentrations ranging from 0.34 to 30 μg/ml. Proliferation was assessed by 3H incorporation, and cytokine release was assessed by IL-17A and IFN-γ ELISA of culture supernatants (at 48 h) according to the manufacturer’s recommendations (R&D Systems). Mononuclear cells were isolated from brain and spinal cord as follows. Brain and spinal cord were disrupted with a gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA) and then passed through a 70-μm cell strainer to obtain a single-cell suspension. Cells were centrifuged in 37% Percoll for 20 min at 1000 × g. Cell pellets were collected and washed. These mononuclear infiltrates as well as splenocytes and cells isolated from inguinal lymph nodes were stimulated with PMA (50 ng/ml) and ionomycin (1 μM) for 2 h, and 10 μg/ml brefeldin A (Epicenter Technology, Madison, WI) was added for an additional 2 h. Intracellular cytokine staining was performed with the BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s directions. The following Abs were used: PerCP-conjugated anti-mCD3, allophycocyanin-conjugated anti-mCD4, PE-conjugated anti-murine IL-17A (TC11-18H10, BD Biosciences), FITC-conjugated anti-murine IFN-γ (XMG1.2; ebiosciences, San Diego, CA).
Brain and spinal cord were harvested from each mouse that survived to day 15. Tissues were fixed in neutral buffered formalin and paraffin-embedded, and sections were cut at 4 μm. Four sections of spinal cord from each of three different levels were examined. All of the spinal cord sections were stained with H&E to evaluate lesion severity and with luxol fast blue (LFB) to document demyelination. H&E-stained histologic sections of spinal cord were scored as follows for inflammation: 0 = no lesions; 1 = mild, ≤3 small foci; mostly meningeal; no or minimal myelinopathy or superficial mild myelinopathy without inflammatory cell infiltration; 2 = moderate, ≤5 foci meningeal and deep or multifocally extensive superficial; mild to moderate accompanying myelinopathy; 3 = severe, multifocally extensive and deep. LFB-stained histologic sections of spinal cord were scored as follows for demyelination: 0 = no lesions; 1 = mild, ≤3 small foci of subpial and/or perivascular attenuation of staining; 2 = moderate, ≤5 foci of subpial/perivascular and deep attenuation of staining, but not extending to gray matter; or multifocally extensive subpial attenuation of staining; 3 = severe, multifocally extensive and deep attenuation of staining, at least focally reaching gray matter. For each spinal cord slide, average scores (averages of four sections on each slide) for both overall lesion severity and demyelination were reported.
We calculated statistical significance by t test with Prism software (GraphPad, La Jolla, CA) and paired t test with JMP software (JMP, Cary, NC). We considered all p values ≤ 0.05 significant. Unless otherwise specified, all studies for which data are presented are representative of at least two independent experiments.
Mouse IL-17RC is required for the signaling of IL-17A and IL-17F, but not for IL-17E/IL-25, in vitro
To critically examine the roles of IL-17RC in the IL-17 pathway and in the development of autoimmune diseases, we generated Il-17rc–deficient mice. Il-17rc−/− mice develop normally and have leukocyte subset counts that are comparable to those of WT mice in their peripheral blood, lymph nodes, spleen, and bone marrow (Supplemental Fig. 1). Fibroblasts were generated from lung (Fig. 1A), mouse embryo (Fig. 1B), and tail tip (23) of WT and Il-17rc−/− mice. IL-17A and, to a lesser extent, IL-17F induced the production of IL-6, KC/CXCL1 (Fig. 1), and G-CSF (data not shown) in WT fibroblasts. The induction of these cytokines and chemokines was completely abolished in fibroblasts generated from Il-17rc−/− mice (Fig. 1A, 1B) (23). IL-17A activated both NF-κB and C/EBP pathways (16) and also augmented the expression of C/EBPδ and IκB-ζ from MEFs (27) (Fig. 1C). Consistently, the increased expression induced by IL-17A was abolished in MEFs from Il-17rc–deficient mice. In WT MEFs, IL-17 treatment led to a degradation of IκB-α within 5 min, and resynthesis of IκB-α could be detected after 6 h (27) (Fig. 1D). However, the degradation of IκΒ-α protein was not detected in Il-17rc−/− MEFs (Fig. 1D). Taken together, these data support a critical role for IL-17RC in mediating the functions of IL-17A and IL-17F on murine fibroblasts.
IL-17A has been reported to inhibit IFN-γ production during Th1 differentiation (48). Naive T cells from both WT and Il-17rc−/− spleens were differentiated into Th1 cells with or without IL-17A treatment. IFN-γ was measured by ELISA under both primary and recall responses. We did not observe any differences in terms of IFN-γ protein production under these conditions in the presence of IL-17A (Supplemental Fig. 2). Furthermore, Th1 development in both WT and Il-17rc−/− mice were comparable (Supplemental Fig. 2). Our data suggest that IL-17A does not elicit robust responses from T cells. However, we cannot exclude the functioning of the IL-17 pathway on other leukocytes.
It has been reported that IL-17RA, in addition to IL-17RB, is required for IL-25/IL-17E–induced signaling (49). Because IL-17RA can form a complex with IL-17RC to transduce signals for IL-17 (A and F), we examined the role of IL-17RC in IL-25 signaling. Splenocytes were isolated from Il-17rb−/− (Supplemental Fig. 3) and Il-17rc−/− mice and treated with IL-25. Induction of IL-5 was measured and is shown in Fig. 1E. Although IL-5 induction was abolished in Il-17rb−/− splenocytes, it was maintained in Il-17rc–deficient splenocytes, indicating that IL-17RC is not required for IL-25 signaling.
IL-17RC is required for osteoclastogenesis induced by IL-17A
In addition to fibroblasts, other mesenchymal cells, such as osteoblasts, can respond to both IL-17A and IL-17F. In a coculture system consisting of BMCs and osteoblasts, IL-17A elicits the production of RANKL from osteoblasts (50). RANKL can further promote the differentiation of osteoclasts from monocytes/macrophages in the bone marrow (51). To examine the role of IL-17RC in mediating the functions of IL-17A and IL-17F in this system, osteoclast precursors derived from bone marrow were cocultured with osteoblasts isolated from WT or Il-17rc–deficient mice as previously described (51). IL-17A or IL-17F was added to the culture to promote osteoclast differentiation (Fig. 2). Multinucleated osteoclasts were readily detected by TRAP staining when either IL-17A or IL-17F was added to the culture (Fig. 2B, 2C, 2G). This effect was RANKL-dependent because addition of osteoprotegerin, a decoy receptor for RANKL, along with IL-17A (Fig. 2D) or IL-17F (data not shown), completely abolished this promotion of osteoclast differentiation. IL-17A– (Fig. 2F, 2G) and IL-17F–induced (data not shown) osteoclast differentiation was completely abolished when osteoblasts were derived from Il-17rc–deficient mice, supporting a critical role for IL-17RC in IL-17–induced osteoclast differentiation.
The essential role of IL-17RC in the function of IL-17 is preserved in human cells
Next, we asked whether IL-17RC is also essential for mediating the signaling of IL-17 in human fibroblasts. Primary HFFs were transfected with siRNA oligonucleotides that specifically targeted human Act1, IL-17RA, and IL-17RC, as well as nontargeting siRNA as negative control. The levels of transcript for these genes, when compared with that in control siRNA transfected cells, were reduced to 40, 16, and 13% respectively (Fig. 3A). The siRNA-mediated downregulation of IL-17RA and IL-17RC cell surface expression was confirmed by FACS analyses with Abs specifically recognizing each protein (Fig. 3B). Finally, IL-17A– (Fig. 3C, left panel) and IL-17F–induced (Fig. 3C, right panel) G-CSF production from HFFs was greatly reduced following siRNA knockdown of Act1, IL-17RA, or IL-17RC expression in comparison with that from control siRNA transfected cells, indicating that these molecules are indispensable for IL-17A– and IL-17F–induced functions in human cells (Fig. 3C). In conclusion, these data support that IL-17RC serves as a crucial functional receptor chain for the signaling of IL-17A and IL-17F.
SEFIR domain of IL-17RC is required for the signaling of IL-17A and IL-17F but not for the association of IL-17RC with IL-17RA upon ligand binding
The SEFIR domain is found in all IL-17R family molecules (25). To explore the role of the IL-17RC SEFIR domain, we reconstituted MEFs from Il-17rc−/− mice with FLAG-tagged full-length IL-17RC as well as with truncated IL-17RC lacking the SEFIR domain (IL-17RCΔ; Fig. 4A) and made them into stable cell lines. FACS analysis using anti-FLAG Ab showed that expression levels of exogenous FLAG-tagged IL-17RC and IL-17RCΔ were similar (Fig. 4A).
Using this system, we first examined whether IL-17RA and IL-17RC are preassembled on the cell surface prior to ligand binding. By using the FLAG-tagged IL-17RC overexpression system, we confirmed that the endogenous IL-17RA chain cannot be coimmunoprecipitated with IL-17RC in the absence of IL-17A (Fig. 4B) (18). Interestingly, when IL-17A was added, IL-17RA was readily detected in the protein complex with FLAG-tagged IL-17RC (Fig. 4B, left panel). These data support that IL-17A induces or enhances the association of IL-17RA and IL-17RC on the cell surface. Furthermore, this ligand-induced association of IL-17RA and IL-17RC is not dependent on the SEFIR domain of the IL-17RC chain, because deletion of the SEFIR domain in IL-17RC did not disrupt this association (Fig. 4B, right panel). The ratios of IL-17RA to IL-17RC-FLAG are similar in both cell lines (Fig. 4B and data not shown).
Next, we investigated whether the SEFIR domain of IL-17RC is required for downstream signaling. The two different MEF cell lines were treated with either IL-17A or IL-17F, and the induction of IL-6 was assessed by ELISA. As shown in Fig. 4C, although KO MEFs overexpressing full-length IL-17RC were able to restore IL-17 responsiveness, KO MEFs transfected with IL-17RCΔ failed to respond. This result demonstrates that the SEFIR domain is essential for IL-17RC–mediated functions. It is interesting to note that when IL-17RC is exogenously overexpressed IL-17F can induce IL-6 production to levels similar to those induced by IL-17A (Fig. 4C), whereas in WT MEFs, IL-17F is much less potent than IL-17A (Fig. 1A).
IL-17RC deficiency results in delayed disease onset as well as reduced disease severity in the MOG-EAE model of autoimmune CNS inflammation
To further examine the role of IL-17RC in vivo during the development of autoimmune diseases, we challenged Il-17rc−/− mice with MOG35–55 peptide emulsified in CFA to induce CNS inflammation. Il-17rc–deficient mice had a delay in the onset of neurologic disease as well as greatly reduced clinical disease severity when compared with those of WT mice. The average clinical scores were 0.4 ± 0.1 for Il-17rc−/− mice and 2.3 ± 0.5 for WT mice (p < 0.0007) (Fig. 5A). Histological analyses of spinal cord sections demonstrated that leukocytic infiltration (Fig. 5B–E, 5J) and demyelination (Fig. 5F–I, 5K) were greatly reduced in Il-17rc–deficient mice. These data support an important role for IL-17RC in the development of immune-mediated CNS inflammation and demyelination.
IL-17RC is not required for Th17 cell differentiation in the MOG-induced EAE model
IL-17A produced by Th17 cells has important pathogenic roles in murine EAE models (52, 53). To better understand the mechanisms by which Il-17rc deficiency protects mice from the development of EAE, the differentiation of Th17 cells in the periphery was examined. Splenocytes of Il-17rc−/− and WT mice at day 21 after MOG immunization (MOG35–55/CFA) were tested with recall analysis. T cell proliferation in response to MOG peptide was comparable in WT and Il-17rc−/− groups (Fig. 6A). In addition, T cells from WT and Il-17rc−/− mice produced similar levels of IL-17A and IFN-γ protein as measured by ELISA (Fig. 6B). Next, we analyzed IL-17A and IFN-γ production in inguinal lymph nodes (Fig. 6C, 6D, Supplemental Fig. 4) and splenocytes (Supplemental Fig. 5) via intracellular cytokine staining. CD4+ T cells numbers were comparable in the lymph nodes and spleen from Il-17rc−/− and WT groups (Fig. 6C, Supplemental Fig. 5A). The IL-17A–producing T cells in Il-17rc−/− were significantly increased in lymph nodes (Fig. 6D) and spleens (Supplemental Fig. 5B), whereas the fractions of IFN-γ–producing cells were comparable in WT and Il-17rc−/− groups (Fig. 6E, Supplemental Fig. 5C). These data suggest that IL-17RC is not required for Th1 or Th17 cell differentiation in vivo.
Reduced Th17 cell infiltration in the CNS correlates with decreased chemokine expression in the CNS of Il-17rc−/− mice
The normal Th17 responses in the periphery prompted us to further isolate and analyze mononuclear immune infiltrates from the brain and spinal cord. There was a significant decrease in the total number of immune cells infiltrating the brain and spinal cord in the KO group compared with that of the WT group (Fig. 7A). In addition, the fraction of IL-17A–producing cells was also decreased in the Il-17rc−/− group (Fig. 7B, left panel), resulting in a significant reduction of the total IL-17A–producing cell count in the Il-17rc−/− group (Fig. 7B, right panel). IL-17A induces chemokine production from various tissue epithelial cells and fibroblasts. Therefore, we examined CXCL1, CXCL2, CCL2, CCL5, and CCL20 expression in brains isolated from Il-17rc−/− and WT animals challenged with MOG peptide. The expression of CXCL1, CXCL2, CCL2, and CCL5, but not CCL20, was significantly reduced in Il-17rc−/− mice (Fig. 7C). Taken together, these data suggest that IL-17RC expressed in brain may amplify inflammation induced by the initial infiltration of Th17 cells. The reduced chemokine expression in the Il-17rc−/− mice may diminish further immune infiltration into the CNS and progression of EAE.
IL-17RA was first identified as a functional receptor for IL-17A. It has also been identified as an essential receptor chain for IL-17F, IL-17A/F, and IL-25/IL-17E (7, 49, 54). IL-17RC was later found to be the second functional receptor chain for IL-17A and IL-17F, based on its indispensable role in rescuing the signaling of IL-17A and IL-17F when it is coexpressed with IL-17RA in Il-17ra−/− MEF cells (18). Furthermore, in human synoviocytes, both IL-17RA and IL-17RC are required for IL-17A–induced chemokine expression (55). The necessity of IL-17RC for the functions of IL-17A and IL-17F is still under debate because of two observations. First, IL-17RA only binds to IL-17A with high affinity, whereas IL-17RC can bind to IL-17A, IL-17F, and IL-17A/F with similar affinities (21). Second, IL-17RA and IL-17RC have different expression patterns. IL-17RC is highly expressed in tissues, such as colon, whereas IL-17RA is expressed in a variety of leukocyte subsets (16). It has been suggested that, in addition to forming the IL-17RA/IL-17RC heterodimeric complex, IL-17RA and IL-17RC may also be able to independently deliver signals for IL-17A and IL-17F, respectively, especially in cells where only one receptor chain is expressed (20, 21). In this study, for the first time, we critically examined the requirement of IL-17RC in the signaling of IL-17 ligands by generating Il-17rc−/− mice. We found that IL-17RC is essential for mediating the functions of all IL-17A/F ligands, both in vitro and in vivo. Our data, in conjunction with data derived from Il-17ra−/− mice, support the hypothesis that both IL-17RA and IL-17RC chains are required to form the functional receptor for IL-17 ligands. This premise is also further strengthened by the structure of the IL-17F/IL-17RA complex recently solved by Ely et al. (22). The asymmetric IL-17RA/IL-17F complex indicates a preference in forming the heterodimeric receptor complex.
IL-17RA can form multimers on the cell surface without ligand binding (24). It is unclear, however, whether IL-17RA and IL-17RC can form a preassembled complex on the cell surface. Previous studies have suggested that this may be the case, based on the coimmunoprecipitation results from cellular overexpression of both IL-17RA and IL-17RC (18). In the same study, however, it was shown that overexpression of a single IL-17RA or IL-17RC chain on the cells could not coimmunoprecipitate the endogenous counterpart in the cells, suggesting that IL-17RC and IL-17RA do not preassociate on the cell surface at the physiological expression level. We confirmed this latter observation in our FLAG-tagged IL-17RC–overexpressing MEF cells. Without adding IL-17A, anti-FLAG Ab fails to precipitate endogenous IL-17RA, supporting that there is no or very weak interaction between IL-17RA and IL-17RC. Interestingly, addition of IL-17A is sufficient to trigger the tight association of IL-17RA and IL-17RC, as demonstrated by the detection of the IL-17RA chain in the co-IP complex (Fig. 4B). Finally, we have demonstrated that the ligand-induced formation of the receptor complex is not dependent on the intracellular domain of IL-17RC. Computational modeling has helped to identify two fibronectin III (FN)-like domains (FN1 and FN2) in IL-17RA connected by a nonstructured linker. FN2 mediates ligand-independent preassembly, and FN1 mediates conformational alteration upon ligand binding (56). Structural studies show that IL-17RA binds to IL-17F in a 1:2 stoichiometry (22). Engagement of IL-17RA or IL-17RC by IL-17A or IL-17F encourages a “preference” for the second receptor binding site to engage a different receptor and thereby to form a heterodimeric receptor complex (22). These data, together with our results, strongly suggest that IL-17A or IL-17F may bind to either IL-17RA or IL-17RC first and that this binding then induces a conformational change that enables the recruitment of second heterodimeric chain into this receptor complex and hence downstream signal transduction.
The intracellular SEFIR domain in IL-17RA is essential for mediating the stimulatory signals induced by IL-17A. The SEFIR domain recruits the key adaptor molecule, ACT1, to the receptor complex and triggers the downstream signal cascade. The SEFIR domain is a conserved feature in all IL-17R family members, including IL-17RC. IL-17RC with its SEFIR domain truncated still mediates ligand binding and the formation of the receptor complex. However, it fails to mediate the IL-17–induced production of IL-6, implying that the SEFIR domain in IL-17RC is not redundant. It is unclear whether the SEFIR domain binds to ACT1 as well or it recruits different signaling molecules instead.
Many studies have established the indispensable role of the IL-17 pathway in host defense and in the development of autoimmune diseases (14). Elevated IL-17A has been detected in a number of human autoimmune diseases, including multiple sclerosis (57). In EAE models, blocking the IL-17A pathway has been shown to ameliorate disease severity (43). In this study, we demonstrate for the first time that IL-17RC also directly participates in the pathogenesis of autoimmune CNS inflammation. Il-17rc–deficient mice have delayed disease onset and develop significantly milder clinical disease, resulting from substantial reduction of leukocytic infiltration/inflammation in the CNS and concomitant reduction in demyelination. Intriguingly, peripheral Th17 cells are greatly increased in Il-17rc–deficient mice. This observation, together with the fact that there are fewer Th17 cells in the CNS in Il-17rc–deficient mice, suggests that the peripheral development of Th17 cells in Il-17rc–deficient mice is normal but there are fewer Th17 cells entering the CNS, resulting in the accumulation of Th17 cells in the peripheral lymphoid tissues. It is unclear why blocking the IL-17 pathway in Il-17rc−/− mice results in reduced leukocyte infiltration into the CNS. A report by Reboldi et al. (58) suggests a two-stage model for the development of EAE in which CCR6-expressing autoreactive T cells enter the CNS through the choroid plexus, where epithelial cells constitutively express CCL20, the ligand of CCR6, and disseminate into the subarachnoid space. These T cells are locally activated by resident APCs that display self Ags, thus triggering the recruitment of a second wave of T cells that enter the inflamed brain in a CCR6-independent manner. Our results are consistent with this model. First, we observe unchanged CCL20 expression in both WT and Il-17rc−/− mice. Second, the expression of many other chemokines, including CXCL1, CXCL2, CCL2, and CCL5 which are induced by IL-17A (59–61), is greatly diminished in Il-17rc−/− mice. CCL2 and CCL5 mediate leukocyte adhesion in EAE (61). Mice lacking the receptor of CCL2, CCR2, are resistant to EAE development (62). Our data suggest that IL17RC present in the resident cells of the CNS may participate in the second wave of leukocyte infiltration through the induction of chemokines essential for the development of EAE. IL-17A has been shown to induce chemokine expression/production in astrocytes and microglia (59, 60). It is conceivable that IL-17 produced by the initial infiltrating T cells stimulates brain resident cells to make chemokines that help to recruit additional autoreactive T cells and other leukocytes, perpetuating the positive feedback loop and inflammatory process. Alternatively or in addition, the potential role of the IL-17 pathway in promoting blood–brain barrier disruption (63), synergizing with pertussis toxin in this model, may also account for fewer Th17 infiltrates in the CNS of Il-17rc−/− mice. In summary, our data not only confirm and extend the role of IL-17RC in mediating the functions of IL-17 ligands in vivo but also suggest that, in addition to IL-17A and IL-17RA, IL-17RC may also be a potential therapeutic target for the treatment of autoimmune diseases.
We thank J. Ding at Genentech for providing IL-17RC FLAG-tagged constructs, J. Eastham-Anderson for assistance with image quantification, and the Genentech Histology Laboratory for technical assistance.
Disclosures All authors are employees of Genentech.
The online version of this article contains supplemental material.
The sequence presented in this article has been submitted to GenBank protein database under accession number CAD23360.
Abbreviations used in this paper:
bone marrow cell
experimental autoimmune encephalomyelitis
human foreskin fibroblast
luxol fast blue
mouse embryonic fibroblast
myelin oligodendrocyte glycoprotein
similar expression to fibroblast growth factor genes and IL-17Rs
small interfering RNA
tartrate-resistant acid phosphatase