Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F

The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.     

Sensitivity and resistance to regulation by IL-4 during Th17 maturation

Th17 cells are highly pathogenic in a variety of immune-mediated diseases, and a thorough understanding of the mechanisms of cytokine-mediated suppression of Th17 cells has great therapeutic potential. In this article, we characterize the regulation of both in vitro- and in vivo-derived Th17 cells by IL-4. We demonstrate that IL-4 suppresses reactivation of committed Th17 cells, even in the presence of TGF-β, IL-6, and IL-23. Downregulation of IL-17 by IL-4 is dependent on STAT6 and mediated by inhibition of STAT3 binding at the Il17a promoter. Although Th1 cytokines were shown to induce IFN-γ expression by Th17 cells, IL-4 does not induce a Th2 phenotype in Th17 cells. Suppression by IL-4 is stable and long-lived when applied to immature Th17 cells, but cells that have undergone multiple rounds of stimulation, either in vivo during a Th17-mediated inflammatory disease, or in vitro, become resistant to suppression by IL-4 and lose the ability to signal through IL-4R. Thus, although IL-4 is a potent suppressor of the Th17 genetic program at early stages after differentiation, prolonged stimulation renders Th17 cells impervious to regulatory cytokines.     

The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.     

Immunolocalization of IL-17A, IL-17B, and their receptors in chondrocytes during fracture healing.

Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in the epiphyseal growth plate.     

IL-17A and IL-17F repair HIV-1 gp140 damaged Caco-2 cell barriers by upregulating tight junction genes

It is widely accepted that impairment of the intestinal epithelial barrier from HIV/AIDS contributes significantly to microbial translocation and systemic immune activation. Such factors present potential targets for novel treatments aimed toward a functional cure. However, the extracellular mechanisms of intestinal barrier repair are poorly understood. In the current study, we investigated the abilities of IL-17A and IL-17F to repair the damaged barrier caused by HIV-1 gp140 using Caco-2 monolayers. It was found that HIV-1 gp140 downregulated the expression of tight junction-associated genes and disrupted the barrier integrity of Caco-2 monolayers. However, IL-17A and IL-17F treatment reversed the HIV-1 gp140-induced barrier dysfunction by upregulating the expression of tight junction-associated genes, the combination of which resulted in a stronger induction of barrier repair. Furthermore, the effects of IL-17A and IL-17F were reduced by downregulation of Act1 with siRNA and inhibition of NF-κB and MAPK pathways with BAY11-7082 and U0126, respectively. These data indicated that the NF-κB and MAPK pathways are involved in the repair of barrier integrity mediated by IL-17A and IL-17F, and IL-17 pathways are potential targets for gut barrier restoration therapies during HIV/AIDS.     

A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses

CD4＋ helper T （TH） cells play crucial roles in immune responses. Recently a novel subset of TH cells, termed THIL-17, TH 17 or inflammatory TH （THi）, has been identified as critical mediators of tissue inflammation. These cells produce IL-17 （also called IL-17A） and IL-17F, two most homologous cytokines sharing similar regulations. Here we report that when overexpressed in 293T cells, IL-17 and IL-17F form not only homodimers but also heterodimers, which we name as IL-17A/F. Fully differentiated mouse THi cells also naturally secrete IL-17A/F as well as IL-17 and IL-17F homodimeric cytokines. Recombinant IL-17A/F protein exhibits intermediate levels of potency in inducing IL-6 and KC （CXCL 1） as compared to homodimeric cytokines. IL-17A/F regulation of IL-6 and KC expression is dependent on IL-17RA and TRAF6. Thus, IL-17A/F cytokine represents another mechanism whereby T cells regulate inflammatory responses and may serve as a novel target for treating various immune-mediated diseases.     

IL-17A inhibits the expansion of IL-17A-producing T cells in mice through "short-loop" inhibition via IL-17 receptor

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4(-)CD8(-)alphabeta(low), CD4(+)CD8(-)alphabeta(+) (Th17), and gammadelta(+) T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra(-/-)) mice and Il17ra(-/-)Itgb2(-/-) mice that lack both IL-17R and all four beta(2) integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra(-/-) and Il17ra(-/-)Itgb2(-/-) mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.     

Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1(-/-) calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.     

重组人IL-17A和IL-17F的制备方法及活性测定

人IL-17A和IL-17F具有很高的同源性,在炎症性疾病、自身免疫性疾病和肿瘤中都发挥着重要的作用,是当前研究的热点.应用原核表达系统在大肠杆菌BL21(DE3)中高效表达了人IL-17A和IL-17F;经培养条件的优化,未发现可溶性目的蛋白的表达,免疫印记分析显示,重组蛋白位于包涵体中;对包涵体进行洗涤、凝胶过滤层析纯化和柱上复性,获得重折叠的可溶性蛋白;随后用SDS-PAGE对蛋白样品进行了纯度分析、采用免疫印记和质谱的方法鉴定蛋白产物成分、用ME3T3-E1和RAW264.7两个细胞株对IL-17A、IL-17F的生物学活性进行测定.结果显示,柱上复性的方法制备的谊重组蛋白具有较高的纯度和活性.建立的重组人IL-17A和IL-17F的制备方法可为相关研究中细胞因子的大量应用提供参考.     

Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. While it is well-accepted that inflammation is central to NAFLD pathogenesis, the immune pathway(s) orchestrating disease progression are poorly defined. Notably, IL-17RA signaling, via IL-17A, plays an important role in obesity-driven NAFLD pathogenesis. However, the role of the IL-17F, another IL-17RA ligand, in NAFLD pathogenesis has not been examined. Further, the cell types expressing IL-17RA and producing IL-17RA ligands in the pathogenesis of NAFLD have not been defined. Here, IL-17RA^-/-, IL-17A^-/-, IL-17F^-/- and wild-type (WT) mice were fed either standard chow diet or methionine and choline deficient diet (MCDD)—a diet known to induce steatosis and hepatic inflammation through beta-oxidation dysfunction—and hepatic inflammation and NAFLD progression were subsequently quantified. MCDD feeding augmented hepatic IL-17RA expression and significantly increased hepatic infiltration of macrophages and IL-17A and IL-17F producing CD4⁺ and CD8⁺ T cells in WT mice. In contrast, IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice, despite increased steatosis, exhibited significant protection from hepatocellular damage compared to WT controls. Protection from hepatocellular damage correlated with decreased levels of hepatic T-cell and macrophage infiltration and decreased expression of inflammatory mediators associated with NAFLD. In sum, our results indicate that the IL-17 axis also plays a role in a MCDD-induced model of NAFLD pathogenesis. Further, we show for the first time that IL-17F, and not only IL-17A, plays an important role in NAFLD driven inflammation.     

IL-17A suppresses the expression of bone resorption-related proteinases and osteoclast differentiation via IL-17RA or IL-17RC receptors in RAW264.7 cells

Interleukin-17 (IL-17) is produced exclusively by activated T cells and neutrophils, and stimulates osteoclastic bone resorption via osteoblasts by inducing the expression of “receptor activator of NF-κB (RANK) ligand” (RANKL). However, the direct effects of IL-17 on the differentiation of osteoclast precursors into osteoclasts and on the function of osteoclasts have not been clarified. Therefore, we examined the effects of IL-17A on the differentiation of osteoclast precursors using RAW264.7 cells and also on the expression of carbonic anhydrase II (CA II), cathepsin K, matrix metalloproteinases-9 (MMP-9), RANK, c-fms, and IL-17 receptors in these cells. The cells were cultured with or without 0.1, 1.0, 10 or 50 ng/mL IL-17 in the presence of soluble RANKL for up to 10 days. The CA II, cathepsin K, and MMP-9 mRNA and protein expression levels were examined using real-time PCR and Western blotting, respectively. The mRNA expression levels of RANK, c-fms, and IL-17 receptors were monitored by real-time PCR. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of the cells. TRAP-positive cells were observed after day 5 of culture, and the number of cells decreased in the presence of 10 and 50 ng/mL IL-17A at days 5 and 7. In the presence of IL-17A, the expressions of cathepsin K, MMP-9 and c-fms decreased markedly on days 5 and/or 7 of culture, whereas the expression of CA II and IL-17 receptor (type A) increased remarkably at days 3 and 7, respectively. The expression of RANK and IL-17 receptor (type C) was not affected by the addition of IL-17A. These results suggest that the differentiation of osteoclast precursors into osteoclasts is suppressed at high concentrations of IL-17A. Furthermore, IL-17A suppresses the hydrolysis of matrix proteins during bone resorption by decreasing the production of cathepsin K and MMP-9 in osteoclasts.     

Association analysis of IL-17A and IL-17F polymorphisms in Chinese Han women with breast cancer

Background

Research into the etiology of breast cancer has recently focused on the role of the immunity and inflammation. The proinflammatory cytokines IL-17A and IL-17F can mediate inflammation and cancer. To evaluate the influences of IL-17A and IL-17F gene polymorphisms on the risk of sporadic breast cancer, a case-control study was conducted in Chinese Han women.

Methodology and Principal Findings

We genotyped three single-nucleotide polymorphisms (SNPs) in IL-17A (rs2275913, rs3819025 and rs3748067) and five SNPs in IL-17F (rs7771511, rs9382084, rs12203582, rs1266828 and rs763780) to determine the haplotypes in 491 women with breast cancer and 502 healthy individuals. The genotypes were determined using the SNaPshot technique. The differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed with the Chi-square test for trends. For rs2275913 in IL-17A, the frequency of the AA genotype was higher in patients than controls (P = 0.0016). The clinical features analysis demonstrated significant associations between IL-17 SNPs and tumor protein 53 (P53), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her-2) and triple-negative (ER-/PR-/Her-2-) status. In addition, the haplotype analysis indicated that the frequency of the haplotype A_rs2275913G_rs3819025G_rs3748067, located in the IL-17A linkage disequilibrium (LD) block, was higher in patients than in controls (P = 0.0471 after correction for multiple testing).

Conclusions and Significance

Our results suggested that SNPs in IL-17A but not IL-17F were associated with the risk of breast cancer. Both IL-17A and IL-17F gene polymorphisms may provide valuable information for predicting the prognosis of breast cancer in Chinese women.     

High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism

Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-alpha failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.     

Carbonic anhydrase activity in peripheral T-lymphocytes and appearance of the activity during their maturation in the thymus

Summary Carbonic anhydrase (CA) activity is demonstrated in lymphoid tissue for the first time using the histochemical (Hansson's) method. A CA-positive reaction was seen in lymphocytes present in T-lymphocyte areas in both the lymph node and the spleen. The most intense staining was seen in the small T-lymphocytes, whereas the mediumsized T-lymphocytes were less markedly stained. The cortical lymphocytes in the thymus were completely devoid of staining, but the small medullary T-lymphocytes stained intensely. The results suggest that peripheral and thymic medullary T-lymphocytes contain CA activity, which appears in these cells during their maturation in the thymus.     

Interleukin-17F signaling requires ubiquitination of interleukin-17 receptor via TRAF6

Interleukin-17F (IL-17F), together with interleukin-17A (IL-17 or IL-17A), is a marker of T(H)17 cells, a new lineage of effector CD4(+) T cells to contribute to pathogenesis of a growing list of autoimmune and inflammatory diseases, such as experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA). IL-17F, similar to IL-17A, was reported to employ interleukin-17 receptor (IL-17R or IL-17RA) for signaling but the downstream cascades remain largely elusive. Here we report that TRAF6 interacts with IL-17R and mediates ubiquitination of the receptor. We observed that IL-17F and IL-17A could induce IL-17R ubiquitination and DN-TRAF6, a dominant-negative mutant, could block IL-17F- but not IL-17A-triggered ubiquitination of IL-17R. Moreover, we showed that the ubiquitination of IL-17R was positively correlated with the downstream signaling, as evaluated by a luciferase reporter driven by a putative native promoter of 24p3, an IL-17 targeted gene. Our results indicate that ubiquitination of IL-17R mediated by TRAF6 plays a critical role in IL-17F signaling. This study, for the first time, reveals a possible molecular mechanism that the initiation of the IL-17F/IL-17R signaling pathway requires the receptor ubiquitination by TRAF6.     

Molecular mechanisms of cytokine and chemokine release from eosinophils activated by IL-17A, IL-17F, and IL-23: implication for Th17 lymphocytes-mediated allergic inflammation

IL-17A and IL-17F are members of the IL-17 family that play crucial roles in allergic inflammation. Recent studies reported that IL-17A and IL-17F production from a distinct Th lymphocyte subset, Th17, was specifically induced by IL-23, which was produced by dendritic cells and macrophages in response to microbial stimuli. The IL-23-IL-17 axis might therefore provide a link between infections and allergic diseases. In the present study, we investigated the effects of IL-17A, IL-17F, and IL-23, alone or in combination, on cytokine and chemokine release from eosinophils and the underlying intracellular mechanisms. Human eosinophils were found to constitutively express receptors for IL-17A, IL-17F, and IL-23 at the protein level. IL-17A, IL-17F, and IL-23 could induce the release of chemokines GRO-alpha/CXCL1, IL-8/CXCL8, and MIP-1beta/CCL4 from eosinophils, while IL-17F and IL-23 could also increase the production of proinflammatory cytokines IL-1beta and IL-6. Synergistic effects were observed in the combined treatment of IL-17F and IL-23 on the release of proinflammatory cytokines, and the effects were dose-dependently enhanced by IL-23, but not IL-17F. Further investigations showed that IL-17A, IL-17F, and IL-23 differentially activated the ERK, p38 MAPK, and NF-kappaB pathways. Moreover, inhibition of these pathways using selective inhibitors could significantly abolish the chemokine release induced by IL-17A, IL-17F, and IL-23 and the synergistic increases on IL-1beta and IL-6 production mediated by combined treatment of IL-17F and IL-23. Taken together, our findings provide insight for the Th17 lymphocyte-mediated activation of eosinophils via differential intracellular signaling cascades in allergic inflammation.     

CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages

Classically activated macrophages (CAMphi) have been described as a major effector cell on the host's innate immunities. However, CAMphi are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMphi) predominate. In this study, the mechanism by which AAMphi suppress the ability of resident macrophages (RMphi) to generate CAMphi was investigated. AAMphi were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMphi were generated from RMphi (peritoneal Mphi from normal mice) through stimulation with CpG DNA, a typical CAMphi inducer. RMphi did not polarize to CAMphi when they were cultured with AAMphi in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMphi stimulated with CpG DNA did not convert to CAMphi when they were cultured with the culture fluids of AAMphi (AAMphi Culture-Sup). AAMphi Culture-Sup contained IL-6, IL-10, CCL17, PGE(2), and TGF-beta. Among these, CCL17 and IL-10 inhibited CAMphi generation. The ability of AAMphi Culture-Sup to inhibit CAMphi generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMphi inhibit CAMphi generation from RMphi stimulated with CpG DNA.