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IL-17 activates NF-κB and induces expression of proinflammatory genes. IL-17 drives disease in autoimmune conditions, and anti–IL-17 Abs have shown impressive success in the clinic. Although produced by lymphocytes, IL-17 predominantly signals in fibroblasts and epithelial cells. IL-17–driven inflammation is kept in check by negative feedback signaling molecules, including the ubiquitin editing enzyme A20, whose gene TNFAIP3 is linked to autoimmune disease susceptibility. The A20 binding inhibitor of NF-κB activation 1 (ABIN-1) is an A20-binding protein encoded by the TNIP1 gene, which is also linked to autoimmune disease susceptibility including psoriasis. Accordingly, we hypothesized that ABIN-1 might play a role in negatively regulating IL-17 signaling activity. Indeed, ABIN-1 enhanced both tonic and IL-17–dependent NF-κB signaling in IL-17–responsive fibroblast cells. Interestingly, the inhibitory activities of ABIN-1 on IL-17 signaling were independent of A20. ABIN-1 is a known NF-κB target gene, and we found that IL-17–induced activation of NF-κB led to enhanced ABIN-1 mRNA expression and promoter activity. Surprisingly, however, the ABIN-1 protein was inducibly degraded following IL-17 signaling in a proteasome-dependent manner. Thus, ABIN-1, acting independently of A20, restricts both baseline and IL-17–induced inflammatory gene expression. We conclude that IL-17–induced signals lead to degradation of ABIN-1, thereby releasing a constitutive cellular brake on NF-κB activation.

Interleukin (IL)-17 is the founding member of a novel family of inflammatory cytokines. While the proinflammatory properties of IL-17 are key to its host-protective capacity, unrestrained IL-17 signaling is associated with immunopathology, autoimmune disease, and cancer progression. In this review we discuss both the activators and the inhibitors of IL-17 signal transduction, and also the physiological implications of these events. We highlight the surprisingly diverse means by which these regulators control expression of IL-17-dependent inflammatory genes, as well as the major target cells that respond to IL-17 signaling.

The IL-17 family cytokines IL-17A and IL-17C drive the pathogenesis of psoriatic skin inflammation, and anti–IL-17A Abs were recently approved to treat human psoriasis. Little is known about mechanisms that restrain IL-17 cytokine-mediated signaling, particularly IL-17C. In this article, we show that the endoribonuclease MCP-1–induced protein 1 (MCPIP1; also known as regnase-1) is markedly upregulated in human psoriatic skin lesions. Similarly, MCPIP1 was overexpressed in the imiquimod (IMQ)-driven mouse model of cutaneous inflammation. Mice with an MCPIP1 deficiency (Zc3h12a+/−) displayed no baseline skin inflammation, but they showed exacerbated pathology following IMQ treatment. Pathology in Zc3h12a+/− mice was associated with elevated expression of IL-17A– and IL-17C–dependent genes, as well as with increased accumulation of neutrophils in skin. However, IL-17A and IL-17C expression was unaltered, suggesting that the increased inflammation in Zc3h12a+/− mice was due to enhanced downstream IL-17R signaling. Radiation chimeras demonstrated that MCPIP1 in nonhematopoietic cells is responsible for controlling skin pathology. Moreover, Zc3h12a+/−Il17ra−/− mice given IMQ showed almost no disease. To identify which IL-17RA ligand was essential, Zc3h12a+/−Il17a−/− and Zc3h12a+/−Il17c−/− mice were given IMQ; these mice had reduced but not fully abrogated pathology, indicating that MCPIP1 inhibits IL-17A and IL-17C signaling. Confirming this hypothesis, Zc3h12a−/− keratinocytes showed increased responsiveness to IL-17A and IL-17C stimulation. Thus, MCPIP1 is a potent negative regulator of psoriatic skin inflammation through IL-17A and IL-17C. Moreover, to our knowledge, MCPIP1 is the first described negative regulator of IL-17C signaling.

The cytokine IL-17, and signaling via its heterodimeric IL-17RA/IL-17RC receptor, is critical for host defense against extracellular bacterial and fungal pathogens. Polarized lung epithelial cells express IL-17RA and IL-17RC basolaterally. However, their contribution to IL-17-dependent pulmonary defenses in vivo remains to be determined. To address this, we generated mice with conditional deletion of Il17ra or Il17rc in Scgb1a1-expressing club cells, a major component of the murine bronchiolar epithelium. These mice displayed an impaired ability to recruit neutrophils into the airway lumen in response to IL-17, a defect in bacterial clearance upon mucosal challenge with the pulmonary pathogen Klebsiella pneumoniae, and substantially reduced epithelial expression of the chemokine Cxcl5. Neutrophil recruitment and bacterial clearance were restored by intranasal administration of recombinant CXCL5. Our data show that IL-17R signaling in the lung epithelium plays a critical role in establishing chemokine gradients that are essential for mucosal immunity against pulmonary bacterial pathogens.

Summary Signaling through the IL-17 receptor (IL-17R) is required to prevent oropharyngeal candidiasis (OPC) in mice and humans. However, the IL-17-responsive cell type(s) that mediate protection are unknown. Using radiation chimeras, we were able to rule out a requirement for IL-17RA in the hematopoietic compartment. We saw remarkable concordance of IL-17-controlled gene expression in C. albicans -infected human oral epithelial cells (OECs) and in tongue tissue from mice with OPC. To interrogate the role of the IL-17R in OECs, we generated mice with conditional deletion of IL-17RA in superficial oral and esophageal epithelial cells ( Il17ra ΔK13 ). Following oral Candida infection, Il17ra ΔK13 mice exhibited fungal loads and weight loss indistinguishable from Il17ra −/− mice. Susceptibility in Il17ra ΔK13 mice correlated with expression of the antimicrobial peptide β-defensin 3 (BD3, Defb3 ). Consistently, Defb3 −/− mice were susceptible to OPC. Thus, OECs dominantly control IL-17R-dependent responses to OPC through regulation of BD3 expression.

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency.

Interleukin-17A (IL-17A) not only stimulates immunity to fungal pathogens but also contributes to autoimmune pathology. IL-17 is only a modest activator of transcription in experimental tissue culture settings. However, IL-17 controls posttranscriptional events that enhance the expression of target mRNAs. Here, we showed that the RNA binding protein (RBP) Arid5a (AT-rich interactive domain-containing protein 5a) integrated multiple IL-17-driven signaling pathways through posttranscriptional control of mRNA. IL-17 induced expression of Arid5a, which was recruited to the adaptor TRAF2. Arid5a stabilized IL-17-induced cytokine transcripts by binding to their 3' untranslated regions and also counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Arid5a inducibly associated with the eukaryotic translation initiation complex and facilitated the translation of the transcription factors (TFs) IκBζ (Nfkbiz ) and C/EBPβ (Cebpb). These TFs in turn transactivated IL-17-dependent promoters. Together, these data indicated that Arid5a orchestrates a feed-forward amplification loop, which promoted IL-17 signaling by controlling mRNA stability and translation.

Background & Aims GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. Methods Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. Results GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors – although not APOBEC3A or the Smc5/6 complex – and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. Conclusions Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. Lay summary GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Following the discovery of T helper 17 (TH17) cells, the past decade has witnessed a major revision of the TH subset paradigm and substantial progress has been made in deciphering the molecular mechanisms of T cell lineage commitment and function. In this Review, we focus on the recent advances that have been made regarding the transcriptional control of TH17 cell plasticity and stability, as well as the effector functions of TH17 cells, and we highlight the mechanisms of IL-17 signalling in mesenchymal and barrier epithelial tissues. We also discuss the emerging clinical data showing that IL-17-specific and IL-23-specific antibody treatments are remarkably effective for treating many immune-mediated inflammatory diseases.

Conti et al. show that IL-17 is produced by tongue-resident populations of γδ T cells and nTh17 cells in response to oropharyngeal candidiasis in mice., Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα−/−, and Rag1−/− mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1–2 d by tongue-resident populations of γδ T cells and CD3+CD4+CD44hiTCRβ+CCR6+ natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β−/− and TCR-δ−/− mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1−/−, IL-7Rα−/−, and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens.

Candida is the third most common cause of bloodstream infections in hospitalized patients. Immunity to C. albicans, the most frequent species to be isolated in candidiasis, involves a well-characterized Dectin-1/caspase-associated recruitment domain adaptor 9 (CARD9)/IL-17 signaling axis. Infections caused by non-albicans Candida species are on the rise, but surprisingly little is known about immunity to these pathogens. In this study, we evaluated a systemic infection model of C. tropicalis, a clinically relevant, but poorly understood, non-albicans Candida. Mice lacking CARD9 were profoundly susceptible to C. tropicalis, displaying elevated fungal burdens in visceral organs and increased mortality compared with wild-type (WT) controls. Unlike C. albicans, IL-17 responses were induced normally in CARD9(-/-) mice following C. tropicalis infection. Moreover, there was no difference in susceptibility to C. tropicalis infection between WT and IL-23p19(-/-), IL-17RA(-/-), or Act1(-/-) mice. However, TNF-α expression was markedly impaired in CARD9(-/-) mice. Consistently, WT mice depleted of TNF-α were more susceptible to C. tropicalis, and CARD9-deficient neutrophils and monocytes failed to produce TNF-α following stimulation with C. tropicalis Ags. Both neutrophils and monocytes were necessary for defense against C. tropicalis, because their depletion in WT mice enhanced susceptibility to C. tropicalis. Disease in CARD9(-/-) mice was not due to defective neutrophil or monocyte recruitment to infected kidneys. However, TNF-α treatment of neutrophils in vitro enhanced their ability to kill C. tropicalis. Thus, protection against systemic C. tropicalis infection requires CARD9 and TNF-α, but not IL-17, signaling. Moreover, CARD9-dependent production of TNF-α enhances the candidacidal capacity of neutrophils, limiting fungal disease during disseminated C. tropicalis infection.

SUMMARY Inerleukin-23 (IL-23) is required for inflammatory Th17 cell function in experimental autoimmune encephalomyelitis (EAE), and IL-23 blockade reduces the number of effector Th17 cells in the CNS. We report that pro-inflammatory Th17 cells express high integrin β3 that is IL-23 dependent. Integrin β3 was not upregulated on all activated T cells; rather, integrin β3 was upregulated along with its functional partner integrin αv on effector Th17 cells and “ex-Th17” cells, and αvβ3hi RORγt+ cells expanded during EAE. Integrin αvβ3 inhibitors ameliorated clinical signs of EAE, and integrin β3 deficiency on CD4+ T cells alone was sufficient to block EAE induction. Furthermore, integrin-β3-deficient Th17 cells, but not Th1 cells, were impaired in their ability to induce EAE. Integrin β3−/− T cells induced smaller demyelinated lesions and showed reduced spread and accumulation within the CNS, corresponding with impaired extracellular-matrix-mediated migration. Hence, integrin β3 is required for Th17 cell-mediated autoimmune CNS inflammation., Graphical abstract

Candida albicans is a commensal fungal microbe of the human orogastrointestinal tract and skin. C. albicans causes multiple forms of disease in immunocompromised patients, including oral, vaginal, dermal and disseminated candidiasis. The cytokine IL-17 (IL-17A) and its receptor subunits, IL-17RA and IL-17RC, are required for protection to most forms of candidiasis. The importance of the IL-17R pathway has been observed not only in knockout mouse models, but also in humans with rare genetic mutations that impact generation of Th17 cells or the IL-17 signaling pathway, including Hyper-IgE Syndrome (STAT3 or TYK2 mutations) or IL17RA or ACT1 gene deficiency. The IL-17 family of cytokines is a distinct subclass of cytokines with unique structural and signaling properties. IL-17A is the best-characterized member of the IL-17 family to date, but far less is known about other IL-17-related cytokines. In this study, we sought to determine the role of a related IL-17 cytokine, IL-17C, in protection against oral, dermal and disseminated forms of C. albicans infection. IL-17C signals through a heterodimeric receptor composed of the IL-17RA and IL-17RE subunits. We observed that IL-17C mRNA was induced following oral C. albicans infection. However, mice lacking IL-17C or IL-17RE cleared C. albicans infections in the oral mucosa, skin and bloodstream at rates similar to WT littermate controls. Moreover, these mice demonstrated similar gene transcription profiles and recovery kinetics as WT animals. These findings indicate that IL-17C and IL-17RE are dispensable for immunity to the forms of candidiasis evaluated, and illustrate a surprisingly limited specificity of the IL-17 family of cytokines with respect to systemic, oral and cutaneous Candida infections.

“For every action, there is an equal and opposite reaction.” This axiom is true not only for physical processes, but also for biological activation events, where inhibitory mechanisms are in place to reestablish equilibrium. Nowhere is this more evident than during acute inflammation, where cytokines or pathogen-associated stimuli ramp up a rapid and powerful inflammatory cascade, followed by an equally important phase of resolution. Keeping inflammation in check avoids collateral tissue damage, ultimately preventing autoimmune and hyperproliferative disorders. A20 is a vital inhibitor of inflammation encoded by the gene TNFα-induced protein 3 (TNFAIP3). The TNFAIP3 locus is linked to autoimmune diseases and B-cell lymphomas.1 A20 is a deubiquitinating enzyme that reverses Lysine (K)63-linked ubiquitination. A20 was initially described as a negative feedback inhibitor of TNFα-induced signaling, where it acts principally by targeting RIP1, and thereby terminating activation of the NFκB transcription factor. A20 also inhibits the TLR, IL-1R, and Nod-like receptor pathways, where it targets other signaling intermediates such as TRAF6, NEMO, and Ubc13 (Fig. 1).2 Mathematical modeling of the TNFα pathway led to the insight that A20 restricts activation of the second, not the first phase of classical NFκB activation. Moreover, due to its long protein half-life, steady-state levels of A20 dictate the magnitude of inflammatory signaling. Notably, induction of A20 by any stimulus is predicted to exert a regulatory impact on the global response; thus, A20 is considered a “rheostat” that maintains equilibrium following NFκB activation.3 Figure 1. Schematic diagram depicting A20-mediated regulation of inflammatory pathways. The model represents the two phases of feedback inhibition of inflammation with A20 (tnfaip3) as a central player. In the first phase, several stimuli such as TNFα, ... Confirming the importance of A20 in restricting inflammation, A20−/− mice die prematurely of multi-organ inflammation and cachexia. Surprisingly, despite the prominent role of A20 in tempering TNFα signaling, inflammation in A20−/− mice was not alleviated by crossing to mice lacking the TNF receptor. Subsequent in vivo studies revealed that the baseline inflammation in A20-deficient mice is due to signals from commensal microbiota via TLR/MyD88 signaling. A number of subsequent studies examined cell type-specific effects of A20 using conditionally targeted mice, demonstrating important activities for this protein in numerous immune subtypes.1 In recent years IL-17 has come to the limelight due the recognition of Th17 cells and their connection to autoimmune disease. Clinically, antibodies against IL-17 or its receptor are showing promise in treating a variety of other conditions.4 The IL-17R belongs to a distinct subfamily of receptors, with many unique structural and functional properties. Nonetheless, the IL-17 signaling pathway shares many downstream adaptors and transcription factors with the TNFα, IL-1, and TLR pathways.5 Given these commonalities, we predicted that A20 might also serve as an inhibitor of IL-17 signaling. Indeed, IL-17 induced expression of the TNFAIP3 gene in an NFκB-dependent manner. A20 deficiency upon siRNA silencing or in knockout cells led to enhanced IL-17-dependent expression of inflammatory genes. Mechanistically, A20 binds to and dequbitinates TRAF6, ultimately inactivating IL-17-mediated NF-κB and MAPK (particularly JNK) pathways.6 Although the mechanism used by A20 to inhibit IL-17 signaling has parallels with the IL-1R pathway, there are striking differences. In other pathways, A20 interacts with accessory proteins such as Tax1bp1 and Itch to efficiently hydrolyze K63-linked substrates. Deletion of these proteins results in a phenotype similar to the lack of A20, though less severe.2 Unexpectedly, Tax1bp1 and Itch were dispensable for A20s activity in IL-17 signaling, as siRNA-mediated silencing of Tax1bp1 or Itch did not affect IL-17-dependent gene expression.7 This finding raised the possibility that A20 either functions solo or uses alternate adaptor molecules. Accordingly, we found that A20 associates with AnapC5 (APC5), a subunit of a multi-protein E3 ubiquitin ligase complex known as the Anaphase Promoting Complex (APC). APC subunits have not been previously linked with the regulation of inflammatory signaling, suggesting a novel role for AnapC5 outside of regulating the cell cycle (Fig. 1). In analyzing how A20 is recruited to the IL-17R signaling complex, we found that A20 associates directly with the IL-17RA subunit.6 Notably, A20 binds to a C-terminal domain of IL-17RA that was previously shown to downmodulate IL-17 signaling.8 To our knowledge, this is the first demonstration that A20 associates with an inducing receptor. A20 interaction with receptors may also occur in other systems, potentially serving as a mechanism to recruit this inhibitor after a signaling event is triggered. These discoveries about A20 function in the context of IL-17 suggest there are distinct mechanisms by which A20 restricts inflammatory pathways. It will be key to define the contribution of A20 in regulating IL-17 in vivo. As noted, SNPs in the TNFAIP3 gene locus are associated with autoimmune diseases that are linked to IL-17 activity. Therefore, it would not be surprising if patients with TNFAIP3 polymorphisms are more likely to be responsive to anti-IL-17 therapy, as has been demonstrated for TNF inhibitors. To date, therapeutic strategies to block IL-17 have mainly focused on biologic drugs that target Th17 cells (anti-IL-6 or anti-IL-12/23) or directly neutralize this cytokine (anti-IL-17, anti-IL-17RA). Development of small-molecule inhibitors will be aided by a better understanding of how IL-17 pathways are regulated in nature.

The proinflammatory cytokine interleukin-17 (IL-17) is the signature cytokine of the T helper 17 (TH17) subset of CD4(+) T cells, and antibodies targeting IL-17 or the IL-17 receptor (IL-17R) show clinical efficacy in several autoimmune diseases. Although important for protective immunity against microorganisms, IL-17 causes collateral damage in inflammatory settings. TNFAIP3 encodes the deubiquitinase A20 and is genetically linked to numerous autoimmune syndromes. A20, a potent inhibitor of tumor necrosis factor-α signaling, removes ubiquitin from signaling intermediates upstream of nuclear factor κB (NF-κB), thereby dampening NF-κB-mediated inflammation. We demonstrated that IL-17 stimulates TNFAIP3 expression. Enhanced IL-17-mediated induction of genes encoding proinflammatory factors, including IL-6 and various chemokines, occurred upon knockdown of A20 with short inhibitory RNA or in A20(-/-) cells. A20 associated with the E3 ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6) in an IL-17-dependent manner and restricted the IL-17-dependent activation of NF-κB and mitogen-activated protein kinases. A20 interacted directly with the distal domain of IL-17RA, a previously defined inhibitory domain. Together, these data describe a mechanism of restraining IL-17 signaling and reveal an aspect of A20 activity that may help to explain its role in autoimmunity in humans.

The incidence of life-threatening disseminated Candida albicans infections is increasing in hospitalized patients, with fatalities as high as 60%. Death from disseminated candidiasis in a significant percentage of cases is due to fungal invasion of the kidney, leading to renal failure. Treatment of candidiasis is hampered by drug toxicity, the emergence of antifungal drug resistance and lack of vaccines against fungal pathogens. IL-17 is a key mediator of defense against candidiasis. The underlying mechanisms of IL-17-mediated renal immunity have so far been assumed to occur solely through the regulation of antimicrobial mechanisms, particularly activation of neutrophils. Here, we identify an unexpected role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in C. albicans-infected kidney, and we show that the KKS provides significant renal protection in candidiasis. Microarray data indicated that Klk1 was upregulated in infected kidney in an IL-17-dependent manner. Overexpression of Klk1 or treatment with bradykinin rescued IL-17RA-/- mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following C. albicans infection. Furthermore, combining a minimally effective dose of fluconazole with bradykinin markedly improved survival compared to either drug alone. These results indicate that IL-17 not only limits fungal growth in the kidney, but also prevents renal tissue damage and preserves kidney function during disseminated candidiasis through the KKS. Since drugs targeting the KKS are approved clinically, these findings offer potential avenues for the treatment of this fatal nosocomial infection., Author Summary Candida albicans is the causative agent of oropharyngeal candidiasis (OPC, thrush), dermal and vaginal candidiasis. However, the most severe C. albicans-induced disease is disseminated candidiasis, a frequent nosocomial infection associated with a high mortality rate. During disseminated candidiasis, C. albicans form invasive hyphae that damage target organs, particularly kidney and liver. Previous studies have identified an essential role of interleukin-17 (IL-17) in controlling systemic infection through regulation of neutrophils. We show here for the first time that IL-17 also regulates the renal protective Kallikrein-kinin system (KKS). Our discovery of a connection between IL-17 and the KKS suggests a new, previously unanticipated avenue for the treatment of renal damage in disseminated candidiasis. These findings have potential translational significance, as agonists of the KKS are in routine clinical use. Therefore, these results not only identify downstream mediators that could serve as novel drug targets, but could possibly be used to guide decisions on whether targeting these mediators could be a useful therapeutic option in conjunction with current antifungal therapies.

Disseminated candidiasis is a life threatening nosocomial infection with 40–60% mortality. Fatality from candidiasis is due to fungal invasion of the kidney leading to renal failure. IL-17 is a key cytokine important in the defense against candidiasis, but mechanisms of IL-17-mediated renal immunity remain unexplored. We studied the mechanism of renal protection offered by IL-17 in an intravenous infection model with Candida albicans . We identified a previously unknown role for IL-17 in inducing the renal-protective kallikrein 1(Klk1), a member of a family of serine proteases that constitute the Kallikrein-Kinin system (KKS). IL-17 driven Klk1 is expressed in the kidneys of C. albicans infected kidney. Overexpression of Klk1 and treatment with a bradykinin receptor agonist rescued IL-17RA−/− mice from disseminated candidiasis. Therapeutic manipulation of IL17-KKS pathways using bradykinin prevented apoptosis of renal cells. Moreover, this treatment prevented kidney damage and prolonged survival following C. albicans infection. These data demonstrate that IL-17 driven kallikrein expression contributes to the renal protection against disseminated candidiasis. Thus, exploiting IL-17-KKS pathways with approved drugs may offer avenues for treatment of this fatal infection.

The Interleukin-17A (IL-17A) is related to the development of autoimmune diseases as well as to the control of Candida albicans infections. IL-17 triggers signaling by binding to the IL-17 receptor (IL-17R). The IL-17R recruits adaptor proteins activating downstream effector pathways such as NF-κB and MAPK to induce inflammatory gene expression. To identify IL-17 signaling intermediates we screened candidate genes by siRNA knockdown discovering several novel inhibitors. Based on evidence from human genetic studies and the known importance of A20 as an inhibitor of IL-17R signaling we focused on ABIN1, an A20-interacting protein, which has not previously implicated in this pathway. Since many inhibitors work in a feedback manner to block the pathways that induce their expression, we asked whether IL-17R signaling regulates expression of ABIN1. Indeed, mRNA expression of ABIN1 was induced by IL-17A signaling. Upon ABIN1 knockdown, the IL-17-dependent gene Lipocalin2 (Lcn2) showed increased expression, confirming that ABIN1 is an inhibitor of the IL-17R signaling. Consistently, reconstitution of ABIN1-deficient fibroblast with ABIN1 suppressed Lcn2 expression, and IL-17-induced activation of Lcn2 promoter was downregulated when ABIN1 was ectopically expressed in ST2 cells. However, not all IL-17-dependent target genes were regulated by ABIN1. We are currently dissecting the mechanisms by which ABIN1 inhibits IL-17R signaling. Together, these findings suggest an unexpected complexity in the regulation of IL-17-mediated inflammation.

IL-17A (IL-17) is a proinflammatory cytokine that mediates host defense against extracellular pathogens but also contributes to the pathogenesis of various autoimmune diseases. Despite advances in our understanding of IL-17 signal transduction mechanisms, it is far less clear how IL-17R activity is kept in check to prevent harmful bystander inflammation. In an effort to identify novel, physiologically relevant regulators of IL-17 signal transduction, we evaluated microarray data of IL-17RA-dependent genes that mediate antifungal immunity. We thus identified the gene Zc3h12a (encoding MCPIP1/Regnase-1) as a possible feedback restriction element. MCPIP1 is an endoribonuclease (RNase) that inhibits TLR signaling in part by degrading cytokine mRNA transcripts such as Il6 . Since IL-17 and TLR pathways share multiple target genes including Il6 , we investigated the role of MCPIP1 in IL-17 signal transduction. Here, we demonstrate that MCPIP1 is upregulated by IL-17 and serves as a potent feedback inhibitor of IL-17 signal transduction. IL-17 signaling inhibition required MCPIP1’s RNase activity, but not its deubiquitinase activity. In vivo , resistance to disseminated Candida albicans infection was enhanced in MCPIP1 haploinsufficient mice; resistance was reversed in an IL-17R-deficient background. Conversely, IL-17-dependent pathology in MCPIP1+/− mice was dramatically exacerbated during EAE and pulmonary inflammation. In vitro , MCPIP1 strongly inhibited the Lcn2 promoter activation by regulating the mRNA stability of the I κ B ζ transcription factor. Unexpectedly, MCPIP1 also degraded Il17ra and Il17rc mRNA, independently of the 3’ UTR. The cumulative impact of MCPIP1 on IL-6, I κ B ζ and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.

IL-17 is a proinflammatory cytokine that promotes autoimmunity and protects against certain pathogens. Computational modeling of signaling pathways is a powerful tool to help understand the complexities of these dynamic processes. Here, we present a detailed computational model of signaling downstream of the Interleukin-17 receptor (IL-17R). To manage the complexity of the system we use a rule-based modeling approach, in which signaling proteins are modeled as structured objects and rules describe their biochemical interactions. The model includes 19 molecules and 46 reaction rules, which expand to a reaction network with 168 distinct chemical species – complexes and phosphoforms of the basic molecules – which are connected through a network of 5145 unidirectional reactions. The model is defined and simulated using the BioNetGen software. Dynamical behavior of the network is simulated using ordinary differential equations. This model encompasses major signaling components downstream of IL-17R, including the adaptor molecule Act1 and signaling intermediates TRAF6, TAB, TAK1, and IKK, which are activated through site-specific ubiquitination and phosphorylation. These components work in concert to activate NF- κ B, which in turn promotes the production of pro-inflammatory cytokines and chemokines that are critical for controlling infections by extracellular pathogens. NF- κ B also promotes production of A20, a deubiquitinating enzyme that inhibits NF- κ B activation in multiple settings. We recently showed that A20 acts as a negative regulator of the IL-17 signaling pathway, and here by modeling A20 interactions with TRAF6 and IKK, we are able to recapitulate experimentally-observed oscillations in NF- κ B–dependent A20 expression at both the mRNA and protein levels. Further expansion of the model will enable us to develop and explore novel hypotheses about the role of A20 and other potential regulators of IL-17-mediated signaling.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

Introduction IL-17A (IL-17) is a proinflammatory cytokine that mediates host defense against extracellular pathogens but also contributes to the pathogenesis of various autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis (MS) and psoriasis. Despite some homology with the toll-like receptors, IL-17 receptor subunits (IL-17RA and IL-17RC) do not recruit adaptors such as MyD88, but instead associate with an adaptor protein, Act1. Upon recruitment, Act1 activates Traf6, leading to a subsequent activation of a variety of transcription factors such as NF- κ B, C/EBP β and MAPKs. Despite advances in identifying molecular events regulating IL-17 signal transduction, the details of the inhibitory mechanisms of IL-17 signaling are still not well understood. Given the implication that unregulated IL-17 signaling results in enhanced autoimmune disease susceptibility, it is extremely important to understand the inhibitory mechanisms of the pathway. Recent studies have identified that post-translational modifications such as phosphorylation and ubiquitination are central to the regulation of IL-17 signal transduction. Ubiquitination is a stepwise process of linking ubiquitin chains on substrate proteins through any of the seven lysine residues on ubiquitin. Of the three enzymes, E1, E2 and E3 ubiquitin ligases, E3 ligases provide substrate specificity to the process. Interestingly, both Act1 and Traf6 have E3 ubiquitin ligase activity and lysine-63 linked ubiquitination of Traf6 is key in activating IL-17 pathway. Therefore, targeting ubiquitination as a mechanism for downregulating these pathways is a plausible inhibitory strategy. Genome analysis has identified over 100 deubiquitinases (DUBs) in humans but their role in IL-17 signaling is unknown. Mutations in the enzyme A20 (Tnfaip3) are associated with several autoimmune diseases including RA and psoriasis. Moreover, A20 inhibits a variety of proinflammatory signaling pathways such as TNF-alpha and IL-1, making it a compelling target. Methods Function of A20 in IL-17 signaling was tested by either siRNA-mediated knockdown of A20 or overexpressing A20 and measuring subsequent effect on IL-17 target gene expression by ELISA and quantitative PCR. Immunoprecipitation of A20 with different IL-17RA truncations was performed to map the association domain on IL-17RA. Results We demonstrate a functional role of A20 in the inhibition of IL-17 signaling. We observed an IL-17-dependent increase in A20 expression, and transient knockdown of A20 resulted in increased IL-17-dependent proinflammatory gene expression. Consistently, overexpression of A20 inhibited IL-17 target gene expression, which was associated with reduced NF- κ B activity. Interestingly, A20 associates with IL-17RA at its distal C-terminal domain, a motif that has been previously linked to negative regulation of the IL-17 signaling pathway. Conclusion We show for the first time that A20 can inhibit IL-17 signaling, which provides an intriguing mechanistic approach for inhibiting this pathway and could be a potential target for therapeutic intervention in IL-17-mediated autoimmune diseases.

SummaryInterleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1’s endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra−/− background. Conversely, IL-17-dependent pathology in Zc3h12a+/− mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3′ UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.