IL-17 Receptor Signaling in the Lung Epithelium Is Required for Mucosal Chemokine Gradients and Pulmonary Host Defense against K. pneumoniae.
ABSTRACT: 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.
Project description:IL-17 mediates essential inflammatory responses in host defense and autoimmunity. The IL-17A-IL-17F signaling complex is composed of IL-17RA and IL-17RC, both of which are necessary for signal transduction. To date, the specific contribution of IL-17RC to downstream signaling remains poorly understood. To define the regions within the IL-17RC cytoplasmic tail required for signal transduction, we assayed signaling by a panel of IL-17RC deletion mutants. These findings reveal that IL-17RC inducibly associates with a specific glycosylated IL-17RA isoform, in a manner independent of the IL-17RC cytoplasmic tail. Using expression of the IL-17 target genes IL-6 and 24p3/lipocalin-2 as a readout, functional reconstitution of signaling in IL-17RC(-/-) fibroblasts required the SEF/IL-17R signaling domain (SEFIR), a conserved motif common to IL-17R family members. Unexpectedly, the IL-17RC SEFIR alone was not sufficient to reconstitute IL-17-dependent signaling. Rather, an additional sequence downstream of the SEFIR was also necessary. We further found that IL-17RC interacts directly with the adaptor/E3 ubiquitin ligase Act1, and that the functional IL-17RC isoforms containing the extended SEFIR region interact specifically with a phosphorylated isoform of Act1. Finally, we show that IL-17RC is required for in vivo IL-17-dependent responses during oral mucosal infections caused by the human commensal fungus Candida albicans. These results indicate that IL-17RC is vital for IL-17-dependent signaling both in vitro and in vivo. Insight into the mechanisms by which IL-17RC signals helps shed light on IL-17-dependent inflammatory responses and may ultimately provide an avenue for therapeutic intervention in IL-17-mediated diseases.
Project description: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 TNF? 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.
Project description:The interleukin (IL)-17 cytokine family members IL-17A and IL-17F mediate inflammatory activities via the IL-17 receptor (IL-17R) complex, comprised of the IL-17RA and IL-17RC subunits. Proper regulation of the IL-17 signaling axis results in effective host defense against extracellular pathogens, while aberrant signaling can drive autoimmune pathology. Elucidating the molecular mechanisms underlying IL-17 signal transduction can yield an enhanced understanding of inflammatory immune processes and also create an avenue for therapeutic intervention in the treatment of IL-17-dependent diseases. To date, the fundamental signaling mechanisms used by the IL-17R complex are still incompletely defined. While current structure-function studies have primarily focused on the IL-17RA subunit, recent research indicates that the IL-17RC subunit plays a key role in modulating IL-17 responses. This review will examine what is known regarding IL-17RC function and provide a framework for future work on this subunit and its impact on human health.
Project description: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.
Project description:<h4>Introduction</h4>Tumor-associated macrophages (TAMs) are divided into M1 and M2 macrophages. M1 macrophages inhibit tumor growth, whereas M2 macrophages promote tumor growth and metastasis. The aim of this study was to examine the possible causes leading to the formation of an M2-macrophage-dominant tumor microenvironment in non-small-cell lung cancer.<h4>Methods</h4>Forty-eight archived lung tumor samples were examined for the expression of interleukin-17 (IL-17) receptors, IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC), and the number of TAMs using immunohistochemical staining. Twenty fresh lung tumors and matched normal lung tissues were examined for expression of IL-17, cyclooxygenase-2, and prostaglandin E2 (PGE2), using enzyme-linked immunosorbent assay and Western blot analysis. Macrophage-migration assays were performed using fresh lung tumor tissues and IL-17 as chemoattractants. Induction of M2-macrophage differentiation was analyzed using real-time quantitative polymerase chain reaction.<h4>Results</h4>TAMs expressed IL-17RA and IL-17RC. Lung tumors expressed higher levels of IL-17, cyclooxygenase-2, and PGE2, compared with normal lung tissues. Lung tumor tissues attracted migration of mouse RAW264.7 macrophages and primary peritoneal macrophages through IL-17, which was mediated by IL-17RA and IL-17RC. IL-17 did not induce either M1- or M2-macrophage differentiation. However, human lung cancer A549 cells strongly induced M2-macrophage differentiation of RAW264.7 macrophages when the two cell lines were cocultured. The inductive factor secreted by A549 cells was identified to be PGE2.<h4>Conclusions</h4>IL-17 recruits macrophages, and PGE2 induces M2-macrophage differentiation, hence the increased levels of IL-17 and PGE2 in lung cancer contribute to the formation of an M2-macrophage-dominant tumor microenvironment.
Project description: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.
Project description:The specific contribution of interleukin-17/interleukin-17 receptor (IL-17/IL-17R)-mediated responses in regulating host susceptibility against obligatory intracellular Chlamydia infection was investigated in C57BL/6 and C3H/HeN mice during Chlamydia muridarum respiratory infection. We demonstrated that Chlamydia stimulated IL-17/IL-17R-associated responses in both Chlamydia-resistant C57BL/6 and Chlamydia-susceptible C3H/HeN mice. However, C3H/HeN mice developed a significantly greater IL-17/IL-17R-associated response than C57BL/6 mice did. This was reflected by an increase in IL-17 mRNA expression, a higher recall IL-17 production from splenocytes upon antigen restimulation, and higher production of Th17-related cytokines (IL-23 and IL-6) and chemokines (chemokine [C-X-C motif] ligand 2 [CXCL1]/keratinocyte-derived chemokine [KC] and CXCL2/macrophage inflammatory protein 1 [MIP2]) in C3H/HeN mice than in C57BL/6 mice. Furthermore, C3H/HeN mice displayed a massive accumulation of activated and preactivated neutrophils in the airway and lung parenchyma compared to their C57BL/6 counterparts. We further demonstrated that the skewed IL-17/Th17 profile in C3H/HeN mice was predisposed by a higher basal level of IL-17 receptor C (IL-17RC) expression and then further amplified by a higher inducible IL-17RA expression in lungs. Most importantly, in vivo delivery of IL-17RA antagonist that resulted in a 50% reduction in the neutrophilic infiltration in lungs was able to reverse the susceptible phenotype of C3H/HeN mice to respiratory Chlamydia infection. Thus, our data for the first time have demonstrated a critical role for the IL-17/IL-17R axis in regulating host susceptibility to Chlamydia infection in mice.
Project description:Acute ethanol intoxication suppresses the host immune responses against Streptococcus pneumoniae. As interleukin 17 (IL-17) is a critical cytokine in host defense against extracellular pathogens, including S. pneumoniae, we hypothesized that ethanol impairs mucosal immunity against this pathogen by disrupting IL-17 production or IL-17 receptor (IL-17R) signaling. A chronic ethanol feeding model in simian immunodeficiency virus (SIV)-infected rhesus macaques and acute ethanol intoxication in a murine model were used. Transcriptome analysis of bronchial brushes in the nonhuman primate model showed downregulation of the expression of IL-17-regulated chemokines in ethanol-fed animals, a finding also replicated in the murine model. Surprisingly, recombinant CXCL1 and CXCL5 but not IL-17 or IL-23 plus IL-1? rescued bacterial burden in the ethanol group to control levels. Taken together, the results of this study suggest that ethanol impairs IL-17-mediated chemokine production in the lung. Thus, exogenous luminal restoration of IL-17-related chemokines, CXCL1 and CXCL5, improves host defenses against S. pneumoniae.
Project description:Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including interleukin (IL)-17-driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17-mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL-1 suppression, we employed unbiased microarray analysis to identify FSTL-1-regulated genes and pathways that could influence IL-17-dependent production of IL-6 and granulocyte colony-stimulating factor. We discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. This work identifies a mechanism by which FSTL-1 influences IL-17-driven inflammatory signaling in vitro and reveals a novel function for FSTL-1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL-1 and IL-17-mediated inflammation may provide insight into potential therapeutic targets of IL-17-mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL-1-influenced diseases.
Project description:IL-17A and IL-17F are prominent members of the IL-17 family of cytokines that regulates both innate and adaptive immunity. IL-17A has been implicated in chronic inflammatory and autoimmune diseases, and anti-IL-17A antibodies have shown remarkable clinical efficacy in psoriasis and psoriatic arthritis patients. IL-17A and IL-17F are homodimeric cytokines that can also form the IL-17A/F heterodimer whose precise role in health and disease remains elusive. All three cytokines signal through the assembly of a ternary complex with the IL-17RA and IL-17RC receptors. Here we report the X-ray analysis of the human IL-17A/F heterodimer that reveals a two-faced cytokine closely mimicking IL-17A as well as IL-17F. We also present the crystal structure of its complex with the IL-17RA receptor. Unexpectedly in view of the much higher affinity of this receptor toward IL-17A, we find that IL-17RA is bound to the "F-face" of the heterodimer in the crystal. Using site-directed mutagenesis, we then demonstrate that IL-17RA can also bind to the "A-face" of IL-17A/F with similar affinity. Further, we show that IL-17RC does not discriminate between the two faces of the cytokine heterodimer either, thus enabling the formation of two topologically-distinct heterotrimeric complexes with potentially different signaling properties.