Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET.
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SUBMITTER: Lay CS
PROVIDER: S-EPMC8790524 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and purposeInterleukin-23 (IL-23) and its receptor are important drug targets for the treatment of auto-inflammatory diseases. IL-23 binds to a receptor complex composed of two single transmembrane spanning proteins IL23R and IL12Rβ1. In this study, we aimed to gain further understanding of how ligand binding induces signalling of IL-23 receptor complexes using the proximity-based techniques of NanoLuc Binary Technology (NanoBiT) and Bioluminescence Resonance Energy Transfer (BRET).Experimental approachTo monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerisation of receptor subunits can be measured through luminescence. The affinity of NanoBiT complemented complexes for IL-23 was measured using NanoBRET, and cytokine-induced signal transduction was measured using a phospho-STAT3 AlphaLISA assay.Key resultsNanoBiT measurements demonstrated that IL-23 receptor complexes formed to an equal degree in the presence and absence of ligand. NanoBRET measurements confirmed that these complexes bound IL-23 with a picomolar binding affinity. Measurement of STAT3 phosphorylation demonstrated that pre-formed IL-23 receptor complexes induced signalling following ligand binding. It was also demonstrated that synthetic ligand-independent signalling could be induced by high affinity (HiBit) but not low affinity (SmBit) NanoBiT crosslinking of the receptor N-terminal domains.Conclusions and implicationsThese results indicate that receptor complexes form prior to ligand binding and are not sufficient to induce signalling alone. Our findings indicate that IL-23 induces a conformational change in heteromeric receptor complexes, to enable signal transduction. These observations have direct implications for drug discovery efforts to target the IL-23 receptor.
| S-EPMC10953408 | biostudies-literature
Project description:Interleukin-23 (IL-23) is known to play a crucial role in the development and maintenance of T helper 17 cells. It has been previously demonstrated that IL-17 is involved in experimental Lyme arthritis, caused by Borrelia burgdorferi bacteria. However, the precise role of the IL-23 receptor (IL-23R) for the B. burgdorferi-induced IL-17 responses or human Lyme disease has not yet been elucidated. IL-23R single nucleotide polymorphism (SNP) rs11209026 was genotyped using the TaqMan assay. Functional studies were performed using peripheral blood mononuclear cells, and cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Dose-dependent production of IL-23 and IL-17 by B. burgdorferi could be observed. Interestingly, when IL-23 bioactivity was inhibited by a specific antibody against IL-23p19, IL-17 production was significantly downregulated. In contrast, production of gamma interferon (IFN-?) was not affected after the blockade of IL-23 activity. Moreover, individuals bearing a single nucleotide polymorphism in the IL-23R gene (Arg381Gln) produced significantly less IL-17 after B. burgdorferi stimulation compared with that of the individuals bearing the wild type. Despite lower IL-17 production, the IL-23R gene polymorphism did not influence the development of chronic Lyme disease in a cohort of patients with Lyme disease. This study demonstrates that IL-23R signaling is needed for B. burgdorferi-induced IL-17 production in vitro and that an IL-23R gene SNP leads to impaired IL-17 production. However, the IL-23R gene polymorphism is not crucial for the pathogenesis of chronic Lyme.
| S-EPMC3257938 | biostudies-literature
Project description:PurposeRecent studies identified an association between Behcet's disease (BD) and the IL-23R gene polymorphism (rs17375018) in different populations. This study examined whether this IL-23R gene polymorphism is associated with enhanced inflammatory responses.MethodsWe recruited 27 BD patients and 32 controls with three genotypes. Peripheral blood mononuclear cells (PBMCs) were seeded with or without anti-CD3 and CD28. Cells were incubated for 24 hours, and then supernatants were collected and stored at -20◦C until analyzed. Levels of interferon (IFN)-γ, tissue necrosis factor (TNF)-α, interleukin (IL)-17 and IL-6 were detected by ELISA. IL-23R expression was assessed by quantitative real-time polymerase chain reaction (RT-PCR).ResultsThe expression of IL-23R was significantly higher in both BD patients and healthy controls with the GG genotype compared to the AG and AA genotype with anti-CD3 and CD28 stimulation (all P-value < 0.05). Among the PBMCs cultured with anti-CD3 and CD28 stimulation, there was an elevated secretion of TNF-α, IL-6 and IL-17 in BD patients and healthy controls with the GG genotype. However, there was no significant change in secretion of IFN- γ in BD patients and healthy controls among the genotype of this IL-23R gene polymorphism.ConclusionsThe results suggest that the GG genotype of the rs17375018 variant in the IL-23R gene enhances pro-inflammatory cytokine responses.
| S-EPMC4519128 | biostudies-literature
Project description:Background/aim:IL-23R gene polymorphisms and the association of these polymorphisms with serum IL-23 levels were investigated in patients with psoriasis in the current study. Materials and methods:Sixty-seven patients with psoriasis who were admitted to our dermatology outpatient clinic and 67 healthy controls were included in the study. Polymorphisms of the IL-23R gene were determined by KASP-PCR method, and serum IL-23 levels were determined by ELISA method. Results:The distribution of IL-23R gene polymorphisms rs2201841, rs11209026, rs7530511, rs1343152, and rs11465804 was not significantly different in the patient and control groups. The AA genotype of the rs2201841 locus in males and the GA genotype in females, as well as the AA genotype of the rs1343152 locus in males and the CA genotype in females, were statistically significant in patients with psoriasis. The mean serum IL-23 level was significantly lower in the patient group (42.62 ± 5.96) compared to the control groups (75.76 ± 13.24). Conclusion:IL-23R gene polymorphisms including rs2201841, rs11209026, rs7530511, rs11465804, and rs1343152 were not found to be significantly related to psoriasis. Different genetic polymorphisms may play a role in the development of psoriasis in female and male populations. Ethnic differences between different populations may have led to differences in the distribution of polymorphisms in the current study with compared to other published studies. Additionally, many different genes, polymorphisms, and environmental factors that have an effect on the development of psoriasis may affect the disease process.
| S-EPMC7018327 | biostudies-literature
Project description:Acquired aplastic anemia (AA) is a rare disease with a complex pathogenesis. In most cases, T cell-mediated immune destruction of hematopoietic cells results in peripheral blood pancytopenia and bone marrow hypoplasia. A subset of the heterodimeric interleukin-23 receptor gene (IL-23R) is significantly associated with autoimmune-mediated diseases. To examine whether IL-23R single nucleotide polymorphisms (SNPs) might contribute to AA, we selected three IL-23R SNPs with amino acid changes (rs11209026: p.Arg381Gln; rs41313262: p.Val362Ile; and rs11465797: p.Thr175Asn) and compared their frequencies in 279 AA patients and 184 ethnically matched healthy controls. The three SNP prevalences were similar between the AA patients and controls. The Arg381Gln variant, which has a strong protective effect against inflammatory bowel disease, showed no association with AA. Furthermore, IL-23 levels in sera were measured in the AA patients and in controls, and there were no significant differences among them. Our results indicate that these three IL-23R SNPs and serum IL-23 level have no apparent impact on susceptibility to AA.
| S-EPMC3438141 | biostudies-literature
Project description:Interleukin (IL)-10 plays an important role in immune regulation in the intestine. Immune deregulation is suggested in the pathogenesis of inflammatory bowel disease (IBD). This study aims to elucidate the role of IL-23 in the suppression of IL-10 in the IBD intestinal mucosa. Surgically removed colon specimens were obtained from 16 IBD patients. The expressions of IL-10, IL-23, and IgA in the specimens were examined at the protein and gene transcriptional levels. The gene transcription of IL-10 was assessed by chromatin immunoprecipitation assay and promoter accessibility assay. The levels of IgA and IL-10 were significantly lower, whereas the levels of IL-23 were higher, in IBD specimens than in normal controls. The levels of IgA and IL-10 were negatively correlated with the infiltration of inflammatory cells in the IBD mucosa. The production of IL-10 by lamina propria mononuclear cells was lower in the IBD group than in the control group, and these levels could be enhanced by blocking IL-23. The gene transcription of IL-10 was significantly suppressed in CD4(+) T cells of IBD mucosa; this phenomenon could be replicated in vitro by adding IL-23 in the culture of polarized Th2 cells. Overexpression of IL-23 in the intestinal mucosa suppresses the production of IL-10, which weakens the defensive barrier by reducing the production of IgA in the gut.
| S-EPMC3271012 | biostudies-literature
Project description:ContextIL-23 and its receptor (IL-23R) guide T cells toward the T-helper 17 phenotype. IL-23R single nucleotide polymorphisms (SNPs) have been associated with several autoimmune diseases, including Crohn's disease and rheumatoid arthritis.ObjectiveOur objective was to determine whether variants in the IL-23R gene are associated with Graves' disease (GD) and Graves' ophthalmopathy (GO).Design and participantsA total of 216 North American Caucasian GD patients and 368 healthy controls were genotyped for four SNPs spanning the IL-23R gene. SNPs rs11209026 and rs7530511 were genotyped using the TaqMan allelic discrimination assays (Applied Biosystems, Foster City, CA), and SNPs rs2201841 and rs10889677 were genotyped using a fluorescent-based restriction fragment length polymorphism method.ResultsThe A allele of rs2201841 was present in 78.8% of GD patients with GO and 64.7% of controls [P=1.1x10(-4); odds ratio (OR)=2.04]; the AA genotype was also significantly increased in GO patients compared with controls (62.5 and 41%, respectively; P=1.0x10(-4); OR=2.4). The C allele of rs10889677 was present in 78.6% of GO patients and 64.5% of controls (P=1.3x10(-4); OR=2.03), and the CC genotype was also significantly increased in GO patients vs. controls (62.1 and 41.0%, respectively; P=1.4x10(-4); OR=2.36). The TT genotype of rs7530511 was significantly associated with GD, but not specifically with GO; it was present in 2.5% of GD patients and 0.3% of controls (P=0.02; OR=9.4). The rs11209026 SNP, which is the most strongly associated with Crohn's disease, was not associated with GD or GO in our data set.ConclusionsVariants in the IL-23R gene are strongly associated with GO. These variants may predispose to GO by changing the expression and/or function of IL-23R, thereby promoting a proinflammatory signaling cascade.
| S-EPMC2266952 | biostudies-literature
Project description:Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.
| S-EPMC4285857 | biostudies-literature
Project description:The balance of proinflammatory cytokines interleukin (IL)-12 and IL-23 plays a key role in shaping the development of antitumor or protumor immunity. In this review, we discuss the role IL-12 and IL-23 plays in tumor biology from preclinical and clinical data. In particular, we discuss the mechanism by which IL-23 promotes tumor growth and metastases and how the IL-12/IL-23 axis of inflammation can be targeted for cancer therapy.
| S-EPMC6028064 | biostudies-literature
Project description:Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) ?? T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing ?? T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.
| S-EPMC6044435 | biostudies-literature