IL-17A Receptor Research Articles

IL-17A exacerbates diabetic retinopathy by impairing Müller cell function via Act1 signaling.

Diabetic retinopathy (DR), one of the most serious complications of diabetes, has been associated with inflammatory processes. We have recently reported that interleukin (IL)-17A, a proinflammatory cytokine, is increased in the plasma of diabetic patients. Further investigation is required to clarify the role of IL-17A in DR. Ins2Akita (Akita) diabetic mice and high-glucose (HG)-treated primary Müller cells were used to mimic DR-like pathology. Diabetes induced retinal expression of IL-17A and IL-17 receptor A (IL-17RA) in Müller cells in contrast to ganglion cells. Further evidence demonstrated that retinal Müller cells cultured in vitro increased IL-17A and IL-17RA expression as well as IL-17A secretion in the HG condition. In both the HG-treated Müller cells and Akita mouse retina, the Act1/TRAF6/IKK/NF-κB signaling pathway was activated. IL-17A further enhanced inflammatory signaling activation, whereas Act1 knockdown or IKK inhibition blocked the downstream signaling activation by IL-17A. HG- and diabetes-induced Müller cell activation and dysfunction, as determined by increased glial fibrillary acidic protein, vascular endothelial growth factor and glutamate levels and decreased glutamine synthetase and excitatory amino acid transporter-1 expression, were exacerbated by IL-17A; however, they were alleviated by Act1 knockdown or IKK inhibition. In addition, IL-17A intravitreal injection aggravated diabetes-induced retinal vascular leukostasis, vascular leakage and ganglion cell apoptosis, whereas Act1 silencing or anti-IL-17A monoclonal antibody ameliorated the retinal vascular damage and neuronal cell apoptosis. These findings establish that IL-17A exacerbates DR-like pathology by the promotion of Müller cell functional impairment via Act1 signaling.

Mechanism of Corilagin interference with IL-13/STAT6 signaling pathways in hepatic alternative activation macrophages in schistosomiasis-induced liver fibrosis in mouse model

This study tried to find the mechanism of Corilagin interference with interleukin (IL)-13/signal transducer and activator of transcription (STAT) 6 signaling pathways in IL-13-activated liver alternative activation macrophages in schistosomiasis-induced liver fibrosis in Balb/c mice. As a result, IL-13 in serum and the mRNA expression of IL-13 Receptor α1, IL-4 Receptor α and downstream mediators supressor of cytokine signaling (SOCS) 1, Kruppel-like factor (KLF) 4, peroxisome proliferator-activated receptor (PPAR) δ in the liver tissue were significantly inhibited by Corilagin (P<0.05 or 0.01). The protein expression of IL-13 Receptor α1, IL-4 Receptor α, SOCS1, KLF4, PPARγ, PPARδ and Phospho-STAT6 (P-STAT6) in Corilagin group were also markedly suppressed when compared with the model group (P<0.05 or 0.01). Furthermore, the inhibitory effect was enhanced when the concentration of Corilagin increased (P<0.05). By hematoxylin and eosin (HE) staining, when compared with the model group, the Corilagin group showed smaller granulomas (P<0.05 or 0.01). The area of positive cells and integrated optical density (IOD) of CD68, CD206 and KLF4 was significantly decreased by Corilagin stained by IHC (P<0.05 or 0.01). In conclusion, Corilagin had potential to relieve hepatic fibrosis caused by egg granuloma in Schistosoma japonicum infection by decreasing the expression of molecules associated with IL-13/STAT6 signaling pathway in liver alternative activation macrophages.

Everolimus restrains the IL-17A-dependent osteoclast-like transdifferentiation of dendritic cells in multiple myeloma.

Interleukin-17A (IL-17A) promotes the osteoclast (OC)-like differentiation of dendritic cells (DCs) in multiple myeloma (MM) and contributes to the pathogenesis of myeloma bone disease (MBD). In our study, everolimus (EVR) abrogated the invitro OC-like activity of DCs from 12MM patients significantly. Exploring the EVR effects, we found that the inhibition of the osteoerosive activity of OC-DCs was mostly due to the blockade of signals driven by the IL-17A receptor toward the CCAAT/enhancer-binding protein beta/musculoaponeurotic fibrosarcoma oncogene homolog B axis Therefore, MM patients with MBD would probablybenefit from mammalian target of rapamycin inhibition.

Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.

Oral administration of a novel RORγt antagonist attenuates psoriasis-like skin lesion of two independent mouse models through neutralization of IL-17

BackgroundTargeting the IL-17 pathway represents a highly effective strategy for the treatment of psoriasis, using antibodies against IL-17A and IL-17 receptor, suggesting that Th17 cells essentially contribute to development of psoriasis. Th17 differentiation depends on the key transcription factor, RORγt. ObjectiveTo develop a novel RORγt antagonist which is effective on psoriasis via oral administration. MethodsA chemical library was screened using cell-based high-throughput methods, luciferase reporter assay, competitive binding assay, and T cell differentiation assay. To evaluate in vivo effects of a novel RORγt antagonist, A213, we orally administrated it to two independent mouse models of psoriasis; IL-23-injection model and K5.Stat3C transgenic mouse. ResultsOral administration of A213 resulted in attenuation of skin inflammation in the both mouse models. At the same time, increased levels of IL-17A expression were significantly reduced in the skin lesions and skin-draining lymph nodes. ConclusionThese results implicate a new therapeutic application of RORγt antagonist for the treatment of psoriasis.

Plasma cytokine profiling to predict susceptibility to acute mountain sickness.

Extensive studies have been performed on acute mountain sickness (AMS), but biomarkers predicting AMS are lacking. Presently, the mainstay methods to identify AMS biomarkers include proteomic and genetic methods at high altitudes or in hypoxic simulated chambers. In the present study, we compared plasma cytokine profiles between AMS-susceptible individuals and AMS-resistant individuals at low altitude by cytokine array analysis. In total, 75 differentially expressed cytokines were identified between AMS-susceptible individuals and AMS-resistant individuals, most involved in inflammation. A quantifiable human custom cytokine antibody array was then used to further test results of cytokine array analysis. Compared to AMS-resistant individuals, the level of insulin-like growth factor binding protein 6 (IGFBP-6) was significantly lower in AMS-susceptible individuals (37,318.99 ± 23,493.11 pg/mL and 25,665.38 ± 25,691.29 pg/mL, respectively; P = 0.04). Conversely, the levels of serum amyloid A1 (SAA1), dickkopf WNT signaling pathway inhibitor 4 (Dkk4), and interleukin 17 receptor A (IL-17RA) were significantly higher in AMS-susceptible individuals than in AMS-resistant individuals (SAA1: 4,069.69 ± 2,502.93 pg/mL vs. 2,994.98 ± 2,295.91 pg/mL, P = 0.05; Dkk4: 2,090.00 ± 2,094.89 pg/mL vs. 1,049.88 ± 1,690.93 pg/mL, P = 0.07; IL-17RA: 11.52 ± 8.33 pg/mL vs. 8.67 ± 6.22 pg/mL, P = 0.08). Although further in-depth research is required to examine the possible role of these cytokines in the development of AMS, these four cytokines may be of use in predicting AMS-susceptibility in a low-altitude environment.

IL-13 receptor α2 stimulates human glioma cell growth and metastasis through the Src/PI3K/Akt/mTOR signaling pathway.

Glioma is a malignant tumor that affects all kinds of people all over the world. It demonstrates remarkable infiltrative and invasive features. The high expression of interleukin-13 receptor subunit alpha-2 (IL-13Rα2) reportedly plays a pivotal role in some cancers. However, whether IL-13Rα2 contributes to glioma remains unknown. This study demonstrates that IL-13Rα2 is significantly up-regulated in human glioma tissue samples. It is also associated with late stages of disease progression and diminished survival in glioma patients. Gain- and loss-of-function studies demonstrate that IL-13Rα2 promotes the growth, migration, and invasion of glioma cells. In addition, mechanistic investigations show that IL-13Rα2 activates Scr, phosphatidylinositol 3 kinase (PI3K), Akt, and mTOR. Also, restraining Scr in glioma cells attenuates the activation of Scr/PI3K/Akt/mTOR pathway by IL-13Rα2, whereas the silencing of Scr markedly rescues the pro-invasive effect of IL-13Rα2. In conclusion, our results suggest that the high expression of IL-13Rα2 is significantly associated with the growth and metastasis of human glioma cells via the Scr/PI3K/Akt/mTOR pathway, while IL-13Rα2 may be a potential therapeutic target for glioma treatment.

Efficacy and safety of monoclonal antibodies targeting interleukin-17 pathway for inflammatory arthritis: a meta-analysis of randomized controlled clinical trials

T-helper 17 (Th17) pathway plays an important and distinct role in autoimmunity and inflammation. A growing body of evidence demonstrates that interleukin-17 (IL-17) is also synthesized in inflammatory arthritis tissues and exerts potent proinflammatory and joint-destructive activities. Clinical studies have been performed to evaluate the therapeutic efficacy of antibodies blocking the IL-17 signaling pathway in patients with rheumatoid arthritis (RA). In this study, we performed a meta-analysis to systematically evaluate the clinical effects of IL-17 antibodies in RA patients. By searching PubMed, five randomized, placebo-controlled randomized controlled clinical trials that tested three antibodies against IL-17A (LY2439821 and secukinumab/AIN457) and the IL-17A receptor (brodalumab) were identified. The primary outcomes that were analyzed include American College of Rheumatology (ACR) Improvement Criteria and Disease Activity Score in 28 joints (DAS28). Meanwhile, the safety and adverse effects were also systematically analyzed. The results of the meta-analysis demonstrated that IL-17 antibody is effective in ameliorating the RA symptoms. Specifically, IL-17-blocking antibody significantly reduced ACR20 and ACR50. It also dramatically reduced DAS28, an index that measures tenderness and swelling severity of joints. The side effects of and intolerance to the antibody treatment were higher than those in the placebo control. The analysis result provides evidence-based information for clinical use of these agents in the treatment of inflammatory arthritis.

A study on the establishment of co-culture system of peripheral blood mononuclear cells with orbital fibroblasts and the secretion of IL-6 and IL-17A induced by phytagglutinin in thyroid associated ophthalmopathy

Background The pathogenic mechanism of thyroid associated ophthalmopathy (TAO) is still unclear, which is considered to be an autoimmune disease.It is confirmed that interleukin-17A (IL-17A) plays an important role in the occurrence and development of many autoimmune diseases.It is unclear that whether IL-17A participates in the pathogenesis of TAO. Objective This study was to explore whether IL-17A secreted by co-culture system of peripheral blood mononuclear cells (PBMCs) and orbital fibroblasts (OFs) participates in the pathogenesis of TAO and its possible mechanism. Methods Periphery blood and orbital connective tissue were obtained from 12 patients with TAO and 8 patients who received prosthesis implantation for eyeball atrophy in Xiangya Hospital during April to December 2014.PBMCs were isolated by density gradient centrifugation, and OFs were cultured by explant culture method.The purity of T leukomonocyte in PBMCs was tested by flow cytometry, and OFs were identified by Giemsa staining and immunochemistry.OFs and PMBCs were incubated into 96-well plate in a 1∶20 proportion to establish co-culture system.Different concentrations of phytagglutinin (PHA) (0, 1.0, 2.5, 5.0, 10.0 μg/ml) was added for 72 hours, and IL-6, IL-17A levels in the co-culture system supernatant and IL-17A receptor (IL-17RA) of the total cell membranes in the co-culture system were assayed by ELISA.The differences of IL-6, IL-17A, IL-17RA levels in co-culture system were compared between the TAO group and control group. Results The mean purity of T leukomonocyte in PBMCs was (81.10±0.21)% in the TAO group and (80.05±0.38)% in the control group respectively, with no significant difference between them(t=0.923, P>0.05). Cultured OFs showed the positive response for Vimentin expression and Giemsa staining.After stimulated by 1.0 μg/ml PHA, the proliferation of both PBMCs and OFs were increased in the co-culture system.Apoptosis exist in PBMCs and the number of OFs decreased when PHA was higher than 1.0 μg/ml.The growth of PBMCs and OFs was faster in the TAO group than that in the control group in the same concentration of PHA.The contents of IL-6, IL-17A and IL-17RA in co-culture system were significantly different among various concentrations of PHA subgroups (IL-6: Fgroup=12.561, P=0.000; Fconcentration=23.356, P=0.001.IL-17A: Fgroup= 12.037, P=0.000; Fconcentration =19.206, P=0.000.IL-17RA: Fgroup=16.216, P=0.000; Fconcentration=4.627, P=0.018). The production of IL-6, IL-17A and IL-17RA reached peak in both TAO group and the control group after 1.0 μg/ml PHA stimulated.However, the concentrations of IL-6, IL-17A and IL-17RA reduced with the increase of PHA concentration.The concentrations of IL-6, IL-17A and IL-17RA in co-culture system were significantly higher in the TAO group than those in the control group under the stimulation of the same concentration of PHA (all at P<0.05). Conclusions The co-culture system of PBMCs and OFs stimulated with PHA can be the imitation of TAO pathogenesis in vitro, and PHA can amplify its immune reaction to imitate TAO pathogenic processes intuitively.The IL-6, IL-17A and IL-17RA secreted by PBMCs and induced by PHA are increased in TAO patients, implying that IL-17A participates in the pathogenesis of TAO through magnifying cellular immune response and inflammatory reaction. Key words: Cell culture; Thyroid-associated ophthalmopathy; Peripheral blood mononuclear cells; Orbital fibroblasts; T lymphocytes; Interleukin-6; Interleukin-17A; Human

Interleukin (IL)-17A Promotes Angiogenesis in an Experimental Corneal Neovascularization Model

ABSTRACTPurpose: To explore the roles of interleukin (IL)-17A in alkali-induced corneal neovascularization (CRNV).Methods: Induction of CRNV by alkali injury was performed and compared in recombinant mouse IL-17A protein or anti-mouse IL-17A neutralizing antibody (Ab)–treated mice. The expression of IL-17A, IL-17A receptor (IL-17RA), vascular endothelial growth factor (VEGF), IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) in burned corneas or other cells was examined by RT-PCR and western blot. The infiltration of macrophages, neutrophils, and c-Kit-positive progenitor cells into burned corneas was detected by flow cytometry (FCM). The Raw264.7 cell line and mouse corneal fibroblasts were stimulated by mouse recombinant IL-17A or anti-mouse IL-17A neutralizing Ab in vitro, and the expression of IL-6 and VEGF in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).Results: The alkali-induced CRNV peaked 2 weeks after the alkali burn. Compared with the vehicle-treated group, mouse recombinant IL-17A administration significantly increased the amount of CRNV, and it decreased in mice treated with neutralizing anti-mouse IL-17A Ab. RT-PCR and western blot confirmed that the IL-17A upregulated intracorneal VEGF, IL-6, IL-8, MCP-1, and phosphorylated focal adhesion kinase (pFAK) expression. FCM revealed that the infiltration of intracorneal progenitor cells, M1 macrophages, and neutrophils was also augmented in IL-17A-treated mice. The ELISA showed that IL-17A markedly induced VEGF and IL-6 expression in the Raw264.7 murine macrophage cell line and in the mouse corneal fibroblasts.Conclusions: IL-17A-treated mice exhibited enhanced alkali-induced CRNV through enhanced intracorneal progenitor cell and inflammatory cell infiltration, and increased VEGF and IL-6 expression by fibroblasts and macrophages.

Common variants in IL-17A/IL-17RA axis contribute to predisposition to and progression of congestive heart failure.

Heart failure is characterized by immune activation leading to production and release of proinflammatory cytokines. Interleukin 17A (IL-17A) is a proinflammatory cytokine and multiple lines of evidence from animal and human studies suggest crucial roles of IL-17A in heart failure. Therefore, we investigated whether common polymorphisms of genes IL17A and IL17RA (coding interleukin 17 receptor A) contribute to genetic predisposition to heart failure and adverse clinical outcomes associated with it.A total of 1713 adult patients with congestive heart failure and 1713 age- and sex-matched controls were genotyped for promoter single nucleotide polymorphisms (SNPs), rs2275913 and rs8193037 in IL17A and rs4819554 in IL17RA, to assess the relationship between individual SNPs and the risk of congestive heart failure. Results showed that rs8193037 in IL17A was associated with the risk of congestive heart failure (odds ratio [OR] = 0.76; 95% confidence interval [CI] 0.63-0.90, adjusted P = 0.002) after adjustment for multiple cardiovascular risk factors including age, sex, smoking status, diabetes, hypertension, and dyslipidemia. This association was evident in both ischemic and nonischemic heart failure (P = 0.005 and P = 0.05, respectively). Furthermore, prospective follow-up of 12.7 months for the occurrence of adverse clinical outcomes showed that rs4819554 in IL17RA was significantly associated with cardiovascular mortality (hazard ratio [HR] = 1.28; 95% CI = 1.02-1.59, adjusted P = 0.03) after adjustments for multiple cardiovascular risk factors and New York Heart Association functional class.This study demonstrated associations of rs8193037 in the promoter of IL17A with the risk of congestive heart failure, and of rs4819554 in the promoter of IL17RA with the risk of cardiovascular mortality in patients with congestive heart failure. These data lend further support to the notion that immune activation and genetic polymorphisms contribute to heart failure pathogenesis and progression.

Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma

Obesity increases hepatocellular carcinoma (HCC) risks via unknown mediators. We report that hepatic unconventional prefoldin RPB5 interactor (URI) couples nutrient surpluses to inflammation and non-alcoholic steatohepatitis (NASH), a common cause of HCC. URI-induced DNA damage in hepatocytes triggers inflammation via T helper 17 (Th17) lymphocytes and interleukin 17A (IL-17A). This induces white adipose tissue neutrophil infiltration mediating insulin resistance (IR) and fatty acid release, stored in liver as triglycerides, causing NASH. NASH and subsequently HCC are prevented by pharmacological suppression of Th17 cell differentiation, IL-17A blocking antibodies, and genetic ablation of the IL-17A receptor in myeloid cells. Human hepatitis, fatty liver, and viral hepatitis-associated HCC exhibit increased IL-17A correlating positively with steatosis. IL-17A blockers may prevent IR, NASH, and HCC in high-risk patients.

IL-17A Exacerbates Fibrosis by Promoting the Proinflammatory and Profibrotic Function of Orbital Fibroblasts in TAO.

The development of thyroid-associated ophthalmopathy (TAO) is associated with self-immune dysfunction. Recent findings in TAO and Graves' disease indicate that IL-17A may also be involved in the autoimmunity of TAO. We sought to investigate the pathogenic function of IL-17A-producing T cells in TAO. Blood samples and orbital fibroblasts (OFs) were collected from TAO patients and healthy subjects. Flow cytometry, real-time PCR, cytokine-specific ELISA, and Western blotting were performed. Here, we showed a significantly higher proportion of IL-17A-producing T cells in TAO patients and the recruitment of both CD4(+) and CD8(+) T cells in TAO orbits. TAO orbital tissues expressed more IL-17A receptor, IL-17A, and its related cytokines, with severe fibrotic change compared with normal controls. Furthermore, we validated that IL-17A could enhance the proinflammatory function of OFs and stimulate the production of extracellular matrix proteins in OFs but not eyelid fibroblasts. The mechanisms involved in this enhancement mainly relied on MAPK activation. Finally, we observed that the deubiquitinase inhibitor vialinin A could down-regulate retinoic acid receptor-related orphan receptor-γt expression and decrease IL-17A level in TAO patients. Our observations illustrate the potential pathogenic role of IL-17A-producing T cells in the inflammatory response and fibrosis of TAO. The effect of vialinin A on the reduction of retinoic acid receptor-related orphan receptor-γt level implicates its potential role as a novel therapeutic agent for TAO and other autoimmune disorders in the future.

Chitinase 3-Like 1 (Chil1) Regulates Survival and Macrophage-Mediated Interleukin-1β and Tumor Necrosis Factor Alpha during Pseudomonas aeruginosa Pneumonia.

Pseudomonas aeruginosa causes hospital-acquired pneumonia and is associated with high mortality. An effective response to such an infection includes efficient clearance of pathogenic organisms while limiting collateral damage from the host inflammatory response, known as host resistance and host tolerance, respectively. P. aeruginosa expresses a type III secretion system (T3SS) needle complex that induces NLRC4 (NOD-like receptor C4) activation, interleukin-1β (IL-1β) production, and host tissue damage. Chitinase 3-like-1 (Chil1) is expressed during infection and binds to its receptor, IL-13 receptor α2 (IL-13Rα2), to regulate the pathogen-host response during Streptococcus pneumoniae infection, but the role Chil1 plays in balancing the host resistance and host tolerance during P. aeruginosa pneumonia is not known. We conducted experiments using C57BL/6 mice with or without a genetic deficiency of Chil1 and demonstrated that Chil1-deficient mice succumb to P. aeruginosa infection more rapidly than the wild type (WT). The decreased survival time in infected Chil1-deficient mice is associated with more neutrophils recruited to the airways, more lung parenchymal damage, and increased pulmonary consolidation while maintaining equivalent bacterial killing compared to WT mice. Infected Chil1-deficient mice and bone marrow-derived macrophages (BMDMs) from Chil1-deficient mice have increased production of tumor necrosis factor alpha (TNF-α) and IL-1β compared to infected WT mice and macrophages. Infection of Chil1-deficient BMDMs with non-NLRC4-triggering P. aeruginosa, which is deficient in the T3SS needle complex, did not alter the excessive IL-1β production compared to BMDMs from WT mice. The addition of recombinant Chil1 decreases the excessive IL-1β production but only partially rescues stimulated BMDMs from IL-13Rα2-deficient mice. Our data provide mechanistic insights into how Chil1 regulates P. aeruginosa-induced host responses.

Disruption of Interleukin-17 receptor A (IL-17RA) gene in mice aggravates renal interstitial fibrosis in obstructive nephropathy

Abstract Renal fibrosis is the final outcome of obstructive nephropathy and a major cause of chronic renal failure. Renal fibrosis results from an imbalance between extracellular matrix (ECM) synthesis and degradation, which is regulated by multiple inflammatory mediators in the kidney. Recently, Interleukin-17 (IL-17) emerged as a key cytokine in lung and liver fibrosis. Although IL-17 producing cells were detected in the obstructed kidney, the role of IL-17 in renal fibrosis is unknown. We sought to define the contribution of IL-17 in renal fibrosis in obstructive nephropathy. Mice deficient in IL-17 receptor signaling (IL-17RA−/−) were assessed for renal fibrosis following unilateral ureteral obstruction (UUO), a well-characterized mouse model of obstructive nephropathy. We determined IL-17 receptor signaling is required for protection against renal fibrosis following UUO. IL-17RA−/− mice developed severe morphological injury and displayed increased deposition of collagen in the obstructed kidney compared to controls. We observed a significant decrease in the level of matrix degrading enzymes in the kidney of IL-17RA−/− mice, despite normal ECM synthesis, correlating with increased fibrosis. Overexpression of IL-17 increased the level of matrix degrading enzymes and protected mice from renal fibrosis. We also show that IL-17R signaling in tubular epithelial cells up regulated multiple genes of Kallikrein-kinin system, known to play a critical role in renal protection against kidney fibrosis. Our data clearly indicate a previously unappreciated role of IL-17 in renal protection against fibrosis. These results offer the opportunity for the development of potential therapies in the treatment of chronic kidney diseases.

275. Optimization of IL13Rα2-Specific CAR T Cells for Clinical Development Using Orthotopic Human Glioblastoma Models in NSG Mice

Standard-of-care therapies for glioblastoma (GBM), the most aggressive and common type of glioma, are inadequate, resulting in an unacceptably poor median overall survival of approximately 15 months following primary diagnosis. Immunotherapeutic targeting of brain tumors offers the opportunity to redirect the potency and specificity of the immune system to improve therapeutic outcomes. Our research program has developed a CAR T cell immunotherapy for GBM targeting IL-13 receptor α2 (IL13Rα2), a cell surface receptor over-expressed by the majority of high-grade gliomas. We have optimized second-generation IL13Rα2-specific CAR T cells, incorporating enhancements in CAR design and T cell engineering to improve T cell persistence and antitumor potency. These include a second-generation CAR containing the 41BB (CD137) costimulatory signaling domain (IL13BBζ-CAR), and a manufacturing strategy using an enriched central memory T cell (TCM) population for genetic engineering. In order to optimize clinical parameters for translating this therapy to patients, we have used orthotopic human GBM models in NSG mice. For these studies we have utilized the IL13Rα2-expressing primary low-passage GBM tumor sphere line PBT030-2, which has been engineered to express both EGFP and firefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc). We first compared the route of delivery, intravenous (i.v.) or intracranial (i.c.), for mediating optimal antitumor activity against invasive TS-initiated PBT030-2. We found that i.c. delivery of the therapeutic CAR T cells elicited superior antitumor efficacy as compared to i.v. administration, which provided no therapeutic benefit. In a multifocal disease model, we also establish that CAR T cells that were injected i.c. at one tumor site were able to traffic to a second tumor site in the contralateral hemisphere. Performing CAR T cell titration studies, we estimate that approximately one CAR T cell is required for every two IL13Rα2 tumor cells based on volumetric analysis of day 8 PBT030-2 tumors. Further, we evaluated whether CAR positivity impacted therapeutic efficacy, as our product release criteria for the clinical trial requires the therapeutic product to be at least 10% CAR positive. Indeed, we found equivalent efficacy for i.c. delivered T cells of either 10% versus 100% CAR positivity when cell doses were standardized by CAR, suggesting that unengineered T cells do not negatively impact the therapeutic potency of the CAR T cells. Finally, we evaluated the impact of corticosteroid, given its frequent use in clinical management of GBM, and demonstrate that low dose dexamethasone does not diminish T cell antitumor activity in vivo. These findings provide the rational for initiating a first-in-human clinical trial with intracranial administration of IL13Rα2-specific CD4+ CD8+ IL13BBζ-CAR T cells for the treatment of GBM.

IL-17A is rapidly triggered by staphylococcal superantigens and is pathogenic in toxic shock syndrome

Abstract Toxic shock syndrome (TSS) is a life-threatening illness characterized by high-grade fever, erythematous rash, hypotensive shock, and multi-organ dysfunction within hours of onset. It is caused by exposure to bacterial exotoxins known as superantigens (SAgs), which are produced by the common bacterium Staphylococcus aureus. During infection, SAgs activate up to 20% of T cells inducing a severe systemic inflammatory response that can lead to vascular leakage, multiple organ damage and death. However, the role of IL-17A, a potent inflammatory cytokine, in this potentially fatal reaction remains virtually unexplored. We have found that human peripheral blood mononuclear cells (PBMCs) stimulated with SAgs in vitro immediately up-regulate IL-17A mRNA (up to 12,000-fold) and produce substantial amounts of IL-17A protein within hours. Utilizing the cutting-edge RNA-Flow Cytometry technique, we identified a subset of memory T cells that was responsible for the rapid IL-17A response. We also investigated the effect of IL-17A signaling on downstream inflammatory cytokine and chemokine gene expression via IL-17A receptor blockade. Lastly, we used HLA-DR4 transgenic mice to demonstrate rapid IL-17A production in response to SAgs, similar to human PBMCs. Neutralizing IL-17A in this model greatly reduced tissue damage, morbidity and mortality. Taken together, our results reveal a novel role for memory T cells in TSS and define a previously unrecognized contribution by IL-17A to the initiation and pathogenesis of the syndrome.

Generation and characterization of ixekizumab, a humanized monoclonal antibody that neutralizes interleukin-17A.

Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.

Sputum Gene Expression of IL-13 Receptor α2 Chain Correlates with Airflow Obstruction and Helper T-Cell Type 2 Inflammation in Asthma.

BRP-39/YKL-40 is a chitinase-like protein that plays a critical role in IL-13-induced inflammation. It correlates positively with asthma severity and airway remodeling ( 1 ) via binding to IL-13 receptor α2 chain (IL-13Rα2). Because the relationship of IL-13Rα2 to human asthma has never been evaluated previously, we sought to determine the relationship between IL-13Rα2, YKL-40, and asthma. We evaluated 112 patients (69% women) with a mean age of 46.7 years. Subjects completed an asthma phenotyping protocol that included analysis of sputum gene expression by Affymetrix 1.0 ST gene array (Affymetrix, Santa Clara, CA) and YKL-40 protein levels. IL-13Rα2 gene expression was readily detectable in the sputum and correlated negatively with prebronchodilator (BD) FEV1 (rs = -0.282, P < 0.01), post-BD FEV1 (rs = -0.268, P < 0.01), pre-BD FEV1/FVC ratio (rs = -0.228, P < 0.05), and post-BD FEV1/FVC ratio (rs = -0.242, P < 0.01). IL-13Rα2 gene expression correlated positively with gene expression of IL-13 (rs = 0.484, P < 0.001), IL-5 (rs = 0.237, P < 0.05), and IL-8 (rs = 0.218, P < 0.05). Regression analysis showed that the post-BD FEV1/FVC ratio is significantly associated with IL-13Rα2 expression and CHI3L1 expression in sputum after controlling for IL-4, IL-5, IL-13, and transforming growth factor-β1 gene expression (all P < 0.01). Sputum YKL-40 gene expression positively correlated with IL-8 expression (rs = 0.357, P < 0.001) and negatively correlated with pre- and post-BD FEV1/FVC ratios (rs = -0.299, P < 0.001 and rs = -0.305, P < 0.01, respectively). Sputum and serum YKL-40 protein levels were not associated with IL-13Rα2 expression. This analysis demonstrates that IL-13Rα2 is associated with reduced lung function, helper T-cell type 2 gene expression, and airflow obstruction in the airway of individuals with asthma, which might in turn be driven by airway remodeling. Future studies will be required to define the proinflammatory and remodeling effects of this receptor that up to now has been considered solely a modulator of IL-13-induced inflammation.

A highly sensitive and selective impedimetric aptasensor for interleukin-17 receptor A

Interleukin-17 receptor A (IL-17RA) has been recognized as a valuable biomarker for diverse diseases, including autoimmune diseases. In this work, an electrochemical biosensor with great sensitivity and selectivity toward IL-17RA was fabricated using an IL-17RA aptamer (Kd=14.00nM) for the first time. The aptasensor was manufactured using electrodeposition of gold nanoparticles, and then quantitative detection of IL-17RA was performed based on impedimetry. The developed sensor exhibited a superior analytical performance for IL-17RA with a wide dynamic range of 10–10,000pg/mL in buffer and a detection limit of 2.13pg/mL, which is lower than that of commercially available ELISA kits. In addition, we validated the high specificity of the designed aptasensor to only IL-17RA, which showed good sensitivity even in human serum solution. Furthermore, the detection of the differentiated HL-60 cells expressing IL-17RA was successfully performed. Clinical applicability of the sensor was also demonstrated utilizing neutrophils separated from asthma patients. It is expected that the fabricated aptasensor will become an excellent diagnostic platform for IL-17RA-mediated diseases.