IL-1 Receptor Signaling Research Articles

Abstract 112: Myeloid Mineralocorticoid Receptor Controls Inflammatory and Fibrotic Responses After Renal Ischemic Injury via Macrophage Interleukin-4 Receptor

Introduction: Patients who survive an episode of acute kidney injury (AKI) are at high risk of de novo chronic kidney disease (CKD) development. Pharmacological mineralocorticoid receptor (MR) antagonism is useful to prevent CKD after a single episode of ischemic AKI in the rat. Objective: Test the involvement of myeloid MR in the development of kidney fibrosis after an ischemic AKI episode. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 22.5 min and IR plus treatment with the non-steroidal MR antagonist finerenone (10 mg/kg) at -48, -24 and -1 h before IR. MR inactivation in myeloid cells (MR MyKO ) was achieved by crossing mice with the MR alleles flanked by loxP sites (MR f/f ) with mice expressing the Cre recombinase under the LysM promoter activity. In MR f/f and MR MyKO mice we induced renal IR of 22.5 min or sham surgery. The mice were followed-up during 4 weeks to test for AKI to CKD transition. In another set of mice, the macrophages were sorted from kidneys after 24 h of reperfusion and flow cytometry characterization or mRNA extraction was performed. Thyoglycolate elicited peritoneal macrophages were used for in vitro studies. Results: The progression of AKI to CKD after 4 weeks of renal ischemia in the untreated C57/B6 and MR f/f mice was characterized by a 50% increase in plasma creatinine, a 2-fold increase in the mRNA levels of TGF-β and fibronectin as well as by severe tubule-interstitial fibrosis. The mice that received finerenone or MR MyKO mice were protected against these alterations. Increased expression of M2-anti-inflamatory markers in kidney-isolated macrophages from finerenone-treated or MR MyKO mice was observed. The inflammatory population of Ly6C high macrophages was reduced by 50%. In peritoneal macrophages in culture, MR inhibition promoted increased IL-4 receptor expression and activation, facilitating macrophage polarization to an M2 phenotype. Conclusion: MR antagonism or myeloid MR deficiency facilitates macrophage polarization to a M2, anti-inflammatory phenotype after kidney IR, preventing maladaptive repair and chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate macrophage phenotype switching.

Loss of thymic innate lymphoid cells leads to impaired thymopoiesis in experimental graft-versus-host disease

AbstractGraft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22–mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22–dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC–IL-22–TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

Restoration of pharyngeal dilator muscle force in dystrophin-deficient (mdx) mice following co-treatment with neutralizing interleukin-6 receptor antibodies and urocortin 2.

What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n=26) and wild-type (WT; n=26) mice received either saline (0.9%w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2mgkg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30μgkg-1 ) over 2weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal density of infiltrates and collagen content were significantly increased in mdx sternohyoid; both indices were unaffected by drug treatment. The abundance of myosin heavy chain type IIb fibres was significantly decreased in mdx sternohyoid; drug treatment preserved myosin heavy chain type IIb complement in mdx muscle. The chemokines macrophage inflammatory protein2, interferon-γ-induced protein10 and macrophage inflammatory protein3α were significantly increased in mdx sternohyoid compared with WT. Drug treatment significantly increased chemokine expression in mdx but not WT sternohyoid. Recovery of contractile function was impressive in our study, with implications for Duchenne muscular dystrophy. The precise molecular mechanisms by which the drug treatment exerts an inotropic effect on mdx sternohyoid muscle remain to be elucidated.

MicroRNA-146a-5p Mediates High Glucose-Induced Endothelial Inflammation via Targeting Interleukin-1 Receptor-Associated Kinase 1 Expression.

Background and Aims: Interleukin-1 receptor-associated kinase-1 (IRAK-1) is critical for mediating toll-like receptor and interleukin-1 receptor signaling. In this study, we have examined whether IRAK-1 expression is altered in high glucose (HG)-stimulated human aortic endothelial cells (HAECs), and whether microRNAs (miRs) target IRAK-1 to regulate HG-induced endothelial inflammation.Methods: HAECs were treated with HG for 24 and 48 h. Real-time PCR, Western blot, monocyte adhesion assay, bioinformatics analysis, TaqMan® arrays, microRNA mimic or inhibitor transfection, luciferase reporter assay and siRNA IRAK-1 transfection were performed. The aortic tissues from db/db type 2 diabetic mice were examined by immunohistochemistry staining.Results: HG time-dependently increased IRAK-1 mRNA and protein levels in HAECs, and was associated with increased VCAM-1/ICAM-1 gene expression and monocyte adhesion. Bioinformatic analysis, TaqMan® arrays, and real-time PCR were used to confirm that miR-146a-5p, miR-339-5p, and miR-874-3p were significantly downregulated in HG-stimulated HAECs, suggesting impaired feedback restraints on HG-induced endothelial inflammation via IRAK-1. However, only miR-146a-5p mimic transfection reduced the HG-induced upregulation of IRAK-1 expression, VCAM-1/ICAM-1 expression, and monocyte adhesion. Additionally, IRAK-1 depletion reduced HG-induced VCAM-1/ICAM-1 gene expression, and monocyte adhesion, indicating that HG-induced endothelial inflammation was mediated partially through IRAK-1. In vivo, intravenous injections of miR-146a-5p mimic prevented endothelial IRAK-1 and ICAM-1 expression in db/db mice.Conclusion: These results suggest that miR-146a-5p is involved in the regulation of HG-induced endothelial inflammation via modulation of IRAK-1; indicating that miR-146a-5p may be a novel target for the treatment of diabetic vascular complications.

Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling.

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.

IL-1 Receptor–Knockout Mice Develop Epidermal Cysts and Show an Altered Innate Immune Response after Exposure to UVB Radiation

In this study, we observed that mice lacking the IL-1 receptor (IL-1R) (IL1r-/-) or deficient in IL1-β developed multiple epidermal cysts after chronic UVB exposure. Cysts that developed in IL1r-/- mice were characterized by the presence of the hair follicle marker Sox 9, keratins 10 and 14, and normal melanocyte distribution and retinoid X receptor-α expression. The increased incidence of cysts in IL1r-/- mice was associated with less skin inflammation as characterized by decreased recruitment of macrophages, and their skin also maintained epidermal barrier function compared with wild-type mice. Transcriptional analysis of the skin of IL1r-/- mice after UVB exposure showed decreased gene expression of proinflammatory cytokines such as tumor necrosis factor-α and IL-6. Invitro, primary keratinocytes derived from IL1r-/- mice were more resistant to UVB-triggered cell death compared with wild-type cells, and tumor necrosis factor-α release was completely blocked in the absence of IL-1R. These observations illustrate an unexpected yet prominent phenotype associated with the lack of IL-1R signaling in mice and support further investigation into the role of IL-1 ligands in epidermal repair and innate immune response after damaging UVB exposure.

The miR-15 family reinforces the transition from proliferation to differentiation in pre-B cells.

Precursor B lymphocytes expand upon expression of a pre-B cell receptor (pre-BCR), but then transit into a resting state in which immunoglobulin light chain gene recombination is initiated. This bi-phasic sequence is orchestrated by the IL-7 receptor (IL-7R) and pre-BCR signaling, respectively, but little is known about microRNAs fine-tuning these events. Here, we show that pre-B cells lacking miR-15 family functions exhibit prolonged proliferation due to aberrant expression of the target genes cyclin E1 and D3. As a consequence, they fail to trigger the transcriptional reprogramming normally accompanying their differentiation, resulting in a developmental block at the pre-B cell stage. Intriguingly, our data indicate that the miR-15 family is suppressed by both IL-7R and pre-BCR signaling, suggesting it is actively integrated into the regulatory circuits of developing B cells. These findings identify the miR-15 family as a novel element required to promote the switch from pre-B cell proliferation to differentiation.

Macrophage dysfunction initiates colitis during weaning of infant mice lacking the interleukin-10 receptor.

Infants with defects in the interleukin 10 receptor (IL10R) develop very early onset inflammatory bowel disease. Whether IL10R regulates lamina propria macrophage function during infant development in mice and whether macrophage-intrinsic IL10R signaling is required to prevent colitis in infancy is unknown. Here we show that although signs of colitis are absent in IL10R-deficient mice during the first two weeks of life, intestinal inflammation and macrophage dysfunction begin during the third week of life, concomitant with weaning and accompanying diversification of the intestinal microbiota. However, IL10R did not directly regulate the microbial ecology during infant development. Interestingly, macrophage depletion with clodronate inhibited the development of colitis, while the absence of IL10R specifically on macrophages sensitized infant mice to the development of colitis. These results indicate that IL10R-mediated regulation of macrophage function during the early postnatal period is indispensable for preventing the development of murine colitis.

Abstract 326: Baseline IL-17 receptor signaling is essential for controlling aberrant JNK-dependent cellular proliferation via maintenance of endogenous level of ubiquitin-editing enzyme A20

Abstract The molecular mechanisms underlying aberrant activation of NF-κB and JNK in cancer remain incompletely understood. Here, we demonstrate that baseline IL-17 receptor (IL-17R) signaling is essential for controlling aberrant NF-κB and JNK activation, and restraining JNK-dependent homeostatic cellular proliferation. Using a shRNA knockdown approach, we demonstrated in B16 melanoma and 4T1 breast carcinoma murine cell lines that IL-17RA silencing markedly enhanced tumor cell growth in vitro and in vivo. Through mapping IL-17R signaling pathways, we further demonstrated that baseline IL-17A/IL-17R signaling actively restrained JNK phosphorylation in vitro and in vivo via the maintenance of basal expression of the ubiquitin-editing enzyme A20, a negative regulator of NF-κB and JNK. Remarkably, IL-17RA reconstitution evidently restored the A20 level, and suppressed cell proliferation and JNK activity in tumor cells. The reconstitution of A20 in IL-17RA knockdown subclones was able to restore the normal rate of cellular proliferation and associated JNK/c-Jun activity. Finally, meta-analysis of human cancer microarray and RNA-Seq datasets confirmed significant co-expression of IL-17RA and A20. Furthermore, alterations (mutation, upregulation or downregulation) of IL-17RA level in melanoma, ER+ breast cancer and colorectal cancer patients were associated with poorer overall survival compared to the respective patients with normal baseline IL-17RA expression. Together, our data demonstrates a previously unrecognized molecular mechanism underlying aberrant activation of NF-κB and JNK in cancer cells. This work highlights the unique biological role of proinflammatory IL-17R signaling in the maintenance of A20 to regulate the pathogenesis of human cancer, which draws caution on the utility of IL-17A neutralizing antibody in cancer therapy. CY is a PhD student supported by the graduate student scholarship from the IWK Health Centre and received a trainee award from the Beatrice Hunter Cancer Research Institute with funds provided by the Cancer Research Training Program as part of The Terry Fox Foundation Strategic Health Research Training Program in Cancer Research in CIHR. Citation Format: Chi Yan, Yang Lei, Anna L. Greenshields, David W. Hoskin, Tong-Jun Lin, Jun Wang. Baseline IL-17 receptor signaling is essential for controlling aberrant JNK-dependent cellular proliferation via maintenance of endogenous level of ubiquitin-editing enzyme A20 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2017-326

Abstract 1168: Efficacy of the IRAK4 inhibitor CA-4948 in patient-derived xenograft models of diffuse large B cell lymphoma

Abstract IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of DLBCL cases. The MYD88 L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL. We are developing an IRAK4 inhibitor, CA-4948, as a therapeutic agent for hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 (previously AU-4948) is a selective and potent IRAK4 kinase inhibitor with in vivo activity in a TLR4-induced cytokine release model. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88-L265P xenograft tumor models using cell lines OCI-LY3 and OCI-LY10. Here, we report the efficacy results from testing CA-4948 in a panel of well characterized, patient-derived DLBCL tumor xenograft (PDX) mouse models. CA-4948 exhibited the greatest efficacy in four of the five ABC-DLBCL PDX models tested as compared to GBC-DLBCL and ABC/GCB DLBCL PDX models. Furthermore, CA-4948 was efficacious in ABC-DLBCL PDX tumors containing activating mutations in both TLR/IL-1R and BCR signaling pathways (MYD88 and CD79B double mutants). Interestingly, the one ABC-DLBCL PDX model that failed to respond to CA-4948 treatment contained a MYD88 L265P mutation as well as a BCL6 translocation. While this particular PDX model was resistant to CA-4948, and showed a weak anti-tumor response to single-agent ibrutinib, the combination treatment of ibrutinib and CA-4948 exhibited a synergistic tumor growth inhibition effect. In summary, CA-4948 exhibited anti-tumor activity in ABC-type DLCBL PDX tumor models including those containing combinations of activating mutations in the TLR/IL-1R and BCR signaling pathways. These results underscore the therapeutic potential of IRAK4 kinase inhibition by CA-4948, as a single-agent or in combination with BCR inhibitors, for the treatment of DLBCL. Citation Format: Robert N. Booher, Maria Elena Samson, Guang-Xin Xu, Hongsheng Cheng, David P. Tuck. Efficacy of the IRAK4 inhibitor CA-4948 in patient-derived xenograft models of diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1168. doi:10.1158/1538-7445.AM2017-1168

A Critical Role for IL-21 Receptor Signaling in the Coxsackievirus B3-Induced Myocarditis

To determine whether IL-21 receptor signaling plays a significant role in promoting Tfh cell-mediated cardiac injury in viral myocarditis (VMC), we compared IL-21R-deficient mice for some parameters of VMC. Balb/c and IL-21R-/- mice were infected with CVB3. Frequencies of splenic Tfh cells were determined by flow cytometric analysis, and productions of anti-adenine nucleotide translocator (ANT) autoantibodies were detected by enzyme-linked immunosorbent assay. To determine the effects of IL-21R signal on the proliferation of B cells, lymphocytes from spleens of the IL-21R-/- and Balb/c mice infected by CVB3 were tagged with carboxyfluorescein succinimidyl ester (CFSE) and then were stimulated with lipopolysaccharides plus IL-21 or anti-IL-21 neutralizing antibody for 3days. The proliferation of B cells was analyzed by flow cytometry. Anti-ANT antibodies in the supernatants were detected by ELISA. Results showed that IL-21R-/- mice developed significantly less inflammation of the myocardium than Balb/c mice. Numbers of the Tfh cells and levels of anti-ANT antibody were decreased in IL-21R-/- mice, indicating IL-21 signaling plays a role on the Tfh cell response. The percentage of CD19+CFSE+ B cells decreased in IL-21R-/- mice compared to VMC mice. And anti-ANT antibodies were detected at lower levels in cultured supernatant from IL-21R-/- mice than in those from VMC mice. These data suggest that IL-21R signal may contribute to anti-ANT antibody production and expansion of B cells in VMC mice.

Host Resistance to Plasmodium-Induced Acute Immune Pathology Is Regulated by Interleukin-10 Receptor Signaling.

ABSTRACTThe resolution of malaria infection is dependent on a balance between proinflammatory and regulatory immune responses. While early effector T cell responses are required for limiting parasitemia, these responses need to be switched off by regulatory mechanisms in a timely manner to avoid immune-mediated tissue damage. Interleukin-10 receptor (IL-10R) signaling is considered to be a vital component of regulatory responses, although its role in host resistance to severe immune pathology during acute malaria infections is not fully understood. In this study, we have determined the contribution of IL-10R signaling to the regulation of immune responses during Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM). We show that antibody-mediated blockade of the IL-10R during P. berghei ANKA infection in ECM-resistant BALB/c mice leads to amplified T cell activation, higher serum gamma interferon (IFN-γ) concentrations, enhanced intravascular accumulation of both parasitized red blood cells and CD8+ T cells to the brain, and an increased incidence of ECM. Importantly, the pathogenic effects of IL-10R blockade during P. berghei ANKA infection were reversible by depletion of T cells and neutralization of IFN-γ. Our findings underscore the importance of IL-10R signaling in preventing T-cell- and cytokine-mediated pathology during potentially lethal malaria infections.

Macrophage-derived IL-1α promotes sterile inflammation in a mouse model of acetaminophen hepatotoxicity

The metabolic intermediate of acetaminophen (APAP) can cause severe hepatocyte necrosis, which triggers aberrant immune activation of liver non-parenchymal cells (NPC). Overzealous hepatic inflammation determines the morbidity and mortality of APAP-induced liver injury (AILI). Interleukin-1 receptor (IL-1R) signaling has been shown to play a critical role in various inflammatory conditions, but its precise role and underlying mechanism in AILI remain debatable. Herein, we show that NLRP3 inflammasome activation of IL-1β is dispensable to AILI, whereas IL-1α, the other ligand of IL-1R1, accounts for hepatic injury by a lethal dose of APAP. Furthermore, Kupffer cells function as a major source of activated IL-1α in the liver, which is activated by damaged hepatocytes through TLR4/MyD88 signaling. Finally, IL-1α is able to chemoattract and activate CD11b+Gr-1+ myeloid cells, mostly neutrophils and inflammatory monocytes, to amplify deteriorated inflammation in the lesion. Therefore, this work identifies that MyD88-dependent activation of IL-1α in Kupffer cells plays a central role in the immunopathogenesis of AILI and implicates that IL-1α is a promising therapeutic target for AILI treatment.

Defective IL-4 signaling in T cells defines severe common variable immunodeficiency

Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and B-cell dysfunction, with significant clinical and immunological heterogeneity. Severe non-infectious complications, such as autoimmunity, granulomatous disease and splenomegaly, constitute a major cause of morbidity in CVID patients. T cells are generally regarded important for development of these clinical features. However, while T-cell abnormalities have been found in CVID patients, functional characteristics of T cells corresponding to well-defined clinical subtypes have not been identified. As common γ-chain cytokines play important roles in survival and differentiation of T cells, characterization of their signaling pathways could reveal functional differences of clinical relevance. We characterized CVID T cells functionally by studies of cytokine-induced signaling, and correlated the findings to defined clinical subtypes. Peripheral blood T cells from 29 CVID patients and 19 healthy donors were analyzed for i) phenotype, ii) cytokine-induced (interleukin (IL)-2, IL-4, IL-7 and IL-21) phosphorylation of signal transducer and activator of transcription (STAT) 3, STAT5 and STAT6, and iii) T-helper (Th)1/Th2 polarization. Expression of IL-4 receptor and downstream signaling molecules was measured.A subgroup of CVID patients (n = 7) was identified by impaired IL-4-induced p-STAT6 in naive and memory CD4 and CD8 T cells. This corresponded to patients with the largest accumulation of severe (non-infectious) complications. The signaling defect persisted over years and was not due to constitutively activated p-STAT6. The CD4 T cells were strongly Th1-skewed, but IL-4 signaling was impaired independently of Th status. However, IL-4Rα and Janus kinase (JAK) 1 mRNA levels were significantly lower than in normal donors, providing a likely mechanism for the defective IL-4-induced p-STAT6 and Th1-bias. In conclusion, we identified a subgroup of CVID patients with defective IL-4 signaling in T cells, with severe clinical features of inflammation and autoimmunity.

Epithelial expression of IL-17 mediates a gut-associated inflammatory response in the purple sea urchin larva.

Abstract Immune response to microbial disturbance in the gut involves coordinated activity of gut epithelial cells, motile immune cells and other cells throughout the organism. Many of these functions are ancient among bilaterians, although it is not yet clear how they relate among animal phyla. As morphologically simple organisms that share an important genetic heritage with vertebrates, sea urchin larvae offer a novel, experimentally tractable system in which to characterize the gut-associated immune response. We have developed a model to characterize gene regulatory control of this response using the marine bacterium Vibrio diazotrophicus. When exposed to Vibrio in the surrounding seawater, bacteria accumulate in the gut and later invade the body cavity. Bacteria that enter the blastocoel are rapidly cleared by a coordinated response of phagocytic and granular immune cells. Peripheral immune cells migrate through the body cavity and accumulate at the gut. RNA-Seq data indicate that the most acutely upregulated gene in the early phase of response are two types of IL17 genes. Whole mount in situ hybridization and BAC-based fluorescent protein reporters indicate that these genes are expressed within gut epithelial cells. Notably, perturbation of IL17 receptor signaling results in reduced levels of tnfaip3 (a negative feedback inhibitor of IL17), nfkbiz (an IL17 target gene in vertebrates), the transcription factors cebpα and cebpγ and soul1 (SOUL domains are evolutionarily widespread and involved in immune responses). These results indicate that the highly regulated IL17 expression in the gut epithelium and signaling through IL17R1 form a central axis of larval gut-associated immunity.

CD001, an active compound from Astragalus membranaceus, downregulates TH17 cells differentiation and attenuates collagen-induced arthritis through inhibition of IL-6/IL-6R signaling

Abstract Rheumatoid arthritis is a common autoimmune disease affecting approximately one percent of the worldwide population. It causes not only life-long discomfort but also disabilities of the patients. Despite the fact that conventional therapies and biological agents are used to modulate the dysregulated immune response and inflammation; there are still some unmet needs and concerns. We aimed to develop a pharmaceutical agent for targeting IL-6 receptor, which is involved in the differentiation of TH17 and pathogenesis of rheumatoid arthritis. Here we demonstrated an active compound, CD001, from Astragalus membranaceus, inhibited TH17 cells differentiation in a concentration-dependent manner in vitro. To further determine its efficacy in vivo, we administered CD001 in a murine model of collagen-induced arthritis. CD001 ameliorated paw edema and disease incidence as well as the production of IL-17, IL-22 and TNF-a in vivo. Molecular docking revealed that CD001 could couple into the binding site of IL-6 receptor. We further confirmed that CD001 interrupted the binding of IL-6 and IL-6 receptor in an in vitro binding assay. CD001 inhibited the downstream signaling pathways of IL-6 receptor, including JAK/STAT3, ErK1/2, and AKT, in TH17 cells. Our data fit the niche that is required to block IL-6 receptor signaling and inhibit TH17 for immunotherapy. We suggest that, to treat autoimmune disease, administering CD001 could be a promising pharmacologic approach to achieve the goal.

Restoration of Immune Tolerance in Type 1 Diabetes by Modulating Interleukin-2 Receptor Signaling

Abstract Type 1 diabetes (T1D) is an organ-specific autoimmune disease, characterized by the destruction of insulin-producing beta cells in pancreatic islets. A variety of lymphocytes, including T cells and natural killer (NK) cells infiltrate into the islets. Their roles in T1D are rather different--effector T cells and NK cells accelerate, while regulatory T cells, dampen the development of T1D. Signaling through interleukin-2 (IL-2) receptor beta (IL-2Rβ, also known as CD122) is critical for the differentiation and function of both T cells and NK cells. However, it remains poorly understood how these cells respond to IL-2Rβ signaling during the development of T1D. Here we show that in non-obese diabetic (NOD) mice, the most commonly used animal model for human T1D, administration of a non-immunogenic monoclonal antibody (mAb) selectively ablates pathogenic cells, including memory CD4+ T cells, CD8+ T cells and NK cells. In contrast, CD4+ Foxp3+ regulatory T (Treg) cells are only mildly affected. This leads to significantly elevated ratios between Treg cells and pathogenic cells. This reset balance preferentially occurs in pancreatic islets, without systemic immunosuppression. Importantly, IL-2Rβ blockade suppresses both insulitis and diabetes in NOD mice. Moreover, IL-2Rβ blockade significantly suppresses interferon gamma (IFN-γ) production in T cells and NK cells, but promotes interleukin-17 (IL-17) production in CD4+ T cells. Therefore, the modulation of IL-2Rβ signaling represents a novel and efficient approach to restore immune tolerance in T1D.

MiR-146a suppresses STAT3/VEGF pathways and reduces apoptosis through IL-6 signaling in primary human retinal microvascular endothelial cells in high glucose conditions

microRNA (miRNA) play critical roles in the pathological processes of diabetic retinopathy, including inflammatory responses, insulin signaling, and angiogenesis.In addition to their regulatory functions on gene expression, miRNA is considered as a potential therapeutic target, as well as a diagnostic marker for many diseases. Our understanding on the pathological mechanisms underlying diabetic retinopathy is still incomplete and additional investigations are required to develop novel therapeutic strategies. The aim of this study was to investigate our hypothesis that miR-146a plays a role in suppressing pro-inflammatory pathways, involving STAT3 and VEGF, through regulating IL-6 signaling to reduce apoptosis of human retinal endothelial cells (REC) in high glucose conditions. Human REC were cultured in normal (5mM) glucose or high glucose medium (25mM) for 3days. We performed transfections on REC with miRNA mimics (hsa-miR-146a-5p). Overexpression of miR-146a reduced IL-6 levels, STAT3 phosphorylation, and VEGF levels in REC cultured in high glucose. Cellular apoptosis was decreased in REC overexpressing miR-146a, as demonstrated by the inhibition of DNA fragmentation. More importantly, we demonstrated that the regulatory role of miR-146a on STAT3/VEGF and apoptosis was mediated by IL-6 receptor signaling in REC.Overall, we report that miR-146a suppressed IL-6 signaling, leading to reduced levels of STAT3 and VEGF in REC in high glucose conditions, leading to decreased apoptosis. The outcome suggests that miR-146a is a potential molecular target for inhibiting inflammation and apoptosis in the diabetic retina through the suppression of the IL-6-mediated STAT3/VEGF pathway.

Microvesicles released by apoptotic human neutrophils suppress proliferation and IL-2/IL-2 receptor expression of resting T helper cells.

Membrane-coated microvesicles (MVs) have been identified as important mediators in intercellular communication. During the process of apoptosis, dying cells dynamically release MVs. Neutrophils are the most abundant type of leukocytes in the circulation. Due to their very short lifespan, it is likely that they are the source of large amounts of apoptotic cell-derived MVs. Here, we show that MVs released by apoptotic human polymorphonuclear neutrophils (apoPMN-MVs), but not the apoptotic neutrophils themselves, selectively suppress the proliferation of CD25 (IL-2Rα)neg CD127 (IL-7Rα)pos Th cells in a dose-dependent manner. In contrast, the proliferation of total T cells is not affected by MVs. Importantly, apoPMN-MVs suppress the secretion of IL-2 as well as the expression of and signaling via the IL-2 receptor (IL-2R) by CD25neg CD127pos Th cells. Addition of IL-7 strongly reduced the suppression of T-cell proliferation by MVs and the addition of IL-2 completely abrogated the suppressive effect. Thus, apoPMN-MVs suppressed a subset of Th cells by downregulating IL-2 and IL-2R expression and signaling. This may represent an important mechanism to prevent the activation and expansion of resting T cells in the absence of sufficient cytokine stimulation, and thereby maintaining immune tolerance.

Act1: A Psoriasis Susceptibility Gene Playing its Part in Keratinocytes

Unchecked inflammation, impaired keratinocyte differentiation, and heightened host defense responses typify psoriasis. Lambert etal. make clever use of psoriasis patient genetics and whole transcriptome RNA-Seq analysis to implicate Act1 in these seemingly variegated processes by keeping IL-17 receptor signaling in check while supporting differentiation and limiting innate immune responses in human keratinocytes.