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BACKGROUND Infantile-onset inflammatory bowel disease (IO-IBD) occurs in very young children and causes severe clinical manifestations, which has poor responses to traditional inflammatory bowel disease (IBD) treatments. At present, there are no simple and reliable laboratory indicators for early screening IO-IBD patients, especially those in whom the disease is caused by monogenic diseases. AIM To search for valuable indicators for early identifying IO-IBD patients, especially those in whom the disease is caused by monogenic diseases. METHODS A retrospective analysis was performed in 73 patients with IO-IBD admitted to our hospital in the past 5 years. Based on the next-generation sequencing results, they were divided into a monogenic IBD group (M-IBD) and a non-monogenic IBD group (NM-IBD). Forty age-matched patients with allergic proctocolitis (AP) were included in a control group. The clinical manifestations and the inflammatory factors in peripheral blood were evaluated. Logistic regression analysis and receiver operating characteristic (ROC) curve analysis were used to identify the screening factors and cut-off values of IO-IBD as well as monogenic IO-IBD, respectively. RESULTS Among the 44 M-IBD patients, 35 carried IL-10RA mutations, and the most common mutations were c.301C>T (p.R101W, 30/70) and the c.537G>A (p.T179T, 17/70). Patients with higher serum tumor necrosis factor (TNF)-α value were more likely to have IBD [odds ratio (OR) = 1.25, 95% confidence interval (CI): 1.05-1.50, P = 0.013], while higher serum albumin level was associated with lower risk of IBD (OR = 0.86, 95%CI: 0.74-1.00, P = 0.048). The cut-off values of TNF-α and albumin were 17.40 pg/mL (sensitivity: 0.78; specificity: 0.88) and 36.50 g/L (sensitivity: 0.80; specificity: 0.90), respectively. The increased ferritin level was indicative of a genetic mutation in IO-IBD patients. Its cut-off value was 28.20 ng/mL (sensitivity: 0.93; specificity: 0.92). When interleukin (IL)-10 level was higher than 33.05 pg/mL (sensitivity: 1.00; specificity: 0.84), or the onset age was earlier than 0.21 mo (sensitivity: 0.82; specificity: 0.94), the presence of disease-causing mutations in IL-10RA in IO-IBD patients was strongly suggested. CONCLUSION Serum TNF-α and albumin level could differentiate IO-IBD patients from allergic proctocolitis patients, and serum ferritin and IL-10 levels are useful indicators for early diagnosing monogenic IO-IBD.

Background Several studies have employed animal models to explore the association between microbiota and interleukin (IL) 10 signaling; however, limited information is available about the human microbiome. Aim To characterize the microbiome in patients with IL10RA mutations and to explore the association between gut dysbiosis and disease severity. Methods Fecal samples were collected from patients who were diagnosed with loss-of-function mutations in the IL10RA gene between January 2017 and July 2018 at the Children's Hospital of Fudan University. Age-matched volunteer children were recruited as healthy controls. Patients with Crohn's disease (CD) were used as disease controls to standardize the antibiotic exposure. Microbial DNA was extracted from the fecal samples. All analyses were based on the 16S rRNA gene sequencing data. Results Seventeen patients with IL10RA mutations (IL10RA group), 17 patients with pediatric CD, and 26 healthy children were included. Both patients with IL10RA mutations and those with CD exhibited a reduced diversity of gut microbiome with increased variability. The relative abundance of Firmicutes was substantially increased in the IL10RA group (P = 0.02). On further comparison of the relative abundance of taxa between patients with IL10RA mutations and healthy children, 13 taxa showed significant differences. The IL10RA-specific dysbiosis indices exhibited a significant positive correlation with weighted pediatric CD activity index and simple endoscopic score for CD. Conclusion In patients with IL10RA mutations and early onset inflammatory bowel disease, gut dysbiosis shows a moderate association with disease severity.

Interleukin-10 (IL-10) is a pleiotropic cytokine produced by a wide variety of cells. It has been implicated in cancer progression, and at times, it has seemingly contradictory effects. The impact of IL-10 on immune components in the context of cancer has been intensively investigated, but its effect on cancer cells remains poorly understood. In this study, we examined the expression of IL-10 and IL-10 receptor 1 (IL-10R1) in resected locally advanced lung adenocarcinoma by immunohistochemistry. IL-10 immunoreactivity was stronger in intraepithelial regions than in stroma. The amount of IL-10 found either in intraepithelial or in stromal regions had no prognostic value, but the relative distribution of IL-10 in these two locations was related to cancer-immune phenotypes. High expression of IL-10R1 by tumor cells was significantly correlated with poor prognosis, suggesting that IL-10-mediated signaling may induce cancer cell intrinsic effects that promote cancer progression. Functional analysis using human lung adenocarcinoma cell lines revealed that IL-10 did not directly affect cell proliferation and migration. Incubation of cancer cells with IL-10 suppressed interferon-γ (IFN-γ)-induced STAT1 phosphorylation and inhibited the transcription of IFN-γ-targeted genes, such as CXCL9, CXCL10, and PD-L1. IL-10 enhanced IFN-γ-induced SOCS1 and SOCS3 expression, an effect that might be responsible for the downregulation of STAT1 activity in cancer cells. Our findings provide a rationale for targeting IL-10 on cancer cells as a potential strategy for treating cancer.

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

BACKGROUND Interleukin 10 receptor alpha subunit (IL10RA) dysfunction is the main cause of very early-onset inflammatory bowel disease (VEO-IBD) in East Asians. AIM To identify disease-causing gene mutations in four patients with VEO-IBD and verify functional changes related to the disease-causing mutations. METHODS From May 2016 to September 2020, four young patients with clinically diagnosed VEO-IBD were recruited. Before hospitalization, using targeted gene panel sequencing and trio-whole-exome sequencing (WES), three patients were found to harbor a IL10RA mutation (c.301C>T, p.R101W in one patient; c.537G>A, p.T179T in two patients), but WES results of the fourth patient were not conclusive. We performed whole-genome sequencing (WGS) on patients A and B and reanalyzed the data from patients C and D. Peripheral blood mononuclear cells (PBMCs) from patient D were isolated and stimulated with lipopolysaccharide (LPS), interleukin 10 (IL-10), and LPS + IL-10. Serum IL-10 levels in four patients and tumor necrosis factor-α (TNF-α) in the cell supernatant were determined by enzyme-linked immunosorbent assay. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and Ser727 in PBMCs was determined by western blot analysis. RESULTS The four children in our study consisted of two males and two females. The age at disease onset ranged from 18 d to 9 mo. After hospitalization, a novel 333-bp deletion encompassing exon 1 of IL10RA was found in patients A and B using WGS and was found in patients C and D after reanalysis of their WES data. Patient D was homozygous for the 333 bp deletion. All four patients had elevated serum IL-10 levels. In vitro, IL-10-stimulated PBMCs from patient D failed to induce STAT3 phosphorylation at Tyr705 and only minimally suppressed TNF-α production induced by LPS. Phosphorylation at Ser727 in PBMCs was not affected by LPS or LPS + IL-10 in both healthy subjects and in patient D. CONCLUSION WGS revealed a novel 333-bp deletion of IL10RA in four patients with VEO-IBD, whereas the WES results were inconclusive.

Coccidiosis is a major gastrointestinal disease caused by several Eimeria species in floor raised chickens. Feeding an antibody to interleukin 10 (aIL-10) ameliorates the negative symptoms of coccidiosis in broilers, i.e., lack of weight gain, decreased feed conversion, and mortality. IL-10 signals by forming a ligand-receptor complex with IL-10 Receptor 1 (IL-10 R1) and IL-10 Receptor 2 (IL-10 R2). In this study, we hypothesize oral antibodies to the IL-10 receptors will neutralize the IL-10 signaling pathway equal to or better than aIL-10 to act as an oral anti-coccidiosis immunotherapy. A total of 5 sequential feed trials, set up as a 4 (diet antibody) × 2 (Eimeria challenge) factorial design, tested oral egg yolk antibodies to a total of 6 IL-10 R1 epitopes and 3 IL-10 R2 epitopes compared to a control antibody diet. A total of 10 pens of 5 chicks/pen/diet antibody/Eimeria challenge were housed for 21 d. On day 3 of age, chicks were either infected or not infected with a 10× dose of an Eimeria vaccine containing Eimeria acervulina, Eimeria tenella, and Eimeria maxima. Pen feed consumption and mean body weights were assessed weekly (d1, d7, d14, and d21); fecal oocyst shedding was assessed on day 10. Data were analyzed using a 2-way ANOVA. No significant interaction on chick weight was observed in chicks fed IL-10 R1 antibodies compared to chicks fed the control antibody was observed. In studies evaluating aIL-10 R2 oral antibodies, infected chicks fed aIL-10 R2: epitope 1 overcame the negative effects of Eimeria infection and had similar 21-d body weight to uninfected chicks (P(4) = 0.07). We hypothesized that feeding oral antibodies to the IL-10 receptors would result in equivalent anti-coccidial benefits to aIL-10. However, none of the 6 antibodies to IL-10 R1 epitopes yielded any benefits during Eimeria infection compared to controls. A total of 2 oral antibodies to IL-10 R2 showed promising results equivalent to the aIL-10 immunotherapeutic. Immunofluorescence staining shows that the IL-10R2 significantly increases in abundance in response to Eimeria infection, whereas IL-10R1 does not.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1β, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

Samples from patients with rare diseases, such as primary immunodeficiencies, are often limited, which hampers careful analysis of the pathomechanisms involved in immune cell dysregulation. To overcome this issue, induced pluripotent stem cells (iPSCs) represent an almost inexhaustible cell source and thus provide an excellent opportunity to generate disease models for rare diseases and to validate new therapeutic approaches. To obtain a better understanding of primary immunodeficiencies associated with the interleukin (IL)-10 signaling pathway, for example, very-early-onset inflammatory bowel disease (VEO-IBD), we generated genetic knockouts (KOs) of IL-10RA (IL-10 receptor a-chain) and IL-10RB (IL-10 receptor v-chain) as well as the downstream targets of the IL-10-receptor (IL-10R) signal transducers and activators of transcription (STAT)1 and STAT3 via an sgRNA (single-guide RNA)-CRISPR-Cas9-expressing lentiviral system. IL-10 signaling-associated KO models and a VEO-IBD patient-derived iPSC clone were differentiated into macrophages for disease models. IL-10R- or STAT3-deficient disease models showed no IL-10-induced BCL3 or SOCS3 expression, whereas lipopolysaccharide (LPS) stimulation induced IL-10R independently of BCL3 and SOCS3 expression. Cytokine secretion profiles from iPSC-derived macrophage disease models showed that IL-10 was involved in many inflammatory cytokine secretions, which indicated formation of both anti- and proinflammatory macrophage phenotypes. Macrophage-secreted cytokines were separated into IL-10R- and STAT3-dependent (IL-6, TNF-a), or into IL-10R-, STAT1-, and STAT3-dependent cytokines (CCL2, CXCL10). Importantly, lentiviral correction restored IL-10-mediated regulation of LPS-induced cytokine secretion in corrected IL-10RB, STAT1, and VEO-IBD patient-derived disease models. Furthermore, treatment of IL-10RB-deficient macrophages with anti-inflammatory small molecules (SB202190, filgotinib) reduced proinflammatory cytokine secretion patterns. Taken together, the described iPSC KO models gave new insights into the pathomechanisms of immune cell dysregulation and served as model systems to test potential therapeutic approaches, including lentiviral gene therapy and targeted small-molecule treatment.

Anaplastic large cell lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive anaplastic lymphoma kinase (ALK) fusion protein. ALK inhibitors, such as crizotinib, provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by nucleophosmin 1 (NPM1)-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54% to 90% in clinical trials; however, a subset of patients progressed within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) activation and knockout screens in ALCL cell lines, combined with RNA sequencing data derived from ALK inhibitor–relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of interleukin 10 receptor subunit alpha (IL10RA). Elevated IL10RA expression rewires the STAT3 signaling pathway, bypassing otherwise critical phosphorylation by NPM1-ALK. IL-10RA expression does not correlate with response to standard chemotherapy in pediatric patients, suggesting that a combination of crizotinib and chemotherapy could prevent ALK inhibitor resistance–specific relapse.

Interleukin (IL)-10, an immune-regulatory cytokine, exerts various biological functions through interaction with IL-10 receptors. In teleost, very limited functional studies on IL-10 receptors have been documented. In this study, we reported the expression patterns of IL-10 receptor 1 (CsIL-10R1) and receptor 2 (CsIL-10R2) of tongue sole (Cynoglossus semilaevis) and examined their biological properties. The expression of CsIL-10R1 and CsIL-10R2 occurred in multiple tissues and were regulated by bacterial challenge. In vitro binding studies showed that recombinant extracellular region of CsIL-10R1 (rCsIL-10R1ex) rather than rCsIL-10R2ex could bind with rCsIL-10. Cellular study showed that both CsIL-10R1 and CsIL-10R2 were expressed on peripheral blood leukocytes (PBLs), and blockade of CsIL-10R1 or CsIL-10R2 by antibody could reduce inhibitory effect of CsIL-10 on ROS production of PBLs. When injected in vivo, anti-rCsIL-10R1 or anti-rCsIL-10R2 antibody dramatically promoted the expression of proinflammatory cytokines and suppressed bacterial dissemination in tongue sole tissues. Consistently, the overexpression of CsIL-10R1 or CsIL-10R2 significantly enhanced bacterial dissemination, and the overexpression of CsIL-10R1M bearing STAT3 site mutation reduced bacterial dissemination. Overall, these results demonstrate for the first time teleost IL-10 receptors play a negative role in antibacterial immunity and add insight into the function of CsIL-10 receptors.

Cryo-EM helps engineer enhanced IL-10 Interleukin-10 (IL-10) binds to the IL-10 receptor (IL-10R), comprising two high-affinity α chains and two low-affinity β chains. Depending on the cellular context of its recognition, IL-10 can either suppress or activate immune responses. This pleiotropic behavior has complicated efforts to use IL-10 as an anti-inflammatory agent. Saxton et al. generated a high-affinity version of IL-10 (super-10), which allowed them to visualize IL-10 in complex with both IL-10Rα and IL-10Rβ by cryo–electron microscopy (cryo-EM). This enabled them to engineer additional variants of IL-10 with variable IL-10Rβ–binding affinities. Some of the partial agonists exhibited biased actions on myeloid cells, which exhibited anti-inflammatory properties without concomitant CD8 T cell activation. These findings may serve as a blueprint for future enhanced cytokine–based therapeutics. Science , this issue p. eabc8433

Induced pluripotent stem cell (iPSC) line (SDQLCHi012-A) was generated from peripheral blood mononuclear cells of an 11-month-old male who was diagnosed as inflammatory bowel disease-28 caused by compound heterozygote for IL10RA mutations (c.188 + 1G > A and c.301C > T). Non-integrating episomal vectors coding OCT4, SOX2, KLF4, BCL-XL and MYC were used for reprogramming. The established iPSC line contained the same mutations identified in the patient, showed a normal karyotype, differentiation potential in vitro and expressed pluripotency markers.

Cytokines and lipid mediators are key regulators of inflammation; but how they are mechanistically linked is poorly understood. Here, Mukhopadhyay et al. show a novel regulation between cytokine IL-10 and lipid mediator PGE2 that functionally connects them to intestinal inflammation., Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB−/− iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB−/− Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB−/− Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.

Rationale: Infantile inflammatory bowel disease (IBD) is an extremely rare subgroup of IBD that includes patients whose age of onset is younger than 2 years old. These patients can have more surgical interventions, and a severe and refractory disease course with higher rates of conventional treatment failure. Monogenic defects play an important role in this subgroup of IBD, and identification of the underlying defect can guide the therapeutic approach. Patient concerns: In 2007, a 4-month-old girl from a nonconsanguineous family presenting with anal fistula, chronic diarrhea, and failure to thrive. She underwent multiple surgical repairs but continued to have persistent colitis and perianal fistulas. Diagnosis: Crohn's disease was confirmed by endoscopic and histologic finding. Intervention: Conventional pediatric IBD therapy including multiple surgical interventions and antitumor necrosis factor alpha agents were applied. Outcomes: The patient did not respond to conventional pediatric IBD therapy. Interleukin-10 (IL-10) receptor mutation was discovered by whole-exome sequencing and defective IL-10 signaling was proved by functional test of IL-10 signaling pathway by the age of 12. The patient is currently awaiting hematopoietic stem cell transplantation. Lessons: Early detection of underlying genetic causes of patients with infantile-IBD is crucial, since it may prevent patients from undergoing unnecessary surgeries and adverse effects from ineffective medical therapies. Moreover, infantile-IBD patients with complex perianal disease, intractable early onset enterocolitis and extraintestinal manifestations including oral ulcers and skin folliculitis, should undergo genetic and functional testing for IL-10 pathway defect.

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of proinflammatory mediators. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IECs), where it is associated with development and maintenance of barrier function. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in interferon-γ (IFNγ)-dominant murine colitis. In parallel, we demonstrated that IFNγ induction of indoleamine 2,3-dioxygenase 1, an enzyme that catalyzes the conversion of Trp to kynurenine (Kyn), induces IL-10R1 expression. Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed a functional aryl hydrocarbon response element, which is induced by Kyn in luciferase-based IL-10R1 promoter assays. Additionally, this analysis confirmed that IL-10R1 protein levels were increased in response to Kyn in IEC in vitro. Studies using in vitro wounding assays revealed that Kyn accelerates IL-10-dependent wound closure. Finally, reduction of murine dextran sodium sulfate colitis through Kyn administration correlates with colonic IL-10R1 expression. Taken together, these results provide evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon.

textabstractOBJECTIVE:: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in “classical” IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS:: Biomaterial was collected from the IL10RA-deficient patient, pediatric IBD patients and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS:: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of TNFα compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T cell cytokines IFNγ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient decreased peripheral blood mononuclear cell-derived TNFα production. CONCLUSIONS:: With time and during immunosuppressive treatment the IL10RA- deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing IFNγ and IL-17-mediated T-cell responses.

Interleukin-10 (IL10) signaling plays an important role in the pathogenesis of very early onset inflammatory bowel disease (VEO-IBD) in children. However, little is known about the role of the IL10 axis in children with VEO-IBD in China. The Chinese VEO-IBD Collaboration Group was created to collect clinical and genetic data from patients deficient in IL10 and the IL10 receptor. High-throughput sequencing was performed to identify mutations in these genes. We identified 32 compound heterozygous mutations and 9 homozygous mutations in IL10 receptor subunit alpha and 1 homozygous mutation in IL10 receptor subunit beta. Among these mutations, 10 novel mutations were identified, and 6 pathogenic mutations had been previously described. In patients with IL10 receptor subunit alpha mutations, c.301C>T (p.R101RW) and c.537 G>A (p.T179T) were the most common mutations. For 88.1% of the patients, the initial symptom was diarrhea, with a time of onset of 10.4 ± 8.0 days. Oral ulcers were the first symptom in 23.8% of the patients, with a time of onset of 9.7 ± 2.8 days. Extraintestinal manifestations included perianal abscesses (22/42), perianal fistulas (23/42), oral ulcers (20/42), and recurrent eczema (15/42). Twelve patients underwent enterostomy. These patients also had lower average Z scores in height-for-age and weight-for-age. Various treatment strategies were used, including fecal microbiota transplantation; however, only hematopoietic stem cell transplantation was efficacious. This study identified genotypes and phenotypes of Chinese VEO-IBD infants with IL10 receptor mutations. Our study expands the current knowledge on the involvement of the IL10 axis in patients with VEO-IBD.

Background & aim: Comparing to adult inflammatory bowel disease (IBD), those with early onset manifestations have different features in terms of the underlying molecular pathology, the course of disease and the response to therapy. We investigated the IL-10 signaling pathway previously reported as an important cause of infantile (Very Early Onset) IBD to find any possible variants. Method: With the next generation sequencing technique we screened IL-10, IL-10RA and IL10RB genes of 15 children affected by very early onset-GI (gastrointestinal) disorders. Additionally, we analyzed them based on Thermo Fisher immune deficiency panel for genes either having a known role in IBD pathogenesis or cause the disorders with overlapping manifestations. We performed multiple functional analyses only for the cases showing variants in IL-10-related genes. Result: In 3 out of 15 patients we identified variants including a homozygous and heterozygote mutations in IL-10RA and a novel homozygous mutation in IL-12RB1. Our functional studies reveal that in contrast to the IL-10RA heterozygote mutation that does not have deleterious effects, the homozygous mutation abrogates the IL-10 signaling pathway. Conclusion: Our study suggests we need to modify the classical diagnostic approach from functional assays followed by candidate-gene or genes sequencing to the firstly parallel genomic screening followed by functional studies. (C) 2017 Elsevier Masson SAS. All rights reserved

Erythema nodosum leprosum (ENL) is an inflammatory complication in leprosy. Yet, the involvement of ENL neutrophils in the inflammatory response against Mycobacterium leprae remains poorly explored. Our primary aim was to investigate the utility of the surface expression of neutrophil IL-10R1 as an ENL biomarker and, secondarily, to evaluate whether leprosy or healthy M. leprae–stimulated neutrophils produce cytokines and are able to respond to IL-10. We, in this study, describe a subpopulation of circulating neutrophils of ENL patients that exclusively expressed IL-10R1, providing evidence that IL-10R1+ neutrophils are present in ENL lesions. It was also found that ENL neutrophils, but not those of nonreactional leprosy controls, were able to secret detectable levels of TNF ex vivo and the addition of IL-10 blocked TNF release. It was likewise observed that M. leprae–stimulated, healthy neutrophils expressed IL-10R1 in vitro, and ENL-linked cytokines were released by M. leprae–cultured neutrophils in vitro. Moreover, consistent with the presence of a fully functional IL-10R, the addition of IL-10 prevented the release of M. leprae–induced cytokines. Most importantly, dead M. leprae revealed its superior capacity to induce CCL4 and IL-8 in primary neutrophils over live Mycobacterium, suggesting that M. leprae may hamper the inflammatory machinery as an immune escape mechanism.

Immune cells are vital constituents of the adipose microenvironment that influence both local and systemic lipid metabolism. Mice lacking IL10 have enhanced thermogenesis, but the roles of specific cell types in the metabolic response to IL10 remain to be defined. We demonstrate here that selective loss of IL10 receptor α in adipocytes recapitulates the beneficial effects of global IL10 deletion, and that local crosstalk between IL10-producing immune cells and adipocytes is a determinant of thermogenesis and systemic energy balance. Single Nuclei Adipocyte RNA-sequencing (SNAP-seq) of subcutaneous adipose tissue defined a metabolically-active mature adipocyte subtype characterized by robust expression of genes involved in thermogenesis whose transcriptome was selectively responsive to IL10Rα deletion. Furthermore, single-cell transcriptomic analysis of adipose stromal populations identified lymphocytes as a key source of IL10 production in response to thermogenic stimuli. These findings implicate adaptive immune cell-adipocyte communication in the maintenance of adipose subtype identity and function.

Summary Background Immune response failure against hepatitis C virus (HCV) has been associated with an increased regulatory T cell (Treg) activity. After liver transplantation (LT), 80% of patients experience an accelerated progression of hepatitis C recurrence. The aim of this work was to assess the involvement of Tregs, T helper (Th) 1, 2 and 17 cells in recurrent hepatitis C. Methods Peripheral blood cells obtained before and one month after LT from 22 recipients were analysed. Forty-four key molecules related to Treg, Th1, 2 and 17 responses, were evaluated using qRT-PCR. Liver recipients were classified in two groups according to graft fibrosis evaluated by the METAVIR score on the biopsy performed one year after LT (mild: F ≤ 1, n = 13; severe: F > 1, n = 9). Patients developing a severe recurrence were compared with patients with a mild recurrence. Results mRNA levels of Treg markers obtained one month after LT were significantly increased in patients with a severe disease course when compared to patients with a mild recurrence. Markers of the Th1 response were elevated in the same group. No differences in the markers determined before LT were observed. Conclusion These findings suggest that Treg, induced by a multifactorial process, which could include a strong Th1 response itself, may play a role in suppressing the early antiviral response, leading to a severe recurrence of hepatitis C.

This study aims to explore the potential functions of miR-137-5p and interleukin-10R1 (IL-10R1) in mediating the immune inflammation after spinal cord injury (SCI).Firstly, primary microglia were isolated from the spinal cord of newborn rats. Expression levels of miR-137-5p and IL-10R1 in LPS-induced microglia were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). In addition, mRNA expressions of Janus kinase (Jak1) and signal transducer and activator of transcription 3 (STAT3) were also examined by qRT-PCR. SCI model in rats was established and randomly assigned to three different groups: Sham group, SCI group and miR-137-5p mimic group. Within one week of spinal injury, relative levels of miR-137-5p and IL-10R1 in rats of different groups were detected by qRT-PCR. The mRNA levels of JAK1, tyrosine kinase (Tyk2) and STAT3 in rats were also measured. Moreover, protein expression of IL-1β, TNF-α and IL-6 in rats was measured by Western blotting. Finally, the improvement of locomotor function in three groups of rats within 4 weeks via BBB rating scale.Transfection of miR-137-5p mimics upregulated relative levels of IL-10R1, JAK1 and STAT3 in in vitro cultured microglia. Similarly, IL-10R1/JAK1/STAT3 pathway was activated in rats administrated with miR-137-5p mimics. Nevertheless, relative levels of classical inflammatory stimulators IL-1β, TNF-α and IL-6 were downregulated accordingly by miR-137-5p overexpression. Moreover, miR-137-5p effectively improved the locomotor function of rats after SCI.MiR-137-5p exerts an anti-inflammatory response by upregulating IL-10R1, thus improving locomotor function and alleviating spinal cord injury.

These experiments aimed to understand the relationship between interleukin 10 (IL10), the IL10 receptor subunits, and progesterone (P4) at the time of parturition. We hypothesized that there is a biologic connection between IL10 and P4, supporting an immunomodulatory mechanism for the onset of labor. Using samples from control and P4-treated pregnant mice, we assessed the production of IL10 and its receptor subunits (IL10Rα and IL10Rβ) in gestational tissues. After preliminary studies, P4-treated pregnant mice were compared with controls to assess for differences in IL10 and IL10 receptor subunit expression throughout gestation. To investigate the contribution of the P4 receptor at the onset of labor, we performed timed studies on pregnant mice after treatment with RU486. Samples collected included placentas, placentation sites, and maternal livers. IL10, IL10Rα, and IL10Rβ levels were measured in homogenized tissue using ELISA assays; the cytokine results were normalized for homogenate protein concentration. Control mice delivered on gd 18-19, and P4 treatment prevented parturition to beyond gd 20, as expected. In treated mice, P4 not only prevented the anticipated nadir of IL10 at term, but maintained elevated levels of IL10 through gd 20 (p0.05). P4 also reversed the anticipated decrease of the IL10Rα, which was increased in P4-treated mice (p0.05). Treatment with RU486 did not modulate the expression of IL10 or IL10Rα, but showed a significant decrease in the level of IL10Rβ (p0.05). Progesterone functions at least in part through the IL10 signaling pathway to prolong gestation.

Objective Although IL-10 is generally considered as an anti-inflammatory cytokine, it was recently shown to have detrimental effects on insulin sensitivity and fat cell metabolism in rodents. Whether this also pertains to human white adipose tissue (hWAT) is unclear. We therefore determined the main cellular source and effects of IL-10 on human adipocytes and hWAT-resident immune cells and its link to insulin resistance. Methods Associations between hWAT IL-10 production and metabolic parameters were investigated in 216 participants with large interindividual variations in body mass index and insulin sensitivity. Adipose cells expressing or secreting IL-10 and the cognate IL-10 receptor α (IL10RA) were identified by flow cytometry sorting. Effects on adipogenesis, lipolysis, and inflammatory/metabolic gene expression were measured in two human primary adipocyte models. Secretion of inflammatory cytokines was investigated in cultures of IL-10–treated hWAT macrophages and leukocytes by Luminex analysis (Luminex Corp.). Results IL-10 gene expression and protein secretion in hWAT correlated positively with body mass index (BMI) and homeostasis model assessment-insulin resistance (HOMA-IR). Gene expression analyses in mature fat cells and flow cytometry–sorted hWAT-resident adipocyte progenitors, macrophages, and leukocytes demonstrated that the expression of IL-10 and the IL10RA were significantly enriched in proinflammatory M1 macrophages. In contrast to murine data, functional studies showed that recombinant IL-10 had no effect on adipocyte phenotype. In hWAT-derived macrophages and leukocytes, it induced an anti-inflammatory profile. Conclusion In hWAT, IL-10 is upregulated in proinflammatory macrophages of obese and insulin-resistant persons. However, in contrast to findings in mice, IL-10 does not directly affect human adipocyte function.

The current era of drug discovery has been marked by a significant increase in the development of immune modulating agents to address a range of diseases such as cancer, chronic inflammation, and other conditions of dysregulated immunity. Non-clinical evaluation of these agents in animal models can be challenging, as the presence of an active immune state is often required in order to detect the effects of the test agent. Modulation of interleukin (IL)-10 signaling represents this type of situation in that altering IL-10 action in vivo can be difficult to appreciate in the absence of an ongoing immune response. The study presented here reports on the use of lipopolysaccharide (LPS) challenge in cynomolgus macaques to induce predictable inflammatory cytokine responses. The results showed that IL-10 receptor (IL-10R) blockade with an antagonist monoclonal antibody (mAb) dramatically enhanced the LPS-induced cytokine response, thus demonstrating in vivo pharmacologic activity of this immunomodulatory antibody. We submit that this approach could be applied to other cases where the intent of a candidate therapeutic is to modulate components of inflammatory cytokine responses.

Pediatric inflammatory bowel disease (IBD) is a heterogeneous disorder caused by multiple factors. Although genetic and immunological analyses are required for a definitive diagnosis, no reports of a comprehensive genetic study of a Japanese population are available. In total, 35 Japanese patients

Immune-activated microglia from aged mice produce exaggerated levels of cytokines. Despite high levels of microglial interleukin (IL)-10 in the aged brain, neuroinflammation was prolonged and associated with depressive-like deficits. Because astrocytes respond to IL-10 and, in turn, attenuate microglial activation, we investigated if astrocyte-mediated resolution of microglial activation was impaired with age. Here, aged astrocytes had a dysfunctional profile with higher glial fibrillary acidic protein, lower glutamate transporter expression, and significant cytoskeletal re-arrangement. Moreover, aged astrocytes had reduced expression of growth factors and IL-10 receptor-1 (IL-10R1). After in vivo lipopolysaccharide immune challenge, aged astrocytes had a molecular signature associated with reduced responsiveness to IL-10. This IL-10 insensitivity of aged astrocytes resulted in a failure to induce IL-10R1 and transforming growth factor β and resolve microglial activation. In addition, adult astrocytes reduced microglial activation when co-cultured ex vivo, whereas aged astrocytes did not. Consistent with the aging studies, IL-10R(KO) astrocytes did not augment transforming growth factor β after immune challenge and failed to resolve microglial activation. Collectively, a major cytokine-regulatory loop between activated microglia and astrocytes is impaired in the aged brain.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4+ T cells differentially express IL-6 and IL-10 receptors in an activation state–dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6–mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10–generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

Background Very early-onset inflammatory bowel disease (VEO-IBD) is often associated with monogenetic disorders. IL-10RA deficiency is one of the major causal mutations in VEO-IBD. Here, we aimed to identify the causal mutation associated with severe IBD in a 1-year-old patient, validate the pathogenicity of the mutation, and characterize the mutant protein. Methods To identify the causal mutation, targeted exome sequencing (ES) was performed using the genomic DNA from the patient. To validate the pathogenicity, IL-10RA functional tests were performed using the patient's peripheral blood mononuclear cells (PBMCs). Additionally, flow cytometry analysis, confocal microscopy on overexpressed green fluorescent protein-fused mutants, and computational analysis on the structures of IL-10RA proteins were performed. Results We identified a novel compound heterozygote mutation p.[Tyr91Cys];[Pro146Alafs*40] in the IL10RA gene of the patient. The missense variant p.Tyr91Cys was previously identified but not functionally tested, and a frameshift variant, p.Pro146Alafs*40, is novel and nonfunctional. PBMCs from the patient showed defective signal transducer and activator of transcription 3 activation. The p.Tyr91Cys mutant protein failed to properly localize on the plasma membrane. The p.Tyr91Cys mutation seems to disrupt the hydrophobic core structure surrounding the tyrosine 91 residue, causing structural instability. Conclusions Targeted ES and linkage analysis identified novel compound heterozygous mutations p.[Tyr91Cys];[Pro146Alafs*40] in the IL10RA gene of a child with severe VEO-IBD. p.Tyr91Cys proteins were functionally defective in IL-10RA signaling and failed to properly localize on the plasma membrane, probably due to its structural instability.

High-Throughput (HT) SELEX combines SELEX (Systematic Evolution of Ligands by EXponential Enrichment), a method for aptamer discovery, with massively parallel sequencing technologies. This emerging technology provides data for a global analysis of the selection process and for simultaneous discovery of a large number of candidates but currently lacks dedicated computational approaches for their analysis. To close this gap, we developed novel in-silico methods to analyze HT-SELEX data and utilized them to study the emergence of polymerase errors during HT-SELEX. Rather than considering these errors as a nuisance, we demonstrated their utility for guiding aptamer discovery. Our approach builds on two main advancements in aptamer analysis: AptaMut-a novel technique allowing for the identification of polymerase errors conferring an improved binding affinity relative to the 'parent' sequence and AptaCluster-an aptamer clustering algorithm which is to our best knowledge, the only currently available tool capable of efficiently clustering entire aptamer pools. We applied these methods to an HT-SELEX experiment developing aptamers against Interleukin 10 receptor alpha chain (IL-10RA) and experimentally confirmed our predictions thus validating our computational methods.

In the present study, we studied a cohort of patients with very early onset inflammatory bowel disease (IBD) to determine the frequency of mutations in the interleukin 10 (IL10) receptor genes as a cause of early-onset IBD.Sanger sequencing was performed to determine the presence of IL10 and/or IL10 receptor mutations in 17 patients with a diagnosis of very early onset IBD (disease onset2 years of age in 15 patients, between 3 and 4 years in the other 2). Mutation screening was performed including all of the coding regions of the IL10, IL10RA, and IL10RB genes. We then compared the follow-up findings of the patients with IL10 receptor mutations in terms of demographic, clinical, laboratory, and treatment response properties with those of patients diagnosed as having very early onset IBD with no mutation.We identified 3 patients bearing mutations in the IL10 or IL10 receptor genes, including 1 mutation in IL10RB that has been described recently (c.G477A, p.Trp159*) and 2 novel mutations affecting the IL10RA gene (c.T192G, p.Tyr64 and c.T133G, p.Trp45Gly). Collectively, these mutations thus provided genetic etiology for 17.6% of the cohort under investigation. The presence of a family history of IBD and the clinical course of Crohn disease differed between patients with mutations in the IL-10 pathway and those without such mutations. Although perianal fistulas were found in all of the patients with IL10 receptor mutations, they were found in only 14.3% of those without such mutations. The lower values of weight-for-age and height-for-age z scores, necessity for more intensive therapy, achievement of longer periods until remission, and frequent relapses in the patients bearing mutations in the IL10 receptor genes all underlined the severity of the disease and its relatively poor response to treatment.In spite of the small number of patients with mutations affecting the IL-10 signaling pathway in our study, in all of the patients with IL10 receptor mutations, the disease onset occurs at an early age, the prognosis is poor, and the response to treatment is insufficient.

Background and aims: Defects in the interleukin 10 (IL-10) signalling pathway have been shown to cause very early onset inflammatory bowel disease (IBD). We report a patient with severe infantile-onset IBD with a compound heterozygous IL-10 receptor alpha subunit (IL-10RA) mutation, one of which was paternally-inherited and the other occurring de novo . Methods: Deep sequencing of IL-10, IL-10RA and IL-10 receptor beta subunit (IL-10RB) were performed. Peripheral blood mononuclear cell (PBMC) surface expression of IL-10RA was analysed by flow cytometry. IL-10 signalling pathway was examined by measuring phosphorylated STAT3 in PBMC cultured in the presence of IL-6 or IL-10. Result: We identified a missense mutation in exon 4 of IL-10RA (c.583T>C) in one allele and a nonsense mutation in exon 7 of IL-10RA (c.1368G>T) in the other allele. Neither mutation has been reported previously. The patient has functional IL-10RA deficiency despite normal IL-10RA expression. Conclusion: This represents the first case report of a de novo mutation of IL-10RA that is associated with very early onset severe IBD. Therefore, IL-10 pathway defect should be considered in patients with infantile-onset IBD even if the parents are non-consanguineous.

Background Th17 cells are central pathogenic mediators of autoimmune disease, including many forms of GN. IL-10 receptor signaling (IL-10R) in regulatory T cells (Tregs) has been implicated in the downregulation of Th17 cells, but the underlying molecular mechanisms and functional relevance of this process remain unclear. Methods We generated mice with Treg-specific IL-10Ra deficiency and subjected these mice to nephrotoxic serum–induced nephritis as a model of crescentic GN. Immune responses and Treg phenotypes were extensively analyzed. Results Compared with controls, mice with IL-10Ra(−/−) Tregs showed a spontaneously overshooting Th17 immune response. This hyper-Th17 phenotype was further boosted during GN and associated with aggravated renal injury. Notably, abrogation of IL-10Ra signaling in Tregs increased dendritic cell activation and production of Th17-inducing cytokines. In contrast, Treg trafficking and expression of chemokine receptor CCR6 remained unaffected, indicating mechanisms of Th17 control, differing from those of previously identified CCR6(+) Treg17 cells. Indeed, the capacity for direct in vitro suppression of Th17 responses by IL-10Ra(−/−) Tregs was significantly impaired. As underlying pathology, analyses conducted in vitro and in vivo using double-fluorescent reporter mice revealed strikingly decreased IL-10 production by IL-10Ra(−/−) Tregs. To assess, whether reduced IL-10 could explain the hyper Th17 phenotype, competitive cotransfer experiments were performed. Supporting our concept, IL-10Ra(−/−) T cells differentiated into Th17 cells at much higher frequencies than wild type T cells did during GN. Conclusions IL-10R engagement optimizes Treg-mediated suppression of Th17 immunity. We hypothesize a feed-forward loop, in which IL-10Ra signaling reinforces IL-10 secretion by Tregs which potently controls Th17 development via direct and indirect mechanisms. IL-10R thus may be a promising therapeutic target for the treatment of GN.

Delayed-onset muscle soreness is typically observed after strenuous or unaccustomed eccentric exercise. Soon after recovery, blunted muscle soreness is observed on repeated eccentric exercise, a phenomenon known as repeated bout effect (RBE). Although regular physical activity decreases muscle hyperalgesia, likely because of increased production of the anti-inflammatory cytokine interleukin-10 (IL-10) in the skeletal muscle, whether IL-10 also contributes to the antinociceptive effect of RBE is unknown. Furthermore, whether IL-10 attenuates muscle hyperalgesia by acting on muscle nociceptors remains to be established. Here, we explored the hypothesis that blunted muscle nociception observed in RBE depends on a local effect of IL-10, acting on IL-10 receptor 1 (IL-10R1) expressed by muscle nociceptors. Results show that after a second bout of eccentric exercise, rats exhibited decreased muscle hyperalgesia, indicative of RBE, and increased expression of IL-10 in the exercised gastrocnemius muscle. Although knockdown of IL-10R1 protein in nociceptors innervating the gastrocnemius muscle by intrathecal antisense oligodeoxynucleotide did not change nociceptive threshold in naive rats, it unveiled latent muscle hyperalgesia in rats submitted to eccentric exercise 12 days ago. Furthermore, antisense also prevented the reduction of muscle hyperalgesia observed after a second bout of eccentric exercise. These data indicate that recovery of nociceptive threshold after eccentric exercise and RBE-induced analgesia depend on a local effect of IL-10, acting on its canonical receptor in muscle nociceptors.

BACKGROUND/AIM One of the current hypotheses assumes that brain tumors exert an immunosuppressive influence on the surrounding cellular environment. Interleukin-10 (IL-10) is one of the immunosuppressive cytokines modifying the biological activity of cancer. The aim of this study was to assess the expression of IL10R in CD45+ cells within primary brain tumors and metastases and establish its association with tumor basic immunophenotype. PATIENTS AND METHODS Tissue samples were obtained intraoperatively during surgeries of 32 patients suffering from meningiomas (n=9), gliomas (n=12) and metastatic tumors (n=11). Expression was assessed with flow cytometry and immunohistochemical reactions. RESULTS Expression of IL10R subunits within the leukocyte population (CD45+ cells) was significantly higher in primary tumors than in metastases. CONCLUSION To the best of our knowledge, this is the first study describing a correlation between the IL10R expression on leukocytes and histological types of brain tumors.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage–Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections., In vitro models to study the role of host genetics in the response to chlamydial infection are limited. Here, Yeung et al. show that macrophages derived from human induced pluripotent stem cells (which can be genetically manipulated) support chlamydial infection and can be used for this purpose.

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from non-autoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages. Impaired apoptotic cell uptake by macrophages also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO, do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal macrophages (LPM). The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes, whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic macrophages resembling small peritoneal macrophages (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco neutralizing antibodies and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ macrophages. CD138+ macrophages expressed IL-10 receptor, CD206, and CCR2 but little TNFα or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated macrophages. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by lipopolysaccharide. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum, and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.

Bone marrow mesenchymal stromal cells (BM-MSCs) display immunomodulatory features, representing a promising tool for cell-based therapies. However, the mechanisms used by MSCs to regulate T cell fate remain unclear.We investigated the potential of BM-MSCs to modulate T cell activation, proliferation, cytokine secretion and immunophenotype.T cells were co-cultured with BM-MSCs to assess their immunomodulatory impact. T cell characterization was performed using cell tracing, ELISA, intracellular and surface staining, flow cytometry analysis and qPCR.The activation and proliferation of T cells were downregulated during coculture with BM-MSCs. We also observed that BM-MSCs upregulated IL-10 secretion as well as the expression of its receptor CD210 on T cells, thus creating a loop favoring the expansion of IL-10-producing T cells. IL-10 neutralization restored T cell proliferation, demonstrating that IL-10 is functionally relevant during immunomodulation. Moreover, BM-MSCs differently modulated CD4 and CD8 T-cell immunophenotype by inducing broad changes in their molecular pattern.We provide a comprehensive functional and molecular characterization of T cells that are immunomodulated by BM-MSCs. Indeed, a better understanding of the immunological interplay between T cells and MSCs will facilitate the development of new efficient approaches to improve cell-based immune therapies.

Adipocytes are one of the primary sources of inflammatory cytokines that drive the low-grade inflammation associated with obesity and obesity-related diseases. Stearoyl-CoA desaturase, a key adipogenic enzyme in rodents and humans, plays significant role in the regulation of adipocyte inflammation via a mechanism that involves the regulation of inflammatory gene expression. In the present study, we tested the hypothesis that the stearoyl-CoA desaturase 1-related regulation of gene expression might be driven by changes in DNA methylation. We showed that stearoyl-CoA desaturase 1 overexpression causes the global hypomethylation of DNA, even as early as 12h after the induction of differentiation, with the greatest difference seen in mature adipocytes. In contrast, both the silencing of stearoyl-CoA desaturase 1 gene expression by siRNA and inhibition of stearoyl-CoA desaturase 1 activity resulted in DNA hypermethylation in 3T3-L1 adipocytes. The analysis of the promoter methylation of 22 genes that are related to the inflammatory response showed that the level of methylation of CpG sites in interleukin-10 receptor a, interleukin-4 receptor a, interleukin-6 signal transducer, and transforming growth factor β 1 promoters was strongly related to stearoyl-CoA desaturase 1 expression or activity. The changes in methylation at CpG promoter sites correlated with differential expression of the aforementioned genes. The results show that stearoyl-CoA desaturase 1 regulates the level of DNA methylation in adipocytes and suggest that the mechanism by which stearoyl-CoA desaturase 1 affects adipocyte inflammation may involve changes in the methylation of inflammatory genes.

Summary Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3′ splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient’s cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1–null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

Primary immunodeficiencies (PIDs) represent a large group of disorders with an increased susceptibility to infections. Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiencies (PIDs) with marked T-cell lymphopenia. Investigation of the genetic aetiology using classical Sanger sequencing is associated with considerable diagnostic delay. We here established a custom-designed, next-generation sequencing (NGS)-based panel to efficiently identify disease-causing genetic defects in PID patients and applied this method in SCID patients of Turkish origin with previously undefined genetic aetiology. We used HaloPlex enrichment technology, a targeted, NGS-based method which was designed to diagnose patients with SCID and other PIDs. Our HaloPlex panel included a total of 356 PID-related genes, and we searched disease-causing mutations in 19 Turkish SCID patients without a genetic diagnosis. The coverage of targeted regions ranged from 97.47% to 99.62% with an average of 98.31% for all patients. All known SCID genes were covered with a percentage of at least 97.3%. We made a genetic diagnosis in six of 19 (33%) patients, including four novel disease-causing mutations identified in RAG1, JAK3 and IL2RG, respectively. We showed that this NGS-based method can provide rapid genetic diagnosis for patients suffering from SCID, potentially facilitating clinical treatment decisions.