Mechanism and Effect of HNF4α Decrease in a Rat Model of Cirrhosis and Liver Failure.

BACKGROUND & AIMS: HNF4α, a master regulator of liver development and the mature hepatocyte phenotype, is down-regulated in chronic and inflammatory liver disease. We used contemporary transcriptomics and epigenomics to study the cause and effects of this down-regulation and characterized a multicellular etiology. METHODS: Progressive changes in the rat carbon tetrachloride model were studied by deep RNA sequencing and genome-wide chromatin immunoprecipitation sequencing analysis of transcription factor (TF) binding and chromatin modification. Studies compared decompensated cirrhosis with liver failure after 26 weeks of treatment with earlier compensated cirrhosis and with additional rat models of chronic fibrosis. Finally, to resolve cell-specific responses and intercellular signaling, we compared transcriptomes of liver, nonparenchymal, and inflammatory cells. RESULTS: HNF4α was significantly lower in 26-week cirrhosis, part of a general reduction of TFs that regulate metabolism. Nevertheless, increased binding of HNF4α contributed to strong activation of major phenotypic genes, whereas reduced binding to other genes had a moderate phenotypic effect. Decreased Hnf4a expression was the combined effect of STAT3 and nuclear factor kappa B (NFκB) activation, which similarly reduced expression of other metabolic TFs. STAT/NFκB also induced de novo expression of Osmr by hepatocytes to complement induced expression of Osm by nonparenchymal cells. CONCLUSIONS: Liver decompensation by inflammatory STAT3 and NFκB signaling was not a direct consequence of progressive cirrhosis. Despite significant reduction of Hnf4a expression, residual levels of this abundant TF still stimulated strong new gene expression. Reduction of HNF4α was part of a broad hepatocyte transcriptional response to inflammation.

Blockade of OSMRß signaling ameliorates skin lesions in a mouse model of human atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe pruritus and eczematous skin lesions. Although IL-31, a type 2 helper T (Th2)-derived cytokine, is important to the development of pruritus and skin lesions in AD, the blockade of IL-31 signaling does not improve the skin lesions in AD. Oncostatin M (OSM), a member of IL-6 family of cytokines, plays important roles in the regulation of various inflammatory responses through OSM receptor ß subunit (OSMRß), a common receptor subunit for OSM and IL-31. However, the effects of OSM on the pathogenesis of AD remain to be elucidated. When AD model mice were treated with OSM, skin lesions were exacerbated and IL-4 production was increased in the lymph nodes. Next, we investigated the effects of the monoclonal antibody (mAb) against OSMRß on the pathogenesis of AD. Treatment with the anti-OSMRß mAb (7D2) reduced skin severity score in AD model mice. In addition to skin lesions, scratching behavior was decreased by 7D2 mAb with the reduction in the number of OSMRß-positive neurons in the dorsal root ganglia of AD model mice. 7D2 mAb also reduced the serum concentration of IL-4, IL-13, and IgE as well as the gene expressions of IL-4 and IL-13 in the lymph nodes of AD model mice. Blockade of both IL-31 and OSM signaling is suggested to suppress both pruritus and Th2 responses, resulting in the improvement of skin lesions in AD. The anti-OSMRß mAb may be a new therapeutic candidate for the treatment of AD.

The clinical relevance of OSM in inflammatory diseases: a comprehensive review.

Oncostatin M (OSM) is a pleiotropic cytokine involved in a variety of inflammatory responses such as wound healing, liver regeneration, and bone remodeling. As a member of the interleukin-6 (IL-6) family of cytokines, OSM binds the shared receptor gp130, recruits either OSMRß or LIFRß, and activates a variety of signaling pathways including the JAK/STAT, MAPK, JNK, and PI3K/AKT pathways. Since its discovery in 1986, OSM has been identified as a significant contributor to a multitude of inflammatory diseases, including arthritis, inflammatory bowel disease, lung and skin disease, cardiovascular disease, and most recently, COVID-19. Additionally, OSM has also been extensively studied in the context of several cancer types including breast, cervical, ovarian, testicular, colon and gastrointestinal, brain,lung, skin, as well as other cancers. While OSM has been recognized as a significant contributor for each of these diseases, and studies have shown OSM inhibition is effective at treating or reducing symptoms, very few therapeutics have succeeded into clinical trials, and none have yet been approved by the FDA for treatment. In this review, we outline the role OSM plays in a variety of inflammatory diseases, including cancer, and outline the previous and current strategies for developing an inhibitor for OSM signaling.

Canine interleukin-31 binds directly to OSMRß with higher binding affinity than to IL-31RA.

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRß). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRß, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRß. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRß. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRß which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRß than to IL-31RA. Additionally, we demonstrated that D1-D4, especially D4 of cOSMRß, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRß with a KD value of 3.59 × 10-8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.

Cloning, expression, purification, and immunoblotting analysis of recombinant type III fibronectin domains of human oncostatin M receptor.

BACKGROUND: The human oncostatin M receptor subunit , commonly known as the oncostatin M receptor (OSMR), is a cell surface protein and belongs to the family of type I cytokine receptors. It is highly expressed in several cancers and is a potential therapeutic target. Structurally, OSMR consists of three major domains: the extracellular, transmembrane, and cytoplasmic domains. The extracellular domain further comprises four Type III fibronectin subdomains. The functional relevance of these type III fibronectin domains is not known yet, and it is of great interest to us to understand their role in OSMR-mediated interactions with other oncogenic proteins. METHODS & RESULTS: The four type III fibronectin domains of hOSMR were amplified by PCR using the pUNO1-hOSMR construct as a template. The molecular size of the amplified products was confirmed by agarose gel electrophoresis. The amplicons were then cloned into a pGEX4T3 vector containing GST as an N-terminal tag. Positive clones with domain inserts were identified by restriction digestion and overexpressed in E. coli Rosetta (DE3) cells. The optimum conditions for overexpression were found to be 1 mM IPTG and an incubation temperature of 37 °C. The overexpression of the fibronectin domains was confirmed by SDS-PAGE, and they are affinity purified by using glutathione agarose beads in three repetitive steps. The purity of the isolated domains analyzed by SDS-PAGE and western blotting showed that they were exactly at their corresponding molecular weights as a single distinct band. CONCLUSION: In this study, we have successfully cloned, expressed, and purified four Type III fibronectin subdomains of hOSMR.

Efficacy and safety of vixarelimab, a human monoclonal oncostatin M receptor ß antibody, in moderate-to-severe prurigo nodularis: a randomised, double-blind, placebo-controlled, phase 2a study.

Background: Prurigo nodularis is a chronic skin disease characterised by intensely pruritic, hyperkeratotic nodules. Vixarelimab, a human monoclonal antibody, binds to the beta subunit of the oncostatin M receptor, inhibiting signalling of both interleukin 31 and oncostatin M, two cytokine pathways that contribute to pruritus and nodule formation in prurigo nodularis. Methods: This double-blind, placebo-controlled, phase 2a trial was done at both private and academic dermatology outpatient research clinics across the United States and Canada (n = 40). Patient eligibility criteria included age 18-75 years, physician-documented diagnosis of prurigo nodularis minimum 6 months duration of prurigo nodularis, presence of at least 10 pruritic nodules approximately 0.5-2 cm in size on at least two different anatomical locations (excluding face and scalp) and involving the extremities, and presence of normal-appearing skin between nodules; atopic dermatitis as a comorbidity was exclusionary. Patients were required to have moderate-to-severe pruritus, defined as Worst Itch-Numeric Rating Scale (WI-NRS) score ≥7 at screening and LS-mean weekly WI-NRS score ≥5 for each of the 2 consecutive weeks immediately before randomisation. Participants were randomly assigned (1:1) to receive weekly subcutaneous vixarelimab 360 mg (720 mg loading dose) or placebo using stratification factors (sex and presence of atopy) and block size 4 through the IWRS system. Stratification by atopy status was based on the reported high prevalence of atopy in this population and the potential impact of atopy in the immunopathologic process in prurigo nodularis. Patients, investigators, study sponsor, and site staff were masked to study treatment. The primary efficacy endpoint was least squares (LS)-mean percent change from baseline (PCFB) at Week 8 in weekly average Worst Itch-Numeric Rating Scale (WI-NRS) score. The primary efficacy endpoint was analysed with ANCOVA including treatment as fixed effect, with baseline WI-NRS, and randomisation stratification factor as covariates. All randomised patients who had at least 1 dose of study drug or placebo were included in the Safety Analysis Set. This trial is registered at ClinicalTrials.gov, NCT03816891. Findings: Of 50 patients randomised between March 11, 2019 and January 31, 2020, 23 vixarelimab recipients and 26 placebo recipients comprised the modified intent-to-treat analysis population (baseline LS-mean [SD] WI-NRS score, 8.3 [1.05]). Outcomes at Week 8 for vixarelimab versus placebo included LS-mean PCFB in WI-NRS score, -50.6% versus -29.4% (LS-mean difference [95% CI], -21.2% [-40.82, -1.60]; p = 0.03); ≥4-point reduction in WI-NRS score, 52.2% (12/23) versus 30.8% (8/26) (p = 0.11); PN-IGA score of 0 or 1, 30.4% (7/23) versus 7.7% (2/26) (p = 0.03); LS-mean PCFB in pruritus VAS score, -54.4% versus -32.6% (p = 0.03); and LS-mean PCFB sleep loss reduction (improvement), -56.3% versus -30.0% (p = 0.02). No deaths, serious TEAEs, or TEAEs leading to dose interruption were reported. The percentage of vixarelimab recipients reporting any TEAE was 91.3% (21/23) versus 76.9% (20/26) of placebo recipients; drug-related TEAEs generally were similar between the two groups (vixarelimab, 43.5% [10/23]; placebo, 38.5% [10/26]). Interpretation: Vixarelimab demonstrated rapid reduction of pruritus and achievement of clear/almost clear skin in one-third of the patients by Week 8. Relief of itch and clearing of skin nodules represent two important potential therapeutic advances in the management of patients suffering from the debilitating disease Prurigo Nodularis. Funding: Kiniksa Pharmaceuticals, Ltd.

Development and validation of an inflammatory response-related prognostic model and immune infiltration analysis in glioblastoma.

Background: Despite receiving standard treatment, the prognosis of glioblastoma (GBM) patients is still poor. Considering the heterogeneity of each patient, it is imperative to identify reliable risk model that can effectively predict the prognosis of each GBM patient to guide the personalized treatment. Methods: Transcriptomic gene expression profiles and corresponding clinical data of GBM patients were downloaded from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Inflammatory response-related genes were extracted from Gene Set Enrichment Analysis (GSEA) website. Univariate Cox regression analysis was used for prognosis-related inflammatory genes (P<0.05). A polygenic prognostic risk model was constructed using least absolute shrinkage and selection operator (LASSO) Cox regression analysis. Validation was performed through CGGA cohort. Overall survival (OS) was compared by Kaplan-Meier analysis. A nomogram was plotted to accurately predict the prognosis for each patient. GSEA was used for the pathway enrichment analysis. The single sample GSEA (ssGSEA) algorithm was implemented to conduct the immune infiltration analysis. The potential role of oncostatin M receptor (OSMR) in GBM was investigated through the in vitro experiment. Results: A prognostic risk model consisting of 4 genes (PTPRN, OSMR, MYD88, and EFEMP2) was developed. GBM patients in the high-risk group had worse OS. The time-dependent ROC curves showed an area under the curve (AUC) of 0.782, 0.765, and 0.784 for 1-, 2-, and 3-year survival in TCGA cohort, while the AUC in the CGGA cohort was 0.589, 0.684, and 0.785 at 1, 2, and 3 years, respectively. The risk score, primary-recurrent-secondary (PRS) type, and isocitrate dehydrogenase (IDH) mutation could predict the prognosis of GBM patients well. The nomogram accurately predicted the 1-, 2-, and 3-year OS for each patient. Immune cell infiltration was associated with the risk score and the model could predict immunotherapy responsiveness. The expression of the prognostic gene was correlated with the sensitivity to antitumor drugs. Interference of OSMR inhibited proliferation and migration and promoted apoptosis of GBM cells. Conclusions: The prognostic model based on 4 inflammatory response-related genes had reliable predictive power to effectively predict clinical outcome in GBM patients and provided the guide for the personalized treatment.

Nanoimmunosensor for the electrochemical detection of oncostatin M receptor and monoclonal autoantibodies in systemic sclerosis.

Systemic sclerosis (SSc) is a chronic, autoimmune disease that primarily affects connective tissue. SSc can be classified into limited cutaneous (lSSc) and diffuse cutaneous (dSSc). Oncostatin M receptor (sOSMR) is an important inflammatory biomarker expressed in the serum of patients with autoimmune diseases. A nanoengineered immunosensor surface was developed. The biosensor was composed of a conductive layer of polypyrrole, electrodeposited gold nanoparticles, and sOSMR protein for anti-human OSMR monoclonal antibody biorecognition. The electrochemical response evaluated by cyclic voltammetry and electrochemical impedance spectroscopy indicated the detection of the target analyte present in clinical samples from lSSc and dSSc patients. The voltammetric anodic shift for lSSc specimens was 82.7% ± 0.9-93.6% ± 3.2, and dSSc specimens was 118.7 ± 2.6 to 379.6 ± 2.6, revealing a differential diagnostic character for SSc subtypes. The sensor platform was adapted for identifying sOSMR, using anti-OSMR antibodies as bioreceptors. With a linear response range estimated from 0.005 to 500 pg mL-1 and a limit of detection of 0.42 pg mL-1, the sensing strategy demonstrated high sensitivity in identifying the human OSMR protein in clinical samples. The proposed biosensor is a promising and innovative tool for SSc-related biomarker research.

Oncostatin M receptor regulates osteoblast differentiation via extracellular signal-regulated kinase/autophagy signaling.

BACKGROUND: Oncostatin M receptor (OSMR), as one of the receptors for oncostatin M (OSM), has previously been shown to mediate the stimulatory role of OSM in osteoclastogenesis and bone resorption. However, it remains to be clarified whether and how OSMR affects the differentiation of osteoblasts. METHODS: The expression level of OSMR during osteoblast and adipocyte differentiation was examined. The role of OSMR in the differentiation was investigated using in vitro gain-of-function and loss-of-function experiments. The mechanisms by which OSMR regulates bone cell differentiation were explored. Finally, in vivo function of OSMR in cell fate determination and bone homeostasis was studied after transplantation of OSMR-silenced bone marrow stromal cells (BMSCs) to the marrow of ovariectomized mice. RESULTS: OSMR was regulated during osteogenic and adipogenic differentiation of marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. OSMR suppressed osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. Mechanistic investigations showed that OSMR inhibited extracellular signal-regulated kinase (ERK) and autophagy signaling. The downregulation of autophagy, which was mediated by ERK inhibition, suppressed osteogenic differentiation of progenitor cells. Additionally, inactivation of ERK/autophagy signaling attenuated the stimulation of osteogenic differentiation induced by Osmr siRNA. Furthermore, transplantation of BMSCs in which OSMR was silenced to the marrow of mice promoted osteoblast differentiation, attenuated fat accumulation and osteoclast differentiation, and thereby relieved the osteopenic phenotype in the ovariectomized mice. CONCLUSIONS: Our study has for the first time established the direct role of OSMR in regulating osteogenic differentiation of marrow stromal progenitor cells through ERK-mediated autophagy signaling. OSMR thus contributes to bone homeostasis through dual regulation of osteoblasts and osteoclasts. It also suggests that OSMR may be a potential target for the treatment of metabolic disorders such as osteoporosis.

Oncostatin M Induces Lipolysis and Suppresses Insulin Response in 3T3-L1 Adipocytes.

Oncostatin M (OSM) is an immune cell-derived cytokine that is upregulated in adipose tissue in obesity. Upon binding its receptor (OSMR), OSM induces the phosphorylation of the p66 subunit of Src homology 2 domain-containing transforming protein 1 (SHC1), called p66Shc, and activates the extracellular signal-related kinase (ERK) pathway. Mice with adipocyte-specific OSMR deletion (OsmrFKO) are insulin resistant and exhibit adipose tissue inflammation, suggesting that intact adipocyte OSM-OSMR signaling is necessary for maintaining adipose tissue health. How OSM affects specific adipocyte functions is still unclear. Here, we examined the effects of OSM on adipocyte lipolysis. We treated 3T3-L1 adipocytes with OSM, insulin, and/or inhibitors of SHC1 and ERK and measured glycerol release. We also measured phosphorylation of p66Shc, ERK, and insulin receptor substrate-1 (IRS1) and the expression of lipolysis-associated genes in OSM-exposed 3T3-L1 adipocytes and primary adipocytes from control and OsmrFKO mice. We found that OSM induces adipocyte lipolysis via a p66Shc-ERK pathway and inhibits the suppression of lipolysis by insulin. Further, OSM induces phosphorylation of inhibitory IRS1 residues. We conclude that OSM is a stimulator of lipolysis and inhibits adipocyte insulin response. Future studies will determine how these roles of OSM affect adipose tissue function in health and disease.

Development and characterization of a novel mouse anti-canine oncostatin M receptor beta monoclonal antibody.

Oncostatin M receptor beta (OSMRß) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRß might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRß (cOSMRß) was experimentally expressed as a native antigen to develop an effective cOSMRß-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRß expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRß with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRß (fOSMRß) and human OSMRß (hOSMRß). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623-734) of cOSMRß. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.

Interleukin-31 is overexpressed in skin and serum in cutaneous T-cell lymphomas but does not correlate to pruritus.

Introduction: Cutaneous T-cell lymphomas (CTCL) are malignant lymphoproliferative disorders accompanied by persistent pruritus. Pruritogenic role of interleukin-31 (IL-31) has been studied extensively and was proven in atopic dermatitis (AD), while its role in CTCL is still rather vague. Aim: To investigate IL-31 serum level along with IL-31, IL-31 receptor α (IL-31RA) and oncostatin M receptor ß (OSMR) skin expression in CTCL and compare it to controls: AD and healthy volunteers. Material and methods: The level of IL-31 in serum was measured using ELISA, while IL-31 and receptors' expression in the skin were measured using immunohistochemistry and correlated with the stage of disease and pruritus severity. Results: Expression of IL-31 and IL-31 receptor in serum and skin were significantly higher in CTCL and AD in comparison to healthy controls. No significant correlation between the IL-31 serum level and pruritus severity in CTCL patients was found. There was also no correlation between IL-31/IL-31RA/OSMR expression in the skin and CTCL pruritus, while IL-31 and IL-31RA in CTCL skin negatively correlated with the stage of disease. Conclusions: Our data indicate that IL-31 does not play a crucial role in pruritus in CTCL but it is rather involved in the pathogenesis of the disease. It seems that IL-31 plays an essential role in the pruritus pathomechanism that is unique to AD.

Association of oncostatin M receptor polymorphisms with clinical recurrence of ovarian cancer in the Chinese Han population.

Aim: Ovarian cancer (OC) is a gynecological malignancy with a challenging judgment of prognosis due to complicated etiology and high recurrence rate. The oncostatin M receptor (OSMR) from members of the IL-6 receptor family is associated with tumor development. This study aims to explore the correlations between OSMR gene polymorphisms (rs2278329 [G/A, missense, Asp553Asn], rs2292016 [G/T, promoter, -100G/T]) and OC. Methods: This study enrolled 160 OC patients and 397 healthy controls. Genotypes of two single-nucleotide polymorphisms were distinguished using TaqMan SNP Genotyping Assay, and statistical analysis was performed using SPSS software. Results: A significantly decreased overall survival rate was found in serous OC patients carrying rs2278329 GA/AA genotypes. Meanwhile, TT genotype carriers of rs2292016 had an improved relapse rate, and the GT genotype showed a definitive correlation with a lower relapse rate. Conclusion:OSMR gene polymorphisms may be related to recurrence and overall survival of serous OC patients.

The Role of Oncostatin M and Its Receptor Complexes in Cardiomyocyte Protection, Regeneration, and Failure.

Oncostatin M (OSM), a member of the interleukin-6 family, functions as a major mediator of cardiomyocyte remodeling under pathological conditions. Its involvement in a variety of human cardiac diseases such as aortic stenosis, myocardial infarction, myocarditis, cardiac sarcoidosis, and various cardiomyopathies make the OSM receptor (OSMR) signaling cascades a promising therapeutic target. However, the development of pharmacological treatment strategies is highly challenging for many reasons. In mouse models of heart disease, OSM elicits opposing effects via activation of the type II receptor complex (OSMR/gp130). Short-term activation of OSMR/gp130 protects the heart after acute injury, whereas chronic activation promotes the development of heart failure. Furthermore, OSM has the ability to integrate signals from unrelated receptors that enhance fetal remodeling (dedifferentiation) of adult cardiomyocytes. Because OSM strongly stimulates the production and secretion of extracellular proteins, it is likely to exert systemic effects, which in turn, could influence cardiac remodeling. Compared with the mouse, the complexity of OSM signaling is even greater in humans because this cytokine also activates the type I leukemia inhibitory factor receptor complex (LIFR/gp130). In this article, we provide an overview of OSM-induced cardiomyocyte remodeling and discuss the consequences of OSMR/gp130 and LIFR/gp130 activation under acute and chronic conditions.

Determination of Anti-Phospholipase A2 and Anti-Thrombospondin Type 1 Domain-Containing Protein 7A in Latin Patients with Membranous Nephropathy.

Primary membranous nephropathy (MN) is caused by antibodies against podocyte antigens, especially the type M receptor of phospholipase A2 (PLA2R) and thrombospondin type-1 domain containing 7 A (THSD7A). This study's aim was the determination of anti-PLA2R, anti-THSD7A serum antibodies, and anti-PLA2R renal tissue staining prevalence in a Latin population with MN, as well as evaluating their role as biomarkers for disease activity. The performance of the two anti-PLA2R serum diagnostic methods-ELISA and indirect immunofluorescence (IFI)-was evaluated for the diagnosis of MN. Fifty-nine patients, including 29 with MN, 18 with lupus membranous nephropathy (LMN) and 12 with focal and segmental glomerulosclerosis (FSGS), were evaluated for serum antibodies. Renal biopsies were also evaluated for the presence of anti-PLA2R staining. Twenty-one patients with MN were followed for 1 year. Patients with LMN and FSGS were negative for both antibodies. All 29 MN patients were negative for anti-THSD7A; 16 MN patients were positive for anti-PLA2R by ELISA and/or IFI, and 3 MN patients were positive for anti-PLA2R only by IFI. Thus, the anti-PLA2R ELISA test demonstrated 45% sensitivity and 97% specificity, while the IFI test showed, respectively, 55% and 100% in our MN patients. Among the 28 MN renal biopsies, 20 presented anti-PLA2R positive staining, corresponding to a 72% sensitivity. Positive correlations were observed between the anti-PLA2R ELISA titer and proteinuria. In conclusion, determination of anti-PLA2R antibodies in the MN Latin population showed similar rates to those reported for other populations. The anti-PLA2R serum levels correlated with MN disease activity.

The OSMR Gene Is Involved in Hirschsprung Associated Enterocolitis Susceptibility through an Altered Downstream Signaling.

Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.

Interleukin-31: The "itchy" cytokine in inflammation and therapy.

The cytokine interleukin-31 has been implicated in the pathophysiology of multiple atopic disorders such as atopic dermatitis (AD), allergic rhinitis, and airway hyper-reactivity. In AD, IL-31 has been identified as one of the main "drivers" of its cardinal symptom, pruritus. Here, we summarize the mechanisms by which IL-31 modulates inflammatory and allergic diseases. TH 2 cells play a central role in AD and release high levels of TH 2-associated cytokines including IL-31, thereby mediating inflammatory responses, initiating immunoregulatory circuits, stimulating itch, and neuronal outgrowth through activation of the heterodimeric receptor IL-31 receptor A (IL31RA)/Oncostatin M receptor (OSMRß). IL31RA expression is found on human and murine dorsal root ganglia neurons, epithelial cells including keratinocytes and various innate immune cells. IL-31 is a critical cytokine involved in neuroimmune communication, which opens new avenues for cytokine modulation in neuroinflammatory diseases including AD/pruritus, as validated by recent clinical trials using an anti-IL-31 antibody. Accordingly, inhibition of IL-31-downstream signaling may be a beneficial approach for various inflammatory diseases including prurigo. However, as to whether downstream JAK inhibitors directly block IL-31-mediated-signaling needs to be clarified. Targeting the IL-31/IL31RA/OSMRß axis appears to be a promising approach for inflammatory, neuroinflammatory, and pruritic disorders in the future.

LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. METHODS: Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. RESULTS: Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966-0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. CONCLUSIONS: LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR-extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).

Molecular Simulation of Oncostatin M and Receptor (OSM-OSMR) Interaction as a Potential Therapeutic Target for Inflammatory Bowel Disease.

Therapeutics targeting cytokines such as the oncostatin M (OSM)-mediated inflammation represent a potential strategy for the treatment of inflammatory bowel disease (IBD). Despite the investigation of the specific role of the interactions between OSM and the receptor (OSMR) in IBD pathogenesis, the 3D structure of the OSM-OSMR complex remains elusive. In this work, the interaction mode between OSM and OSMR at atomic level was predicted by computational simulation approach. The interaction domain of the OSMR was built with the homology modeling method. The near-native structure of the OSM-OSMR complex was obtained by docking, and long-time scale molecular dynamics (MD) simulation in an explicit solvent was further performed to sample the conformations when OSM binds to the OSMR. After getting the equilibrated states of the simulation system, per-residue energy contribution was calculated to characterize the important residues for the OSM-OSMR complex formation. Based on these important residues, eight residues (OSM: Arg100, Leu103, Phe160, and Gln161; OSMR: Tyr214, Ser223, Asp262, and Trp267) were identified as the "hot spots" through computational alanine mutagenesis analysis and verified by additional MD simulation of R100A (one of the identified "hotspots") mutant. Moreover, six cavities were detected at the OSM-OSMR interface through the FTMap analysis, and they were suggested as important binding sites. The predicted 3D structure of the OSM-OSMR complex and the identified "hot spots" constituting the core of the binding interface provide helpful information in understanding the OSM-OSMR interactions, and the detected sites serve as promising targets in designing small molecules to block the interactions.

Inhibition of JAK1/2 Tyrosine Kinases Reduces Neurogenic Heterotopic Ossification After Spinal Cord Injury.

Neurogenic heterotopic ossifications (NHO) are very incapacitating complications of traumatic brain and spinal cord injuries (SCI) which manifest as abnormal formation of bone tissue in periarticular muscles. NHO are debilitating as they cause pain, partial or total joint ankylosis and vascular and nerve compression. NHO pathogenesis is unknown and the only effective treatment remains surgical resection, however once resected, NHO can re-occur. To further understand NHO pathogenesis, we developed the first animal model of NHO following SCI in genetically unmodified mice, which mimics most clinical features of NHO in patients. We have previously shown that the combination of (1) a central nervous system lesion (SCI) and (2) muscular damage (via an intramuscular injection of cardiotoxin) is required for NHO development. Furthermore, macrophages within the injured muscle play a critical role in driving NHO pathogenesis. More recently we demonstrated that macrophage-derived oncostatin M (OSM) is a key mediator of both human and mouse NHO. We now report that inflammatory monocytes infiltrate the injured muscles of SCI mice developing NHO at significantly higher levels compared to mice without SCI. Muscle infiltrating monocytes and neutrophils expressed OSM whereas mouse muscle satellite and interstitial cell expressed the OSM receptor (OSMR). In vitro recombinant mouse OSM induced tyrosine phosphorylation of the transcription factor STAT3, a downstream target of OSMR:gp130 signaling in muscle progenitor cells. As STAT3 is tyrosine phosphorylated by JAK1/2 tyrosine kinases downstream of OSMR:gp130, we demonstrated that the JAK1/2 tyrosine kinase inhibitor ruxolitinib blocked OSM driven STAT3 tyrosine phosphorylation in mouse muscle progenitor cells. We further demonstrated in vivo that STAT3 tyrosine phosphorylation was not only significantly higher but persisted for a longer duration in injured muscles of SCI mice developing NHO compared to mice with muscle injury without SCI. Finally, administration of ruxolitinib for 7 days post-surgery significantly reduced STAT3 phosphorylation in injured muscles in vivo as well as NHO volume at all analyzed time-points up to 3 weeks post-surgery. Our results identify the JAK/STAT3 signaling pathway as a potential therapeutic target to reduce NHO development following SCI.