SOCS3 mRNA Expression Research Articles

The Atypical Dual Specificity Phosphatase DUSP15 Regulates Jak1-Mediated STAT3 Activation.

The signal transducer and activator of transcription 3 (STAT3) protein is a key regulator of cell differentiation, proliferation, and survival in hematopoiesis, immune responses, and other biological systems. STAT3 transcriptional activity is strictly regulated through various mechanisms, such as phosphorylation and dephosphorylation. In this study, we attempted to identify novel phosphatases which regulate STAT3 activity in response to cytokine stimulations. To this end, leukemia inhibitory factor (LIF)/STAT3 dependent phosphatase induction was evaluated in the mouse hepatoma cell line Hepa1-6. After LIF stimulation, the expression of several atypical dual specific phosphatases (aDUSPs) was upregulated in Hepa1-6 cells. Among the LIF-induced aDUSPs, we focused on DUSP15 and clarified its functions in LIF/STAT3 signaling using RNA interference. DUSP15 knockdown decreased LIF-induced Socs3 mRNA expression and STAT3 translocation. Furthermore, loss of DUSP15 reduced the phosphorylation of STAT3 at Tyr705 and Janus family tyrosine kinase 1 (Jak1) at Tyr1034/1035 in response to LIF. The interaction between Jak1 and DUSP15 was observed in LIF-stimulated Hepa1-6 cells. We also demonstrated the suppression of granulocyte colony-stimulating factor (G-CSF)-mediated gp130/STAT3-dependent cell growth of Ba/F-G133 cells via DUSP15 knockdown. Therefore, DUSP15 functions as a positive feedback regulator in the Jak1/STAT3 signaling cascade.

Circulating mRNA Expression of VEGF, PTEN, and SOCS1 as Potential Prognostic Predictor for Nasopharyngeal Carcinoma Progression.

The molecular mechanisms underlying nasopharyngeal carcinoma (NPC) progression remain poorly understood. In particular, the roles of circulating mRNAs encoding key regulatory proteins have yet to be explored. This study aimed to identify NPC-associated expression signatures of circulating VEGF, PTEN, and SOCS1 mRNAs and their potential as biomarkers. A case-control study was conducted comprising 160 nasopharyngeal carcinoma (NPC) patients and 80 controls, from whom peripheral blood samples. Total RNA was extracted and the levels of VEGF, PTEN, and SOCS1 mRNAs were quantified using reverse transcription quantitative PCR (RT-qPCR). Relative expression was calculated using the 2-ΔΔCt method. Bioinformatic analyses, including GeneMANIA, Gene Ontology (GO), and KEGG pathway analysis, were performed to predict the functional roles and interactions of these mRNAs. We identified significantly increased circulating VEGF mRNA in lymph node metastases (1.66-fold, p<0.05) and elevated SOCS1 mRNA in late-stage NPC (20-fold, p<0.05). PTEN mRNA was reduced 4.26-fold in NPC patients. These data suggest that circulating VEGF, PTEN, and SOCS1 mRNAs represent signatures of NPC progression and can potentially be biomarkers. Network analyses implicate these mRNAs in mechanisms enabling NPC pathogenesis. Our study reveals NPC-associated expression changes of circulating VEGF, PTEN, and SOCS1 mRNAs. These molecular signatures may serve as biomarkers during NPC progression and provide insights into underlying mechanisms. Further validation of their utility as prognostic indicators of NPC is warranted.

Botulinum neurotoxin serotype A inhibited ocular angiogenesis through modulating glial activation via SOCS3.

Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies.

Disruption of SOCS1 Promotes the Antitumor Activity of CD19-Specific CAR NK Cells Coexpressing IL-15

Background Chimeric antigen receptor (CAR) expressing NK cells exhibit robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. Interleukin-15 (IL-15) promotes NK cells expansion and survival, while IL-15 signaling is often inhibited by the suppressor of cytokine signaling (SOCS) proteins (CIS, SOCS1-7), which is rapidly induced in response to IL-15 and negatively regulates cytokine signaling through the JAK/STAT pathway in NK cells. We hypothesized that SOCS1 would be a logical checkpoint to target in human NK cells to increase their persistence and thereby enhance their antitumor potency. Here, we devised a strategy that couples targeting the SOCS1 protein, with CD19-specific human NK cells that constitutively express IL-15. Method Firstly, we evaluated the SOCS1 mRNA expression levels on NK cells after cultured with or without IL-15 for 6 or 24 hours by reverse transcription polymerase chain reaction (RT-PCR). The retroviral vector encoding CAR19.4-1BB-zeta-2A-IL-15 (named BBz.IL-15) and the production of transient retroviral supernatants have been previously described. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. CAR expression on NK cells was determined by flow cytometry, and IL-15 secretion was measured by ELISA. Knockout (KO) of SOCS1 by CRISPR/Cas9 using the Neon transfection system was performed on day 3 post transduction in both non-transduced NK cells (NT-NK) and CAR-transduced NK cells (CAR-NK). To assess KO efficiency, we used polymerase chain reaction (PCR) gel electrophoresis, western blot, and Sanger sequencing. The degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 7 post electroporation. Results The expression of SOCS1 on NK cells was significantly induced within 6 hours of IL-15 treatment. Our approach for combined retroviral transduction with the anti-CD19 CAR and RNP-mediated gene editing of SOCS1 showed that surface expression of the CAR was maintained on NK cells in culture, with construct containing BBz.IL15 (37.18 ± 11.71%) demonstrating comparable transduction efficiency compared with the BBz construct (45.48 ± 10.54%) (P＞0.05), and the efficiency of SOCS1 KO was high in both the nontransduced (NT) control and CAR-expressing NK cells by TIDE and remained stable over time. When tested against CD19-postive Raji cells, SOCS1 KO BBz.IL-15 NK cells produced more IFN-γ and TFN-α, and displayed greater degranulation (CD107a) against their targets than did their BBz.IL-15 NK controls (BBz.IL-15 SOCS1 KO vs BBz.IL-15 control: 15.75±2.05% vs 7.93±0.47% for IFN-γ, P=0.032; 6.27±1.27% vs 1.88±0.26% for TFN-α, P=0.04; 23.25±2.33% vs 12.35±2.33% for CD107a, P=0.042). However, there was no significant difference between the SOCS1 KO BBz. or NT NK cells and their respective BBz. and NT controls regarding IFN-γ and TFN-α production and degranulation (BBz.SOCS1 KO vs BBz.control: 16.95±5.30% vs 16.85±8.70% for IFN-γ, P＞0.05; 6.83±2.98% vs 4.06±2.47% for TFN-α, P＞0.05; 26.65±7.14% vs 25.8±2.12% for CD107a, P＞0.05) (NT SOCS1 KO vs NT control: 21.75±6.58% vs 7.5±2.18% for IFN-γ, P＞0.05; 7.23±3.46% vs 1.58±0.03% for TFN-α, P＞0.05; 15.05±3.46% vs 6.84±2.15% for CD107a, P＞0.05). To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. SOCS1 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS1 KO NK cells can grow faster than the control group. Conclusion In conclusion, the results demonstrate that SOCS1 might be a potential target for genetic modification to improve activity of CAR- NK cells.

Effects of dietary adjustment of n-3:n-6 fatty-acid ratio to 1:2 on anti-inflammatory and insulin-signaling pathways in ovariectomized mice with high fat diet-induced obesity

Estrogen deficiency increases the secretion of inflammatory mediators and can lead to obesity. Consequently, estrogen deficiency can cause metabolic syndrome, particularly insulin resistance during menopause. Both fish oil and perilla oil contain n-3 fatty acids, which may regulate several inflammatory cytokines. Additionally, adjusting the dietary n-3:n-6 fatty-acid ratio to 1:2 may help treat or prevent chronic diseases. Therefore, we investigated the effect of anti-inflammatory and insulin-signaling pathways, not solely in relation to the (n-3:n-6 fatty-acid ratio at 1:2), but also considering the origin of n-3 fatty acids found in fish oil and perilla oil, in a mouse model of estrogen deficiency induced by ovariectomy and obesity induced by a high-fat diet (HFD). Female C57BL/6J mice were divided into five groups: sham mice on a normal diet; ovariectomized (OVX) mice on a normal diet (OC); OVX mice on a HFD plus lard oil (OL), fish oil (OF), or perilla oil (OP). The dietary n-3:n-6 ratio in the OF and OP groups was adjusted to 1:2. The results showed OF group exhibited significantly lower abdominal adipose tissue weight, fewer liver lipid droplets, and smaller uterine adipocytes, compared with the OL group. Compared with the OL group, the OF and OP groups exhibited higher oral glucose tolerance and lower serum alanine aminotransferase activity, triacylglycerol levels, and total cholesterol levels. Hepatic JAK2, STAT3, and SOCS3 mRNA expression and p–NF–κB p65 and IL-6 levels were significantly lower in the OF and OP groups than in the OL group. Only the OF group exhibited an increase in PI3K and Akt mRNA expression, decrease in GLUT2 mRNA expression, and considerable elevation of p-Akt. Both fish and perilla oil reduced inflammatory signaling markers. However, only fish oil improved insulin signaling (PI3K, Akt, and GLUT2). Our data suggest that fish oil can alleviate insulin signaling through activating the PI3K-Akt-GLUT2 cascade signaling pathway.

Resistin stimulates PC-3 prostate cancer cell growth through stimulation of SOCS3 and SOCS5 genes.

Resistin and suppressors of cytokine signaling (SOCSs) have been reported to regulate prostate cancer (PCa) cell proliferation and survival, respectively. Whether any of the SOCS molecules mediate the mitogenic effect of resistin on PCa cells is unknown. Using PC-3 human PCa cells, we found that resistin upregulates the expression of SOCS3 and SOCS5 mRNA, but not SOCS7 mRNA, in a dose- and time-dependent manner. The resistin-induced increases in SOCS3 and SOCS5 expression and cell proliferation were prevented by pretreatment with specific inhibitors of the TLR4, ERK, p38 MAPK, JNK, PI3K, and JAK2 proteins. However, pretreatment with a TLR2 inhibitor had no effect on resistin-mediated SOCS3 and SOCS5 expression. In addition, the effects of resistin on SOCS3, SOCS5, and SOCS7 mRNA levels were cell type-specific. Overexpression of either SOCS3 or SOCS5 enhanced further resistin-stimulated growth of PC-3 cells, whereas silencing SOCS3 or SOCS5 antagonized resistin-increased cell growth. Further PCa tissue analysis demonstrated higher levels of RETN, TLR4, SOCS3, and SOCS5 mRNAs in cancer tissues than benign prostate hyperplasia and indicated positive correlations among RETN, TLR4, and SOCS5. These data suggest that SOCS5, TLR4, and, to a lesser extent, SOCS3 can mediate the mitogenic effect of resistin on PC-3 PCa cells.

Vinpocetine alleviates intestinal ischemia/reperfusion injury and enhances M2 macrophage polarization in rats: Role of SIRT1/SOCS3/STAT3 signaling pathway

Vinpocetine (Vinpo) is a neuroprotective vasodilator drug. It is an effective therapeutic agent for a variety of cerebrovascular and cognitive disorders. However, its potential protective efficacy on intestinal ischemia/reperfusion (I/R) injury remains elusive. The present study aimed to investigate the effect of Vinpo on intestinal I/R injury and to explore its modulatory effect on sirtuin (SIRT1)/ Suppressor of cytokine signaling (SOCS3)/ Signal Transducer and Activator of Transcription (STAT3) signaling. Twenty-four male Wistar albino rats were randomly allocated into four groups. G1 (sham): rats were subjected to surgical stress without I/R, GII (I/R): rats were subjected to 60 min/2-h I/R, GIII (Vinpo + I/R): rats were pre-treated with Vinpo (20 mg/kg/day, P.O. daily) for 2 weeks before intestinal I/R; GIV (EX527 + Vinpo + I/R): rats received both Vinpo (20 mg/kg/day, P.O.) and EX527 (5 mg/kg, once every 2 days, i.p) for 2 weeks before intestinal I/R. The current results showed that Vinpo improved the intestinal histopathological picture, enhanced M1 to M2 macrophage polarization and alleviated the I/R-induced increase in interleukins (IL-6, IL-1β), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (i-NOS), and nitric oxide (NO). Additionally, Vinpo pretreatment upregulated SIRT1 mRNA expression/protein level and SOCS3 mRNA expression while downregulating P-STAT3 immunoreactivity. The effects of Vinpo were attenuated by the SIRT1 inhibitor EX527. We concluded that Vinpo ameliorated the intestinal I/R injury and enhanced M2 anti-inflammatory macrophage polarization through modulation of SIRT1/SOCS3/STAT3/i-NOS cascade.

Gene variants and mRNA expression analysis of SOCS3 and its association with serum IL-4 levels in atopic diseases

BackgroundThe suppressors of cytokine signaling (SOCS) are a class of negative regulators for several aspects of cytokine signaling that have been attributed to the pathophysiology of inflammatory disorders. Given the role of the SOCS3 gene in regulating Th2 cell proliferation, our study aimed to analyze two SOCS3 SNPs viz. rs8074003 and rs7222391, and their potential influence on IL-4 levels and SOCS3 mRNA expression besides analyzing the interaction of the SOCS3 genotypes with the various clinicopathological parameters. MethodsA total of 314 subjects including 154 atopic cases and 160 healthy controls were genotyped for SOCS3 polymorphisms by PCR-RFLP. SOCS3 mRNA was quantified by Real-Time PCR. The serum IL-4 and total IgE levels were determined by ELISA and Vitamin-D levels were quantified by chemiluminescence. ResultsThe CC genotype of rs8074003 was more frequent in atopic cases and posed a 3- fold risk of atopy (p = 0.001) whereas CG and GG genotypes were widespread in the controls (p = 0.1). For the other SNP rs7222391, there was no difference in genotypic and allelic distribution. The SOCS3 mRNA expression and serum IL-4 levels were substantially increased in the atopic cases with a significant positive correlation between them (p < 0.05). ConclusionSOCS3 SNP rs8074003 poses a convincing risk of atopic disease development. The SOCS3 expression and IL-4 levels were up-regulated in total atopy and in its different presentations. It seems plausible to target SOCS3 and IL-4 as a potential target for the diagnosis of atopy and for the development of reliable personalized therapeutic strategies to control atopic conditions.

Alterations of SOCS1 and SOCS3 transcript levels, but not promoter methylation levels in subcutaneous adipose tissues in obese women

BackgroundAnimal model studies suggest that change in the members of the suppressor of the cytokine signaling (SOCS) family (mainly SOCS1 and SOCS3) is linked to the pathogenesis of obesity-related metabolic disorders. Moreover, epigenetic modification is involved in the transcriptional regulation of the SOCS gene family. Here, we aimed to evaluate the mRNA expression as well as gene promoter methylation of SOCS1 and SOCS3 in subcutaneous adipose tissue (SAT) from obese women compared to normal-weight subjects. We also intend to identify the possible association of SOCS1 and SOCS3 transcript levels with metabolic parameters in the context of obesity.MethodsThis study was conducted on women with obesity (n = 24) [body mass index (BMI) ≥ 30 kg/m 2] and women with normal-weight (n = 22) (BMI < 25 kg/m 2). Transcript levels of SOCS1 and SOCS3 were evaluated by real-time PCR in SAT from all participants. After bisulfite treatment of DNA, methylation-specific PCR was used to assess the putative methylation of 10 CpG sites in the promoter of SOCS1 and 13 CpG sites in SOCS3 in SAT from women with obesity and normal weight.ResultsIt was found that unlike SOCS3, which disclosed an elevating expression pattern, the expression level of SOCS1 was lower in the women with obesity as compared with their non‐obese counterparts (P-value = 0.03 for SOCS1 transcript level and P-value = 0.011 for SOCS3 transcript level). As for the analysis of promoter methylation, it was found that SOCS1 and SOCS3 methylation were not significantly different between the individuals with obesity and normal weight (P-value = 0.45 and P-value = 0.89). Correlation analysis indicated that the transcript level of SOCS1 mRNA expression had an inverse correlation with BMI, hs-CRP levels, HOMA-IR, and insulin levels. However, the SOCS3 transcript level showed a positive correlation with BMI, waist-to-height ratio, waist circumference, hip circumference, hs-CRP, HOMA-IR, insulin, fasting blood glucose, and total cholesterol. Interestingly, HOMA-IR is the predictor of the transcript level of SOCS1 (β = − 0.448, P-value = 0.003) and SOCS3 (β = 0.465, P-value = 0.002) in SAT of all participants.ConclusionsOur findings point to alterations of SOCS1 and SOCS3 transcript levels, but not promoter methylation levels in subcutaneous adipose tissues from women with obesity. Moreover, mRNA expression of SOCS1 and SOCS3 in SAT was associated with known obesity indices, insulin resistance, and hs-CRP, suggesting the contribution of SOCS1 and SOCS3 in the pathogenesis of obesity-related metabolic abnormalities. However, further studies are required to establish this concept.

Effect of HDAC Inhibitor Chidamide on PD-L1 Expression in Peripheral T-Cell Lymphoma

To explore the role of chidamide in the regulatory mechanism of PD-1/PD-L1 immune escape signaling pathway in peripheral T-cell lymphoma. Jurkat cell line was treated with different concentrations of chidamide. The changes of PD-L1 and JAK/STAT pathway gene mRNA expression and PD-L1 protein expression on cell surface were detected by fluorescence quantitative PCR and flow cytometry after treatment. Chidamide upregulated PD-L1 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.989). The mRNA expression of PD-L1 in 5.0 μmol/L group was 15.4 times higher than that in the control group. The proportion of PD-L1 positive cells in Jurkat cell line was less than 0.5%. Chidamide upregulated PD-L1 protein expression on Jurkat cell surface. Chidamide upregulated the mRNA expression of JAK2, STAT1 and STAT3 in Jurkat cell line. The level of up-regulation was obvious in high concentration group (5.0 μmol/L group). Meanwhile, the mRNA expression of SOCS1 and SOCS3, the negative regulatory genes upstream of the JAK/STA T pathway, were up-regulated. In peripheral T-cell lymphoma, chidamide may up-regulate the expression of cell surface PD-L1 and induce T-cell chemokines by upregulation of STAT1 expression, thus improving the reaction rate of PD-1 monoclonal antibody and T-cell toxicity.

MiR-222-3p Aggravates the Inflammatory Response by Targeting SOCS1 to Activate STAT3 Signaling in Ulcerative Colitis.

Ulcerative colitis is characterized by relapsing inflammation in the gastrointestinal tract with limited treatment options. The aim of the present study was to assess the anti-inflammatory effect of Suppressor of cytokine signaling (SOCS1) on lipopolysac- charide-stimulated RAW264.7 cells and to investigate its potential mechanisms. The in vitro ulcerative colitis model was established by using lipopolysaccharide-stimulated RAW264.7 cells. Western blot- ting was used to detect the protein expression levels of SOCS1, JAK2, STAT3, and VDR. Reverse transcription-quantitative polymerase chain reaction was used to measure the mRNA expression of SOCS1, miR-222-3p, and VDR. An enzyme-linked immunosorbent assay was performed to measure the levels of inflammatory cytokines. A luciferase assay assessed the binding of SOCS1 to miR-222-3p. A total of 15 patients with ulcerative colitis and 18 healthy controls were recruited. The expression levels of SOCS1 and miR-222-3p in the colonic mucosa tissues of patients with ulcerative colitis and healthy controls were determined by reverse transcription-quantitative polymerase chain reaction. SOCS1 upregulation inhibited the lipopolysaccharide-induced inflammation in RAW264.7 cells. SOCS1 was confirmed to be tar- geted by miR-222-3p. Silencing SOCS1 significantly abolished the inhibitory effects of miR-222-3p downregulation on inflammation. MiR-222-3p activated STAT3 signaling and reduced VDR expression by targeting SOCS1 in lipopolysaccharide-treated RAW264.7 cells. Additionally, miR-222-3p expression was upregulated in ulcerative colitis patients (P = 5.16E-10), while SOCS1 (P = 2.75E-10) and VDR (P = 52.5E-9) expression was downregulated in ulcerative colitis patients. Endoscopic scores (UCEIS) revealed significant positive cor- relation with miR-222-3p and negative correlation with SOCS1 and VDR. MiR-222-3p targets SOCS1 to aggravate the inflammatory response by suppressing VDR and activating STAT3 signaling in ulcerative colitis.

Simultaneous Detection of RIG-1, MDA5, and IFIT-1 Expression Is a Convenient Tool for Evaluation of the Interferon-Mediated Response.

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.

Differential Expression of Suppressor of Cytokine Signaling and Interferon Gamma in Lean and Obese Patients with Type 2 Diabetes Mellitus.

The model of obesity-induced insulin resistance has long been used to explain the development of type 2 diabetes mellitus (T2DM) in obese individuals (body mass index (BMI) > 25 kg/m2), but this model failed to explain the development of the disease in lean individuals (BMI < 18.5 kg/m2). Defects in the insulin signaling pathway have been postulated to play a role in these patients, particularly in suppressors of cytokine signaling (SOCS) proteins, which are involved in the downregulation of insulin transduction. The expression of SOCS is also known to be induced by cytokines such as interferon gamma (IFN-γ). It is still not clear whether these pathways operate differently in lean versus obese patients with T2DM. Therefore, this pilot study was designed to study the expression of SOCS1, SOCS3, and IFN-γ in lean and obese patients with T2DM. The levels of IFN-γ in serum and the messenger RNA (mRNA) expression of SOCS (SOCS1 and SOCS3) and IFN-γ genes in whole blood in lean and obese patients with T2DM. Sixty newly diagnosed T2DM patients (not on any pharmacotherapy) were enrolled and divided into 2 groups of lean (BMI < 18.5 kg/m2) and obese (BMI > 25 kg/m2) patients (n = 30 per group). Serum IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA), and mRNA expression of IFN-γ, SOCS1, and SOCS3 was measured by real-time polymerase chain reaction (PCR) using the ∆∆ Ct method. Serum IFN-γ levels were 10.83 ± 5.81 pg/mL in the lean group and 9.35 ± 5.14 pg/mL in the obese group (P = 0.02). Fasting serum insulin levels were 16.07 ± 8.39 µIU/mL in the lean group and 27.11 ± 4 .91 µIU/mL in the obese group (P = 0.001). There was a 3.16-fold increase in mRNA expression of IFN-γ and a 1.3-fold increase in mRNA expression of SOCS1 in the lean group compared to the obese group. mRNA expression of SOCS3 was similar in both groups. The level of IFN-γ increased at both transcriptional and translational levels, and mRNA expression of SOCS1 was higher in the lean group than in the obese group. The SOCS protein is a known negative regulator in insulin signaling pathways. Thus, our findings and available scientific literature suggest that IFN-γ might impair the insulin signaling pathway to a greater extent in lean patients than in obese patients via induction of SOCS1. This signaling pathway could be a major contributing factor to hyperglycemia in lean patients with T2DM compared with obese counterparts. This suggests that different therapeutic approaches to these groups might be of greater benefit in the treatment of T2DM.

MicroRNAs modulate Chlamydia trachomatis infection- and vaccine-induced immunity in macrophages

Abstract Recent studies indicate that microRNAs (miRNAs), including immune cell type-specific miRNAs modulate inflammation and immune response to Chlamydia trachomatis (CT) genital infection. However, the role of these miRNAs in modulation of immune cell function following CT infection or vaccine stimulation has not been investigated. We hypothesized that pretreatment of CT-infected and vaccine-stimulated macrophages with miRNA mimics or inhibitors will modulate secretion of inflammatory cytokines and expression of IFN-γ pathway-related genes. Thus, THP-1-differentiated macrophages (TDM) transfected with miRNA mimics or inhibitors were infected with CT or stimulated with a Vibrio cholerae ghost (rVCG)-based vaccine (rVCG-MECA) and the level of inflammatory cytokines secreted in culture supernatants was assessed by cytokine ELISA. Also, TDM expression of IFN-γ pathway-related genes was assessed by RT-qPCR. The results show that CT infection of TDM resulted in the downregulation of miR-15a, miR-29a, miR-146a and miR-222 while stimulation with rVCG-MECA resulted in their upregulation, as we previously reported. The level of TNF-α, IL-8, IL-1β and IFN-γ secreted by miRNA mimic- or inhibitor-treated TDM following infection or vaccine stimulation was dependent on the miRNA type. Furthermore, CT infection of untreated macrophages increased mRNA expression of INFGR1, JAK2, STAT1 and SOCS1. However, pretreatment of CT-infected cells with miR-146a mimic or inhibitor showed significantly decreased expression of these genes. The study demonstrated that miRNAs differentially modulate macrophage secretion of inflammatory cytokines and expression of IFN-γ pathway-related genes following CT infection or vaccine stimulation. Supported by None.

The Effect of SOCS2 Polymorphisms on Type 2 Diabetes Mellitus Susceptibility and Diabetic Complications in the Chinese Han Population.

BackgroundSOCS2 is downregulated in diabetes, which might be related to diabetes. We explored the effect of SOCS2 polymorphisms on the development of type 2 diabetes mellitus (T2DM) and diabetic complications.MethodsThe subjects consisted of 500 patients with T2DM and 501 healthy controls. Five variants in SOCS2 were genotyped by Agena MassARRAY system. RT-qPCR profiling was performed to detect the expression of SOCS2 mRNA. Logistic regression analysis was utilized to calculate odds ratio (OR) and 95% confidence intervals (95% CIs).ResultsRs3825199 (OR = 1.44, p = 0.007), rs11107116 (OR = 1.39, p = 0.014) and rs10492321 (OR = 1.48, p = 0.004) had an increased T2DM risk of T2DM. Moreover, the contribution of SOCS2 polymorphisms to T2DM risk was associated with age, gender, smoking, drinking, and BMI. SOCS2 variants also had a reduced risk for T2DM patients with diabetic nephropathy, diabetic retinopathy and coronary heart disease. SOCS2 rs10492321 was the best single locus model. SOCS2 mRNA was downregulated in patients with T2DM compared to healthy controls (p = 0.029).ConclusionThis study firstly reported that rs3825199, rs11107116 and rs10492321 in SOCS2 conferred to an increased risk for the occurrence of T2DM in the Chinese Han population. Moreover, SOCS2 mRNA was downregulated in patients with T2DM, suggesting that SOCS2 might have an important role in the occurrence of T2DM.

Latent M.tuberculosis infection is associated with increased inflammatory cytokine and decreased suppressor of cytokine signalling (SOCS)-3 in the diabetic host.

Tuberculosis (TB) outcomes are worsened by type II diabetes mellitus (DM). Protective immunity against Mycobacterium tuberculosis (MTB) is driven by cytokines. Latent TB (LTBi) is common but its effect on the diabetic host is not well understood. We investigated mycobacterial antigen-stimulated responses in peripheral blood mononuclear cell (PBMC) isolated from healthy endemic controls (EC), those with LTBi, DM groups with and without LTBi, as compared with TB patients. Cytokines were measured using a Luminex-based assay. Gene expression was determined by RT-PCR. In DM-LTBi cases, PPD-stimulated proinflammatory cytokines; IFN-γ, IL-6, IL-2, TNF-α and GM-CSF and anti-inflammatory cytokines, IL-5 and IL-13 were raised as compared with EC. DM-LTBi PPD-stimulated IFN-γ, IL-6 and TNF-α mRNA titres were found raised in DM-LTBi, whilst suppressor of cytokine signalling (SOCS)-3 expression was lowered. Within DM cases, stratification based on HbA1c levels revealed raised IFN-γ but lowered IL-6 gene expression in those with controlled levels as compared with uncontrolled glycaemic levels. Further, SOCS1 expression levels were found higher in DM cases with controlled glycaemia when compared with EC. Overall, we show that diabetics with LTBi manifest raised levels of inflammatory and anti-inflammatory cytokines concomitant with reduced SOCS3 mRNA expression. Reduced glycaemic control results in further inflammatory dysregulation impacting conversing impacting IFN-γ and IL-6 activation. These results suggest that dysregulated immune activation in diabetes is exacerbated by LTBi, lack of glycaemic control may further compromise immunity against MTB infection.

Transcriptional profiling and network pharmacology analysis identify the potential biomarkers from Chinese herbal formula Huosu Yangwei Formula treated gastric cancer in vivo

Huosu Yangwei (HSYW) Formula is a traditioanl Chinese herbal medicine that has been extensively used to treat chronic atrophic gastritis, precancerous lesions of gastric cancer and advanced gastric cancer. However, the effective compounds of HSYW and its related anti-tumor mechanisms are not completely understood. In the current study, 160 ingredients of HSYW were identified and 64 effective compounds were screened by the ADMET evaluation. Furthermore, 64 effective compounds and 2579 potential targets were mapped based on public databases. Animal experiments demonstrated that HSYW significantly inhibited tumor growth in vivo. Transcriptional profiles revealed that 81 mRNAs were differentially expressed in HSYW-treated N87-bearing Balb/c mice. Network pharmacology and PPI network showed that 12 core genes acted as potential markers to evaluate the curative effects of HSYW. Bioinformatics and qRT-PCR results suggested that HSYW might regulate the mRNA expression of DNAJB4, CALD, AKR1C1, CST1, CASP1, PREX1, SOCS3 and PRDM1 against tumor growth in N87-bearing Balb/c mice.

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting

BackgroundSepsis and inflammation can cause intensive care unit‐acquired weakness (ICUAW). Increased interleukin‐6 (IL‐6) plasma levels are a risk factor for ICUAW. IL‐6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT‐pathway, but its role in sepsis‐induced muscle wasting is uncertain. In a clinical observational study, we found that the IL‐6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT‐pathway activation. We tested the hypothesis that the IL‐6/gp130‐pathway mediates ICUAW muscle atrophy.MethodsWe sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture‐operated (CLP) and sham‐operated wildtype (WT) mice. The effects of the IL‐6/gp130/JAK2/STAT3‐pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h.ResultsAnalyses of differentially expressed genes in RNAseq (≥2‐log2‐fold change, P < 0.01) revealed an activation of IL‐6‐signalling and JAK/STAT‐signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL‐6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three‐fold, P < 0.01) and Socs3 mRNA expression (3.1‐fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL‐6‐induced STAT3 phosphorylation (−30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (− 29.0%; P < 0.01) or STAT3 inhibition (−38.7%; P < 0.05) decreased IL‐6‐induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL‐6. CLP‐operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st‐cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st‐cKO mice (WT: −22.3%, P < 0.001, cKO: −13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin‐1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin‐1 were unaltered between CLP‐treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (−22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin‐1‐mRNA (−81.3%, P < 0.05) and Trim63/MuRF1‐mRNA expression (−77.6%, P < 0.05) and protein content.ConclusionsIL‐6 via the gp130/JAK2/STAT3‐pathway mediates sepsis‐induced muscle atrophy possibly contributing to ICUAW.

Differential immunological response detected in mRNA expression profiles among diverse chicken lines in response to Salmonella challenge

Salmonella enterica serovar Enteritidis is a bacterial pathogen that contributes to poultry production losses and human foodborne illness. The bacterium elicits a broad immune response involving both the innate and adaptive components of the immune system. Coordination of the immune response is largely directed by cytokines. The objective of the current study was to characterize the expression of a select set of cytokines and regulatory immune genes in three genetically diverse chicken lines after infection with S. Enteritidis. Leghorn, Fayoumi and broiler day-old chicks were orally infected with pathogenic S. Enteritidis or culture medium. At 2 and 18 h postinfection, spleens and ceca were collected and mRNA expression levels for 7 genes (GM-CSF, IL2, IL15, TGF-β1, SOCS3, P20K, and MHC class IIβ) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-β1, SOCS3 and P20K in the spleen and on P20K and MHC class IIβ in the cecum. Comparing challenged vs. unchallenged birds, the expression of SOCS3 and P20K mRNA were significantly higher in the spleen and cecum, while MHC class IIβ mRNA was significantly lower in spleen. Combining the current RNA expression results with those of previously reported studies on the same samples reveals distinct RNA expression profiles among the three genetic chicken lines and the 2 tissues. This study illustrates that these diverse genetic lines have distinctively different immune response to S. Enteritidis challenge within the spleen and the cecum.

Porcine reproductive and respiratory syndrome virus increases SOCS3 production via activation of p38/AP-1 signaling pathway to promote viral replication

SOCS3 belongs to the suppressor of cytokine signaling (SOCS) family, which function as negative factors in host immune responses. Prior studies have noted the importance of SOCS family proteins in immunosuppression induced by some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine-borne viruses and has threatened the global swine industry with huge economic losses since it was first described in the 1980s. PRRSV is the etiological agent of PRRS, which causes reproductive failure and respiratory disorders. PRRSV causes immunosuppression thus establishing persistent infection. In this study, it was observed that SOCS3 was upregulated in PRRSV-infected primary porcine alveolar macrophages (PAMs) and Marc-145 cells with dose-dependent effects, which depends on virus replication. Deletion of AP-1 binding motif located in SOCS3 promoter inhibited promoter activities, which indicates that AP-1 is essential for PRRSV-induced SOCS3. This result was confirmed by experiments using AP-1 inhibitor, whose pretreatment suppressed SOCS3 mRNA and protein expression. Further research showed that p38 was crucial for PRRSV-induced SOCS3 production. Importantly, SOCS3 enhanced PRRSV replication during infection. Taken together, this study indicates that PRRSV infection induced SOCS3 expression through p38/AP-1 signaling pathway. These results revealed the molecular basis of SOCS3 upregulation and would advance further understanding of the strategy for viral immune evasion.