SOCS3 mRNA Expression Research Articles

IL‐6 Signaling during Cancer Cachexia Progression: The Female Response

Cachexia arises secondary to many chronic diseases and accounts for about 30% of cancer‐related deaths. Cachexia occurs in both sexes, but its etiology and progression have been almost exclusively examined in the male. Interleukin‐6 (IL‐6) has become an attractive therapeutic target as IL‐6 signaling is a recognized regulator of male ApcMin/+ mouse cancer cachexia progression. Despite established sex‐dependent inflammatory responses, how IL‐6 signaling contributes to female cancer cachexia progression is unknown. Consequently, we examined the role of circulating IL‐6 for female ApcMin/+ mouse cancer cachexia progression. Female ApcMin/+ mice weight and functional testing were monitored until sacrifice at 18 weeks of age. A second group of female ApcMin/+ mice underwent IL‐6 overexpression at 15 weeks of age. Blood and hindlimb muscle were collected to examine IL‐6 levels and downstream signaling. Mice were stratified into weight stable, initially cachectic, and severely cachectic groups. Plasma IL‐6 did not differ between groups (17.6 ± 6.7; 34.4 ± 18.9; 39.3 ± 13.1 pg/ml); however, muscle IL‐6 mRNA correlated with body weight loss and hindlimb muscle mass (p=0.008, 0.019). Muscle IL‐6r, gp130, and SOCS3 mRNA expression did not change with cachexia severity. Muscle STAT3 phosphorylation increased with severe cachexia compared to initial cachexia (14.8 ± 1.6 vs 5.2 ± 2.1 fold change relative to WT levels). Surprisingly, IL‐6 over‐expression did not increase muscle IL‐6 signaling or bodyweight loss. Overall, elevated IL‐6 does not exacerbate cachexia progression in the female ApcMin/+ mouse.

The somatotropic axis during the physiological estrus cycle in dairy heifers—Effect on hepatic expression of GHR and SOCS2

Pituitary growth hormone (GH) release and hepatic insulin-like growth factor-I (IGF-I) production increase after an injection of 17β-estradiol (E2) in ovariectomized dairy cattle. However, whether endogenous sexual steroid hormones also influence the hepatic GH receptor (GHR) signaling pathway during a physiological estrus cycle remains unclear. The aim of this study was to analyze the hepatic GHR signaling pathway during the luteal phase and after a period of increased E2 concentrations (after ovulation) as well as in 7 heifers before ovulation. Ovarian ultrasounds were performed daily during repeated physiological cycles (n=56) of 30 Holstein Friesian heifers to determine ovulation [before ovulation (n=7, bOv) and after ovulation 24–60h after the appearance of estrus signs (n=49, aOv)] and luteal phase (CLP; d 12±1 after ovulation). Blood samples and liver biopsies were obtained, and blood concentrations of E2, P4, insulin-like growth factor (IGF)-I, IGF-II, and GH were measured. In the liver biopsies, we determined mRNA expression of the estrogen receptor α (ERα), GHR, Janus kinase 2 (JAK2), signal transducer and activator of transcription 5B (STAT5B), suppressor of cytokine signaling (SOCS)2 and 3, IGF-I, and IGF-II by quantitative reverse transcription-PCR. The concentration of E2 was higher bOv than aOv and CLP, as expected. The concentrations of IGF-I and GH were higher bOv and aOv compared with CLP. In contrast, concentrations of IGF-II were lower aOv compared with bOv and CLP. The mRNA expression of GHR was higher in liver biopsies obtained bOv compared with aOv and CLP. Notably, the expression of SOCS2 was higher bOv than aOv and in the CLP. Increased hepatic expression of SOCS2 during estrus was detectable when IGF-I concentrations were high; this result might indicate that SOCS2 expression attenuates the GHR signal transduction pathway during the phase of increased pituitary GH release. In conclusion, hepatic GHR and SOCS2 mRNA expression appeared to be promptly and sensitively regulated by increased E2 levels before ovulation of dairy heifers.

Cell-specific effects in different immune subsets associated with SOCS1 genotypes in multiple sclerosis

Background: Single nucleotide polymorphisms (SNPs) near SOCS1 are associated with multiple sclerosis (MS), but the most important SNPs in the area and mechanisms by which they influence the disease are unknown. Methods: A haplotype-tagging association study was performed covering 60.5kbp around SOCS1, and the index SNP was validated in a total of 2292 individuals. mRNA expression of SOCS1 and nearby genes was measured in MS patients with different disease courses and healthy controls. SOCS1 protein expression was studied by flow cytometry in a separate cohort of patients and controls. Differentiation and maturation of monocyte-derived dendritic cells (moDCs) were also studied. Results: One SNP, rs423674, reached genome-wide significance. No genotype-specific mRNA expression differences were seen, but, by flow cytometry, significant interactions were observed between genotypes for rs423674 and disease activity (relapse or remission) in B cells and regulatory T cells. Furthermore, homozygotes for the risk allele (GG) showed higher levels of CD1a and CD86 than carriers of the protective allele (GT) in immature moDCs and a greater increase of HLA-DR+ cell percentage than GT heterozygotes upon maturation. Conclusions: rs423674, or its genetic proxies, may influence MS risk by modulating SOCS1 expression in a cell-specific manner and by influencing dendritic cell function.

Effect of caffeic acid phenethyl ester on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

Caffeic acid phenethyl ester (CAPE) has numerous potentially beneficial properties, including antioxidant, immunomodulatory and anti-inflammatory activities. However, the effect of CAPE on periodontal disease has not been studied before. This study was designed to investigate the efficacy of CAPE in ameliorating the production of proinflammatory mediators in macrophages activated by lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease. LPS from P. intermedia ATCC 25611 was isolated by using the standard hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO), interleukin (IL)-1β and IL-6. We used real-time polymerase chain reaction to quantify inducible NO synthase, IL-1β, IL-6, heme oxygenase (HO)-1 and suppressors of cytokine signaling (SOCS) 1 mRNA expression. HO-1 protein expression and levels of signaling proteins were assessed by immunoblot analysis. DNA-binding activities of NF-κB subunits were analyzed by using the enzyme-linked immunosorbent assay-based kits. CAPE exerted significant inhibitory effects on P. intermedia LPS-induced production of NO, IL-1β and IL-6 as well as their mRNA expression in RAW264.7 cells. CAPE-induced HO-1 expression in cells activated with P. intermedia LPS, and selective inhibition of HO-1 activity by tin protoporphyrin IX attenuated the inhibitory effect of CAPE on LPS-induced NO production. CAPE did not interfere with IκB-α degradation induced by P. intermedia LPS. Instead, CAPE decreased nuclear translocation of NF-κB p65 and p50 subunits induced with LPS, and lessened LPS-induced p50 binding activity. Further, CAPE showed strong inhibitory effects on LPS-induced signal transducer and activator of transcription 1 and 3 phosphorylation. Besides, CAPE significantly elevated SOCS1 mRNA expression in P. intermedia LPS-stimulated cells. Modulation of host response by CAPE may represent an attractive strategy towards the treatment of periodontal disease. In vivo studies are required to appraise the potential of CAPE further as an immunomodulator in the treatment of periodontal disease.

Antiviral effects of Jinxin oral liquid against respiratory syncytial virus infection in the BALB/c mice model

Ethnopharmacological relevanceJinxin oral liquid (JOL) is used in traditional Chinese medicine (TCM) to treat influenza, cough, asthma, and viral pneumonia, on the basis of Ma Xing Shi Gan Tang (MXSGT) and the clinical experience of Professor Wang Shouchuan, one of the most prestigious pediatricians in China. Aim of studyTo investigate the anti-inflammatory and antiviral activities of JOL in mice infected with respiratory syncytial virus (RSV). Materials and methodsMice were orally administered JOL at doses of 27.6gkg−1d−1 and 55.2gkg−1d−1 for 1, 3, or 6d after RSV challenge. The viral loads in the lung tissue were measured by real-time RT-PCR. The levels of IFN-β in bronchoalveolar lavage fluid (BLAF) and lung tissue were detected by ELISA and real-time RT-PCR, respectively. The mRNA and protein expression of TLR3, IRF3, and SOCS1 were detected by real-time RT-PCR and western blot, respectively. The protein expression of phoshorylated-IRF3 (p-IRF3) was detected by western blot. ResultsJOL significantly ameliorated lung inflammation in RSV-infected mice, and significantly reduced the viral load in the lung tissues. On days 2 and 4 after infection, the mRNA and protein expression of IFN-β, TLR3, IRF3 (p-IRF3), and SOCS1 were significantly downregulated in RSV-infected mice treated with JOL. However, 7d after infection, JOL significantly upregulated the RSV-induced decrease in IFN-β, TLR3, and IRF3 (p-IRF3), but reduced SOCS1 expression. ConclusionsJOL ameliorated lung inflammation and inhibited virus replication significantly in RSV-infected mice. During early stage infection, the effect of JOL was improved through inhibition of the TLR3-IRF3-IFN-β signaling pathway and the expression of SOCS1, whereas during the later stage of infection, JOL upregulated the expression of key signaling molecules in the TLR3 signaling pathway and downregulated the expression of SOCS1.

Sex differences in the relationship of IL-6 signaling to cancer cachexia progression

A devastating aspect of cancer cachexia is severe loss of muscle and fat mass. Though cachexia occurs in both sexes, it is not well-defined in the female. The ApcMin/+ mouse is genetically predisposed to develop intestinal tumors; circulating IL-6 is a critical regulator of cancer cachexia in the male ApcMin/+ mouse. The purpose of this study was to examine the relationship between IL-6 signaling and cachexia progression in the female ApcMin/+ mouse. Male and female ApcMin/+ mice were examined during the initiation and progression of cachexia. Another group of females had IL-6 overexpressed between 12 and 14weeks or 15–18weeks of age to determine whether IL-6 could induce cachexia. Cachectic female ApcMin/+ mice lost body weight, muscle mass, and fat mass; increased muscle IL-6 mRNA expression was associated with these changes, but circulating IL-6 levels were not. Circulating IL-6 levels did not correlate with downstream signaling in muscle in the female. Muscle IL-6r mRNA expression and SOCS3 mRNA expression as well as muscle IL-6r protein and STAT3 phosphorylation increased with severe cachexia in both sexes. Muscle SOCS3 protein increased in cachectic females but decreased in cachectic males. IL-6 overexpression did not affect cachexia progression in female ApcMin/+ mice. Our results indicate that female ApcMin/+ mice undergo cachexia progression that is at least initially IL-6-independent. Future studies in the female will need to determine mechanisms underlying regulation of IL-6 response and cachexia induction.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

Primordial follicle activation in the ovary of Ames dwarf mice.

BackgroundThe insulin receptor substrate 1 (IRS1), phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt1), Forkhead Box O3a (FOXO3a) pathway is directly involved in aging and ovarian activation of follicle growth. Therefore, the aim of this work was to measure the expression of genes related to the ovarian pathway for activation of primordial follicles and FOXO3a protein phosphorylation between young and old female Ames dwarf (df/df) and normal (N) mice.MethodsFor this study ovaries from N (n = 10) and df/df (n = 10) female mice were collected at 5–6 months of age and at 21–22 months of age. For immunohistochemistry ovaries from 12 month-old and df/df mice were used.ResultsThe expression of Irs1, Pi3k, Akt1, mammalian target of rapamycin (Mtor), suppressor of cytokine signaling −2 (Socs2), Socs3 was lower (P < 0.05) in older than younger N mice and not different (P > 0.05) between young and old df/df mice. The expression of Foxo3a was also lower (P < 0.05) in old than younger N and df/df mice and was higher (P < 0.05) in old df/df than N mice. Expression of Amh was lower (P < 0.05) in old than young N and df/df mice and was higher (P = 0.0009) in df/df than N mice. Imunnostaining for p-FOXO3 was lower in df/df than N mice (P < 0.001), although FOXO3 immunostaining was not different (P > 0.05) between df/df and N mice.ConclusionsIn sum, the present study indicates that lower expression of Irs1, Socs2, Socs3, Akt1, Pi3k, Mtor and Foxo3a mRNA in the ovaries of older mice of both genotypes is associated to a reduced ovarian activity revealed by lower expression of Amh mRNA. At the same time, ovaries of old df/df mice maintained higher expression of Foxo3a mRNA, which was associated to higher ovarian activity. We have shown that df/df females have a lower level of p-FOXO3 in oocytes from primordial/primary follicles, an important activator of follicular growth. Therefore, this study strongly indicates that Prop1df mutation causes delayed ovarian aging.

Editor’s Letter

<h3>Background</h3> Interferon-gamma (IFN-γ) plays an important role in the development of lupus nephritis (LN). Regulation of IFN-γ signaling that occurs in disease remission and in active LN is herein addressed. <h3>Objectives</h3> To study the impact of IFN-γ on PBMC obtained from patients with LN in remission as compared to active LN. <h3>Methods</h3> Sixteen patients fulfilling the ACR classification criteria for systemic lupus erythematosus were recruited. All patients had a history of LN of whom 10 were in remission (as defined by EULAR criteria) and 6 had active LN (as defined by SLEDAI-2K or BILAG). Healthy subjects (n=10) were included as a control group. Sera and PBMC were obtained from each individual. Flow cytometry, western blots and real time RT-PCR were used in processing and detection of cell subtypes, protein and mRNA levels. Recombinant human IFN-γ (rhIFN-γ) and anti-IFN-γ neutralizing antibody were used in vitro. Mann-Whitney and student t-tests were used for statistical analysis. <h3>Results</h3> In active LN there was a significant 2-fold increase in CD4⁺CD69⁺ activated T cells as compared to healthy subjects and patients in remission. Reactivity to interferon-gamma receptor was determined by the phosphorylation of its predominant transcription factor, signal transducer and activator of transcription 1 (STAT1) in the cells that were incubated with rhIFN-γ or with media alone. In active LN, 3- and 6-fold increase in pSTAT1 occurred with rhIFN-γ incubation during 24h and 48h, respectively, and healthy subjects responded likewise. In patients in remission, pSTAT1 increase was even higher (by 8- and 10-fold at 24h and 48h, respectively). After 24h incubation with rhIFN-g all groups had elevated (mRNA) expression of SOCS1, but at 48h, it significantly decreased in healthy subjects and in patients in remission by 34% and 50%, respectively. Further, at 24h the frequency of CD4⁺CD127^lowFoxP3⁺ regulatory T cells (e.g. Tregs) increased by 27–30% in active LN and in healthy subjects, and in remission it was minimally changed. At 48h, the frequency of Tregs significantly decreased in healthy subjects (to baseline levels before rhIFN-γ challenge) and in patients in remission (by 24%, p=0.003). A challenge of PBMC from LN patients in remission with sera (at 1% concentration) derived from patients with active LN resulted in 7.5-fold increase in pSTAT1 expression and 20–30% decrease in Tregs, however, combination of sera and anti-IFN-g neutralizing antibody resulted in 5-fold decrease in pSTAT1 expression and significantly diminished the decrease in Tregs. <h3>Conclusions</h3> In LN in remission a challenge with IFN-γ could lead to immune activation and a risk of flare-up, as it results in a decrease in both SOCS1 and Tregs and a robust STAT1 phosphorylation. In active LN, STAT1 phosphorylation is less diminishing, as both SOCS1 and Tregs are saturated, which could affect their suppressive effectiveness. <h3>Disclosure of Interest</h3> None declared

Increased miR-155 Expression in Peripheral Blood Mononuclear Cells of Primary Immune Thrombocytopenia Patients Was Correlated with Serum Cytokine Profiles

We investigated the possible pathogenic role of a microRNA (miR-155) in primary immune thrombocytopenia (ITP). We used quantitative real-time PCR to determine the relative expression of miR-155 and SOCS1 (suppressor of cytokine signaling) mRNA in peripheral blood mononuclear cells (PBMCs) from 28 ITP patients and 28 healthy controls. Cytokine plasma levels were determined by ELISA. Possible associations between miR-155 levels and serum cytokine concentrations were assessed using Spearman or Pearson correlation analysis. Seven naive ITP patients were followed and the effects of medical treatment on miR-155 levels were assessed. Compared to healthy controls, ITP patients had increased miR-155 and decreased SOCS1 mRNA levels. ITP patients also had increased plasma IL-17A and decreased IL-4, IL-10 and TGF-β₁ levels. miR-155 levels were negatively correlated with platelet counts, SOCS1 mRNA levels, and the plasma levels of IL-4, IL-10 and TGF-β₁, but positively correlated with plasma IL-17A levels. Medical treatment for ITP decreased miR-155 levels. Thus, our results suggest that miR-155 might be involved in the pathogenesis of ITP by regulating cytokine profiles, which may be mediated by miR-155 targeting SOCS1. © 2014 S. Karger AG, Basel

Abstract 422: DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer

Abstract Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete. Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation. Materials &amp; methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed. Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p≤0,01) in the biopsy samples along the adenoma-carcinoma sequence. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p&amp;lt;0,01) upregulated in the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence. Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422

GH signaling in skeletal muscle and adipose tissue in healthy human subjects: impact of gender and age.

The mechanisms underlying the impact of age and gender on the GH-IGF1 axis remain unclear. We tested the hypothesis that age and gender have impacts on GH signaling in human subjects in vivo. A total of 20 healthy non-obese adults ('young group'<30 years (5F/5M) and 'old group'>60 years (5F/5M)) were studied after: i) an i.v. GH bolus (0.5 mg) and ii) saline. Muscle and fat biopsies were obtained after 30 and 120 min. Total and phosphorylated STAT5B proteins, gene expression of IGF1, SOCS1, SOCS2, SOCS3 and CISH, body composition, VO2max, and muscle strength were measured. In the GH-unstimulated state, women displayed significantly elevated levels of CISH mRNA in muscle (P=0.002) and fat (P=0.05) and reduced levels of IGF1 mRNA in fat. Phosphorylated STAT5B (pSTAT5b) was maximally increased in all subjects 30 min after GH exposure and more pronounced in women when compared with men (P=0.01). IGF1, SOCS1, SOCS2, SOCS3, and CISH mRNA expression increased significantly in muscle after 120 min in all subjects with no impact of age and gender. GH-induced pSTAT5b correlated inversely with lean body mass (LBM; r=-0.56, P=0.01) and positively with the CISH mRNA response (r=0.533, P=0.05). i) GH signaling in muscle and fat after a single GH bolus in healthy human subjects is age independent, ii) we hypothesize that constitutive overexpression of CISH may contribute to the relative GH resistance in women, and iii) experimental studies on the impact of sex steroid administration and physical training on GH signaling in human subjects in vivo are required.

Leptin Inhibits Preproinsulin mRNA Expression Induced by Suppression of Cytokine Signalling 3 in Beta-Cells

Aim: To study the role of crosstalk between SOCS3 and leptin on insulin expression in rat insulinoma (RIN-5AH) cells that inducibly express SOCS3 mRNA. Materials and Methods: SOCS3 and preproinsulin mRNA expression induced by 5 μM ponasterone A, and the effects of leptin on SOCS3 and preproinsulin mRNA levels were detected by RT-PCR and quantitative PCR, respectively. The effects of SOCS3 on STAT3 phosphorylation were investigated by Western blot analysis. Results: We discovered that SOCS3 regulates preproinsulin mRNA levels in a dose-dependent and timedependent manner. The insulin-suppressing effect of leptin appears to be mediated through reducing the suppressive effects of SOCS3 on STAT3 phosphorylation. Conclusion: Our findings suggest that leptin inhibits preproinsulin mRNA expression induced by SOCS3 in RIN-5AH beta-cells.

Reactive oxygen species up-regulates SOCS-3 in 3T3-L1 adipocytes

We investigated the effect of increased intracellular reactive oxygen species(ROS) on SOCS-3 expression in 3T3-L1 adipocytes. Increased intracellular ROS levels in 3T3-L1 adipocytes were achieved by two methods of exposure to H2O2 and the occurrence of oxidative stress in cells was assessed by flow cytometry . Expression of SOCS-3 mRNA and that of some adipokines were measured by real time PCR. The level of SOCS-3 protein was determined by western blot. The effect of the antioxidant alpha-lipoic acid was also investigated. Both the relatively mild increased intracellular ROS and the acute but transient increased ROS elevated the levels of SOCS-3 mRNA and protein in 3T3-L1 adipocytes, acompanied with elevated levels of TNF-α mRNA and resistin mRNA and the decreased levels of adiponectin mRNA and secretory adiponectin in culture medium. α-lipoic acid could attenuate the effects of ROS on 3T3-L1 adipocytes. We hypothesized that in mature 3T3-L1 adipocytes, SOCS-3 could be upregulated directly by the induction of increased intracellular ROS and to some extent which was up-regulated by adipokine modulation.

Simvastatin attenuates rhinovirus-induced interferon and CXCL10 secretion from monocytic cells in vitro

RV infections frequently trigger exacerbations of respiratory diseases, such as asthma, yet treatment and intervention options remain limited. Statin drugs are the treatment of choice for dyslipidemia and can also modulate immune cell function. To determine whether statin drugs modify antiviral responses of human monocytic cells, we obtained blood monocytes from donors with allergies and/or asthma and treated the cells with sim prior to challenge with RV. RV-induced secretion of CXCL10 was attenuated significantly, irrespective of RV type (RV-16, -14, or -1A), which corresponded with decreases in IFN-α secretion and pSTAT1. Sim pretreatment also reduced RV-induced CXCL10 secretion from human alveolar macrophages. The addition of mev and GGPP-two intermediates of the cholesterol biosynthesis pathway-was able to rescue CXCL10 release fully, demonstrating that effects of sim were related to inhibition of cholesterol biosynthesis and not to an off-target effect. In addition, sim pretreatment attenuated IFN-α-induced pSTAT1 and CXCL10 secretion, providing evidence that sim additionally can affect type I IFNR signaling. SOCS1 and 3 mRNA are both induced with RV stimulation, but sim did not elevate SOCS1 or SOCS3 mRNA expression basally or in the presence of RV. Our findings suggest that sim inhibition of the cholesterol biosynthesis pathway leads to decreased RV-induced chemokine secretion in monocytes and macrophages. These findings suggest that statin drugs have the potential to curb the inflammatory response to RV infection.

OP0138 Novel Role of Mast Cells in the Regulation of Large Vessel Vasculitis

BackgroundOur previous reports suggested that mast cell-mediated innate immune up-regulation plays a key role in vascular inflammation (1-2). The literature also document increased presence of mast cells in the temporal...

Apocynin inhibits reactive oxygen species (ROS) and downregulates chemokine and cytokine production induced by avian influenza H5N1and H7N9 virus infection. (INC8P.433)

Abstract Influenza A virus infection causes acute inflammation resulting in increased reactive oxygen species (ROS) production and lung injury. ROS are produced following activation of the NADPH oxidase enzyme complex. Apocynin, an inhibitor of NADPH oxidase complex formation has been shown to reduce the accumulation of inflammatory cells in the lungs of influenza-infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of both H5N1 and H7N9 infection in humans, we hypothesized that apocynin could reduce virus-induced cytokine/chemokine production. Human lung epithelial and chicken cell lines were infected with low pathogenic avian influenza H7N9 (A/Anhui/1/2013) and HPAI H5N1 (A/chicken/Vietnam/0008/2004) viruses in the absence or presence of apocynin. Quantitative real-time PCR demonstrated that apocynin inhibited influenza-induced cytokines/chemokines and ROS production following infection. Interestingly, addition of apocynin to in vitro cultures significantly increased SOCS1 and SOCS3 mRNA and protein expression, contributing to the negative regulation of overall cytokine expression. We suggest that intervention strategies targeting both the virus (current therapeutics) and the host e.g. apocynin (future therapeutics) could be used to ameliorate disease in infected individuals, reducing hypercytokinemia and pathology and allowing full development of adaptive immune responses.

P44 H2S down-regulates expression of hepcidin in response to inflammation via inhibition of IL-6/STAT3 and activin B/SMAD1/5/8 pathway

Hepcidin, which is produced primarily by liver, negatively regulates plasma iron. Its expression is induced in response to inflammation, which leads to anemia of inflammation. In this study, we used an in vitro co-culture model to investigate the regulation effect of H 2 S. HuH7 hepatoma cells were exposed to the conditioned medium (CM) which was collected from LPS treated THP-1 macrophages. NaHS, or l -cysteine, was applied at two different stages. One was “pre-treatment” to THP-1 cells which generated different CM. The other was “post-treatment” to HuH7 cells which were suffered same CM. Both treatments with H 2 S down-regulated LPS-induced hepcidin ( HAMP ) mRNA expression. H 2 S also inhibited the HAMP promoter activity induced by IL-6 in HuH7 cells. IL-6 concentration in CM was significantly reduced by H 2 S pre-treatment, so did the phosphorylation of JAK2 and STAT3. Inhibition of IL-6/JAK2/STAT3 pathway by H 2 S counts for the down-regulation of hepcidin expression in pre-treatment model. In post-treatment model, the transcription function of STAT3 was inhibited, which was proved by decreased SOCS3 mRNA expression and phosphorylation of STAT3. Increased SIRT1 activity decreased the acetylation of STAT3, which was related to decreased phosphorylation of STAT3. In vivo results also indicated H 2 S down-regulated hepcidin expression through inhibition of IL-6/Jak2/Stat3 and activin B/Smad1/5/8 pathway. Our data suggest H 2 S inhibits inflammation hepcidin expression via multi-pathways.

Prognoses of patients with acute-on-chronic hepatitis B liver failure are closely associated with altered SOCS1 mRNA expression and cytokine production following glucocorticoid treatment.

Suppressor of cytokine signaling (SOCS) 1 plays a crucial role in the immune response and might contribute to the prognoses of liver failure treated with glucocorticoid. We recruited 47 acute-on-chronic hepatitis B liver failure (ACHBLF) patients receiving glucocorticoid treatment and 30 healthy controls to determine the potential effects of glucocorticoid on the transcriptional level of SOCS1 in peripheral blood mononuclear cells. On the third and twenty-eighth days of glucocorticoid treatment, SOCS1 expression was negatively correlated with model for end-stage liver disease (MELD) score. Interleukin-6 (IL-6) and tumor-necrosis factor-α (TNF-α) levels were statistically lower, while the SOCS1 transcription level was higher in survivors than non-survivors both in pre- and post-treatment ACHBLF patients. The methylation rate of the SOCS1 promoter in ACHBLF patients was higher than in healthy control patients as determined by methylation-specific polymerase chain reaction. The mRNA level of SOCS1 in methylated promoters was significantly lower than from patients with unmethylated SOCS1 promoters. interferon (IFN)-γ-responsive and STAT1-dependent gene expression was higher in survivors and was dramatically decreased with rising expression of SOCS1 after glucocorticoid treatment. Mortality rates were significantly higher in methylated patients than for those without methylation at the end of a 90-day follow-up. Furthermore, we found that five in six surviving patients displayed demethylated SOCS1 on the twenty-eighth day after treatment, while that number was 3 in 10 in the non-survivors. These findings suggested that ACHBLF patients without SOCS1 methylation may have a favorable response to corticosteroid treatment.

Investigation of Biomarkers in Peripheral Blood Cells of Pakistani Chronic HCV Patients with Genotype 3a: An Assessment of HCV-Induced Pathogenesis

ABSTRACT: Aim: The main objective of the current study was to measure the mRNA expression of potent genes involved in oxidative stress, fibrosis and insulin resistance in chronic HCV patients using peripheral white blood cells. Materials &amp; methods: A total of 50 individuals were selected for this study, including 40 chronic HCV patients and ten healthy individuals. RNA was extracted from human peripheral white blood cells of chronic HCV-infected patients and healthy individuals. Transcript analysis of selected genes was performed using real-time PCR. Results: A significant increase was observed in the mRNA expression of Nox4, TGF-β, TNF-α and collagen, while a significant decrease was observed in mRNA expression of Cu/Zn SOD and IL-10. However, there was no significant change found in mRNA expression of SOCS3 and SOCS7 level. Conclusion: The current study confirms the significant expression of different genes in peripheral white blood cells of chronic HCV patients. This could be an easy, cost-efficient approach for the assessment of HCV-induced pathologies at extrahepatic sites, which could be further validated in comparison with liver tissue biopsies.