SOCS3 Gene Expression Research Articles

Expression of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis

BackgroundAppropriate immune activation of T cells and macrophages is central for the control of Mycobacterium tuberculosis infections. IFN-γ stimulated responses are lowered in tuberculosis (TB), while expression of Suppressor of Cytokine Signaling (SOCS) molecules – 1 and 3 and CD4+CD25+FoxP3+T regulatory cells is increased. Here we investigated the association of these molecules in regard to clinical severity of TB.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from patients with pulmonary TB (PTB, n = 33), extra-pulmonary TB (ETB, n = 33) and healthy endemic controls (EC, n = 15). Cases were classified as moderately advanced or far advanced PTB, and less severe or severe disseminated ETB. M. tuberculosis -stimulated IFN-γ, SOCS1, SOCS3 and FoxP3 gene expression and secretion of Th1 and Th2 cytokines was measured. Statistical analysis was performed using Mann–Whitney U, Wilcoxon Rank and Kruskal Wallis non-parametric tests.ResultsIn un-stimulated PBMCs, IL-6 (p = 0.018) and IL-10 (p = 0.013) secretion levels were increased in PTB while IL-10 was also increased in ETB (p = 0.003), all in comparison with EC. M. tuberculosis-stimulated IL-6 (p = 0.003) was lowered in ETB as compared with EC. SOCS1 mRNA expression in M. tuberculosis stimulated PBMCs levels in moderately advanced PTB (p = 0.022), far advanced (p = 0.014) PTB, and severe ETB (p = 0.009) were raised as compared with EC. On the other hand, SOCS1 mRNA titers were reduced in less severe ETB, in comparison with severe ETB (p = 0.027) and far advanced PTB (p = 0.016). SOCS3 mRNA accumulation was reduced in far advanced PTB (p = 0.007) and FoxP3 mRNA expression was increased in less severe ETB as compared with EC (p = 0.017).ConclusionsThe lowered SOCS1 mRNA levels in patients with less severe extra-pulmonary TB as compared to those with more severe ETB and PTB may lead to elevated IFN-γ pathway gene expression in the latter group. As localized ETB has shown to be associated with more effective Th1 immunity and adaptive responses, this suggests a role for SOCS1 in determining disease outcome in extra-pulmonary TB.

Inhibition of Th17 cells and induction of regulatory T cells is mediated by SOCS-1 in the tumor microenvironment of breast cancer patients

Event Abstract Back to Event Inhibition of Th17 cells and induction of regulatory T cells is mediated by SOCS-1 in the tumor microenvironment of breast cancer patients Arash Pourgholaminejad1, 2*, Rahil Eftekhari1, Keivan Majidzadeh3 and Jamshid Hadjati1 1 School of Medicine- Tehran University of Medical Sciences, Department of Immunology, Iran 2 Faculty of Medical Sciences- Tarbiat Modares University, Department of Immunology, Iran 3 Iranian Center for Breast Cancer (ICBC); Academic Center for Education, Culture and Research (ACECR), Iran Introduction: Suppressors of cytokine signaling (SOCS) are important negative feedback regulators of the JAK/STAT signaling pathway, and have been recently investigated for their role in the development of different cancers. It has been shown that SOCS-1 negatively regulates STAT-3 activation in breast cancer tissues and therefore it may be a regulator of Th17 cells in tumor area. In the preset study, we demonstrated that SOCS-1 is in associated with decrease of Th17 cells and increase of Tregs in breast cancer patients. Methods and Materials: In this study, we examined the expression of SOCS-1 gene in normal and breast cancer tissue and correlated this with factors related to Th17 cells and Tregs. Human breast cancer tissues from 4 stages and normal tissues were collected. Afterward, mRNA was extracted from the tissues and cDNA was synthesized. Subsequently, the mRNA expression of genes related to Th17 cells (IL-17, RORc, STAT-3) and Tregs (FoxP3, IL-10, CTLA-4) and also SOCS-1 were determined by Quantitative Real-time PCR. Results: The results showed that the expression of SOCS-1 gene is increased in breast cancer and it negatively associated with IL-17, RORc and STAT-3 gene expression significantly and also factors related to regulatory T cells such as IL-10, FoxP3 and CTLA-4 is decreased. Conclusion: We concluded that regulatory T cell responses increase in the tumor microenvironment and that inversely correlates with Th17 cells as a potent anti-tumor immune response. It seems that SOCS-1 inhibits Th17 and also induces Tregs in breast cancer patients with advanced stages. Keywords: SOCS-1, Th17, Treg, breast cancer, Tumor Microenvironment Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Pourgholaminejad A, Eftekhari R, Majidzadeh K and Hadjati J (2013). Inhibition of Th17 cells and induction of regulatory T cells is mediated by SOCS-1 in the tumor microenvironment of breast cancer patients. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00088 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Arash Pourgholaminejad, School of Medicine- Tehran University of Medical Sciences, Department of Immunology, Tehran, Iran, arash_pgn@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Arash Pourgholaminejad Rahil Eftekhari Keivan Majidzadeh Jamshid Hadjati Google Arash Pourgholaminejad Rahil Eftekhari Keivan Majidzadeh Jamshid Hadjati Google Scholar Arash Pourgholaminejad Rahil Eftekhari Keivan Majidzadeh Jamshid Hadjati PubMed Arash Pourgholaminejad Rahil Eftekhari Keivan Majidzadeh Jamshid Hadjati Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Long-term fasting in the anadromous Arctic charr is associated with down-regulation of metabolic enzyme activity and up-regulation of leptin A1 and SOCS expression in the liver.

The life strategy of the anadromous Arctic charr (Salvelinus alpinus) includes several months of voluntary fasting during overwintering in freshwater, leading to emaciation prior to seawater migration in spring. In this study we compared changes in condition, substrate utilization and liver metabolism between captive anadromous charr subjected to food deprivation during late winter and spring, and conspecifics fed in excess. In March, nine out of the 10 sampled fed fish had not eaten, indicating that they were in a voluntary anorexic state. In June, the fed fish were eating and all had higher body mass, condition factor and adiposity than in March. In fasted fish there were only small decreases in body mass, condition factor and adiposity between March and May, but all these parameters decreased markedly from May to June. The fasted fish were depleted in fat and glycogen in June, had suppressed activity of hepatic enzymes involved in lipid metabolism (G6PDH and HOAD) and seemed to rely on protein-derived glucose as a major energy source. This was associated with upregulated liver gene expression of leptin A1, leptin A2, SOCS1, SOCS2 and SOCS3, and reduced IGF-I expression. In an in vitro study with liver slices it was shown that recombinant rainbow trout leptin stimulated SOCS1 and SOCS3 expression, but not SOCS2, IGF-I or genes of enzymes involved in lipid (G6PDH) and amino acid (AspAT) metabolism. It is concluded that liver leptin interacts with SOCS in a paracrine fashion to suppress lipolytic pathways and depress metabolism when fat stores are depleted.

Chronic helicobacter pylori infection induces SOCS1 expression but not CISH in a murine model of gastric infection

Background and Aims Helicobacter pylori infections continue to be a major challenge to the field of gastroenterology. Gastric H. pylori infections is a critical cause of chronic atrophic gastritis and peptic ulcer disease and is one of the leading carcinogens that leads to stomach cancer and MALT lymphoma. Helicobacter pylori infection has been demonstrated to modulate the local inflammatory response in the gastric mucosa. In this study, we examine the role of chronic in vivo H. pylori infection in the modulation of inflam-mation controlled by Supressors of Cytokine Signalling (SOCS), a family of anti-inflammatory regulators that act on various cytokine pathways. Methods C57Bl6 mice were infected at 5 weeks of age with the mouse-adapted H. pylori strain SS1 via oral gavage × 3 inoculations. The mice were culled 20 weeks post-infection and the stomach tissue harvested post-mortem and snap frozen. Total RNA was extracted from these tissues and cDNA produced from the mRNA by reverse transcription. A semi-quantitative real time PCR study was conducted examining Socs1 and Cish relative gene expression in infected ( n = 10) and uninfected controls ( n = 10). Results Mice infected with H. pylori had a significantly increased gene expression of Socs1 ( p Cish gene expression was not signifi-cantly altered upon infection. Conclusions Helicobacter pylori may supress localised inflam-mation via increased expression of SOCS1 in a murine model of chronic infection.

Effect of leptin and photoperiod on pituitary SOCS-3 gene expression in sheep – in vitro study

Effect of leptin and photoperiod on pituitary SOCS-3 gene expression in sheep – in vitro study

Cycloheximide stimulates suppressor of cytokine signaling-3 gene expression in 3T3-L1 adipocytes via the extracellular signal-regulated kinase pathway

Suppressor of cytokine signaling (SOCS)-3 can act as a regulator of energy metabolism and cytokine signaling in fat cells. It is regulated by hormones and toxicological factors. However, the action of cycloheximide on expression of adipocyte SOCS-3 gene is unknown. Using 3T3-L1 adipocytes, we found that cycloheximide up-regulated SOCS-3 mRNA expression in dose- and time-dependent manners. Treatment with actinomycin D prevented cycloheximide-stimulated SOCS-3 mRNA expression, suggesting that the effect of cycloheximide requires new mRNA synthesis. While cycloheximide was shown to increase activities of MEK1 and JNK, signaling was demonstrated to be inhibited by pretreatment with either MEK1 inhibitors U0126 and PD98059, or with the JNK inhibitor SP600125. U0126 and PD98059, respectively, reduced cycloheximide-stimulated SOCS-3 mRNA expression, but SP600125 did not antagonize cycloheximide effect. Moreover, cycloheximide was observed to up-regulate expression of other SOCS family members, such as SOCS-1, -2, -4, -5, -6, -7, and cytokine-inducible SH2-containing protein (CIS)-1 mRNAs. Such effects varied with the dosage and duration of cycloheximide treatment. These results imply the functional MEK1/ERK-mediated pathway is necessary for the cycloheximide stimulation of SOCS-3 gene expression.

Simvastatin inhibits secretion of Th17‐polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis

Statins, widely used cholesterol-lowering agents, have also been demonstrated to have antiinflammatory effects. Here, we characterize the capacity of simvastatin to target DCs and modulate T-cell priming and Th17-cell differentiation, in a cohort of patients with relapsing remitting multiple sclerosis (RRMS). We report that simvastatin inhibits IL-1β, IL-23, TGF-β, IL-21, IL-12p70, and induces IL-27 secretion from DCs in RRMS patients, providing an inhibitory cytokine milieu for Th17 and Th1-cell differentiation. The effect on DCs is mediated via induction of SOCS1, SOCS3, and SOCS7 gene expression, which are associated with the inhibition of STAT1, STAT3, and ERK1/2 phosphorylation. A geranylgeranyl transferase inhibitor replicated simvastatin's effects on DC cytokine secretion, implicating that simvastatin-induced depletion of isoprenoids mediates this effect. Simvastatin inhibited antigen presentation by DCs via suppression of the MHC class I expression, costimulatory molecules CD80 and CD40, and by inducing a dramatic loss of dendritic processes. The changes in DC morphology were also mediated via inhibition of geranylgeranylation. The therapeutic use of geranylgeranylation inhibitors may provide selective inhibition of key pathogenic cytokines that drive the autoimmune response in MS; their use represents a promising therapeutic approach that requires further clinical testing.

Expression of SOCS1 and SOCS3 Genes with Human Cytomegalovirus Infection After Allogeneic Hematopoietic Stem Cell Transplantation.

AbstractAbstract 3038 Background:After allogeneic hematopoietic stem cell transplantation (HSCT), cytomegalovirus (CMV) infection is highly prevalent in recipients. Although the suppressor of cytokine signaling (SOCS) genes are mainly regarded as pivotal negative feedback regulators for signaling of cytokines, the expression pattern of SOCS genes in CMV infections remains largely unexplored. To understand the molecular mechanism of cytokine cascades associated with CMV infection, we investigated the expression of SOCS genes from various types of hematologic malignancies after allogeneic HSCT. Methods:In the present study, we investigated SOCS1 and SOCS3 gene expressions, in order to examine the feasibility of SOCS genes as a promising therapeutic target as well as a prognostic predictor in CMV infection. A total of 99 recipients with acute myelogenous leukemia (AML; n=64), acute lymphoblastic leukemia (ALL; n=23), myelodysplastic syndrome (MDS; n=12), who received allogeneic HSCT and 55 normal donors were included. Real-time quantitative polymerase chain reaction (RQ-PCR) was used and all sample analyses were performed in triplicate. Results:The expression levels of the SOCS3 gene were clearly decreased in recipients compared to normal donors, including in the Pre-HSCT, Post-HSCT No CMV, and Post-HSCT CMV subgroups (P=0.0007, P=0.0009, and P=0.0014, respectively). Meanwhile, expressions of SOCS1 gene were significantly decreased in the Pre-HSCT, Post-HSCT No CMV, and normal donors, when compared to Post-HSCT CMV subgroup (P=0.026, P=0.0129, and P<0.0001, respectively), suggesting a correlation between expression of SOCS genes and CMV reactivation. In addition, expression of SOCS1 gene was remarkably increased in patients who received allogeneic HSCT with myeloablative conditioning (MAC), compared to reduced-intensity conditioning transplantation (RIST) and normal donors (P=0.0119, P=0.0002, respectively), while, both regimen with MAC and RIST showed a decreasing pattern with statistical significance compared to normal donors (P=0.0037, P=0.0024, respectively). Notably, SOCS1 gene expression in acute lymphoblastic leukemia and myelodysplastic syndrome recipients were higher than acute myelogenous leukemia, when compare to normal donors (P=0.0239). Furthermore, high expression of SOCS1 gene unarguably showed high mortality (P=0.0068). In AML patients, decreased SOCS3 gene was detected (P=0.0007) and overall high expression of SOCS3 gene was displayed high mortality, but there is no statistical significance. Conclusions:We firstly report that the SOCS genes are differently expressed in human CMV infection after allogeneic HSCT, suggesting a correlation between cytokines by modulation of SOCS genes associated with CMV reactivation. This result provides a new platform for studying CMV immunobiology and these genes may be a potential of diagnostic and therapeutic targets in post-allogeneic HSCT associated with CMV infection. Disclosures:No relevant conflicts of interest to declare.

SOCS1 Gene Expression is Increased in Severe Pulmonary Tuberculosis

Suppressors of cytokine signalling (SOCS) molecules inhibit cytokine signalling and may regulate protective immunity in tuberculosis (TB). We investigated the association of SOCS with disease progression in patients with pulmonary TB. For this purpose, we studied peripheral blood mononuclear cells (PBMCs) and T cells from patients with pulmonary TB (TB, n=33) and healthy endemic controls (EC, n=15). Cases were stratified into those with moderately advanced (Mod-PTB) or far advanced disease (Adv-PTB). Interferon-gamma (IFN-γ), SOCS1 and SOCS3 gene expression was determined by RT-PCR. Statistical analysis was performed using the Mann-Whitney test. Levels of IL6 (P=0.018) and IL10 (P=0.013) were found to be elevated in PBMC supernatants from patients with TB as compared with EC. SOCS1 mRNA gene expression in T cells from patients with TB was increased as compared with that of EC (P=0.02). In addition, levels of SOCS1 mRNA transcripts were found to be elevated in PBMCs of Adv-PTB as compared with Mod-PTB (P=0.008) cases. Our data show that raised SOCS1 levels are associated with increased disease severity in TB. As SOCS1 regulates IFN-γ-driven immunity and SOCS1 can be further upregulated by IL6 levels, the increase in SOCS1 in severe disease indicates a mechanism by which mycobacteria impede disease control in TB.

153P - Tocilizumab can Enhance the Anti-Tumor Effect of IFN-α in Renal Cell Carcinoma

ABSTRACT Background Interleukin 6 (IL-6), one of the proinflammatory cytokines, contributes to the onset and maintenance of various types of cancer. IL-6 signaling induces up-regulation of SOCS3, which leads to interferon (IFN) –α resistance in renal cell carcinoma (RCC) cells. To propose a new treatment for mRCC, we investigated the effect of combination therapy with IFN-α and humanized antihuman IL-6R antibody, tocilizumab. Material and methods Real time PCR (RT-PCR) was used to analyze gene expression of IL-6 and SOCS3 after IFN stimulation and ELISA were used for IL-6 secretion in RCC cell lines. These expression levels were compared among the RCC cell lines, i.e., ACHN, Caki-1, TUHR3TKB, TUHR4TKB and 786-O. Phosphorylation of STAT-1, STAT-3 and ERK were investigated by Western blotting (WB) analysis. We investigated alteration of IL-6 signaling by tocilizumab, and analyzed its impacts on the susceptibility to IFN-α in RCC cells by MTT assay. The effects of tocilizumab on in vivo growth on 786-O xenografts were determined by SOCS3 expression, morphological observation and tumor volume. Results Among the RCC cell lines, the expression levels of IL-6, SOCS3 in RT-PCR and the production level of IL-6 were highest in 786-O. These results revealed a correlation between the expression levels IL-6 and SOCS3 (P = 0.0001, r = 0.99974). Although the 786-O cells had IFN-α resistance, MTT assay showed that the tocilizumab induced a growth inhibitory effect of IFN-α. WB analysis showed that phosphorylation level of STAT-1 was increased and the levels of STAT-3 and ERK were decreased with the simultaneous use of tocilizumab in 786-O cells. An in vivo study demonstrated that tocilizumab promoted IFN-α-induced cell death and growth suppression in 786-O cell xenograft in nude mice. Conclusions IL-6 could be a key component in the resistance to IFN-α treatment of renal cell carcinoma. Antihuman IL-6R antibody can be an effective strategy to enhance the anti-tumor effect of IFN-α, and probably, the effect of angiogenic inhibitors, in human RCC cells. Disclosure All authors have declared no conflicts of interest.

Clinical significance of suppressor of cytokines signalling-3 mRNA expression from patients with non-Hodgkin lymphoma under chemotherapy

To date, little is known about blood immune marker changes that may be related to the development of Non Hodgkin Lymphoma (NHL) and treatment response with few serum biomarkers that could be useful in follow- up of the patients. To quantify the expression of suppressor of cytokine signalling-3-(SOCS-3) gene at the mRNA level in the peripheral blood of patients with NHL and correlate with clinical pathological features and response to treatment. Thirty patients with NHL and 20 healthy controls were enrolled in the study. The SOCS-3 mRNA level in peripheral blood (PB) was detected by semi-quantitative real-time polymerase chain reaction. Quantification of cytokines such as interleukin 6 and tumour necrosis factor alpha (IL-6 & TNF-α) were performed using sandwich enzyme-linked immunosorbent assays (ELISA). Increased expression of SOCS-3 mRNA in peripheral blood plus increased serum levels of IL-6 and TNF alpha from NHL cases with no complete remission after therapy. Higher levels of expression of SOCS-3 are associated with advanced disease, bone marrow involvement, extranodal involvement, poor performance status, B cell symptoms (fever, night sweats and weight loss) and high serum lactate dehydrogenase level which are evaluated by international prognostic index (IPI). Complete responses occur in 60% of patients with normal expression of SOCS-3 gene. Increased expression of SOCS-3 is common in diffuse large B cell lymphoma, CLL/small lymphocytic B cell lymphoma and follicular lymphoma. Over-expression of SOCS-3 mRNA from peripheral blood of NHL patients correlates with advanced disease and poor response to treatment. SOCS-3 mRNA expression in peripheral blood from NHL patients might be used to monitor response during treatment.

Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes

Endothelin (ET)-1 and suppressor of cytokine signaling (SOCS)-3 were respectively found to regulate energy metabolism and hormone signaling in fat cells. Although ET-1 can also regulate the expression of SOCS-3-stimulating hormones, it is still unknown whether ET-1 regulates SOCS-3 gene expression. This study investigated the pathways involved in ET-1’s modulation of SOCS-3 gene expression in 3T3-L1 adipocytes. ET-1 upregulated SOCS-3 mRNA and protein expression in dose- and time-dependent manners. The concentration of ET-1 that increased SOCS-3 mRNA levels by 250–400% was ∼100nM with 2–4h of treatment. Treatment with actinomycin D prevented ET-1-stimulated SOCS-3 mRNA expression, suggesting that the effect of ET-1 requires new mRNA synthesis. Pretreatment with the ET type A receptor (ETAR) antagonist, BQ-610, but not the ET type B receptor (ETBR) antagonist, BQ-788, prevented the stimulatory effect of ET-1 on SOCS-3 gene expression. The specific inhibitors of either MEK1 (U-0126 and PD-98059), JAK (AG-490), JNK (SP-600125), or PI3K (LY-294002 and wortmannin) reduced ET-1-increased levels of SOCS-3 mRNA and respectively inhibited ET-1-stimulated activities of MEK1, JAK, JNK, and PI3K. These results imply that the ETAR, ERK, JAK, JNK, and PI3K are functionally necessary for ET-1’s stimulation of SOCS-3 gene expression. Moreover, ET-1 was observed to upregulate expressions of SOCS-1, -2, -3, -4, -5, and -6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein-1 mRNAs. This suggests that ET-1 selectively affects particular types of SOCS family members. Changes in SOCS gene expressions induced by ET-1 may help explain the mechanism by which ET-1 modulates hormone signaling of adipocytes.

A novel pro-inflammatory mechanism of action of resistin in human endothelial cells: Up-regulation of SOCS3 expression through STAT3 activation

Resistin is a significant local and systemic regulatory cytokine involved in inflammation. Suppressors of cytokine signaling (SOCS) proteins are intracellular regulators of receptor signal transduction induced by several cytokines in a cytokine and cell specific manner. Resistin up-regulates SOCS3 expression in mice adipocytes but it is not known whether this is a common occurrence in other cells. We questioned whether resistin-induces SOCS3 in human endothelial cells and if signal transducer and activator of transcription (STAT) proteins are involved in the process. The Real-Time PCR and Western blot analysis showed that in resistin-activated HEC the gene and protein expression of SOCS3 were significantly increased. Furthermore, resistin induced activation of STAT3 as characterized by increased tyrosine phosphorylation. Resistin-induced SOCS3 expression was blocked by specific inhibitors of STAT3 signaling and by the transfection of siRNA specific for STAT3. Silencing of SOCS3 gene expression by transfection with SOCS3 siRNA reduced the expression of resistin induced-P-selectin and fractalkine in HEC. Together, our results demonstrate that in HEC (1) resistin up-regulates SOCS3 expression and activates STAT3 transcription factor; (2) the increase in SOCS3 mRNA and protein expression as well as STAT3 activation have a long-lasting effect (up to 18h); (3) inhibition of SOCS3 function prevents resistin-induced expression of cell adhesion molecules P-selectin and fractalkine and thus activation of endothelial cells. The data uncover a new resistin-mediated mechanism in human endothelial cells and designate SOCS3 as a novel therapeutic target to modulate resistin-dependent inflammation in vessel wall diseases.

Defective maturation of dendritic cells during HIV-1 infection is associated with increased expression of SOCS-1

Methods DCs from 21 therapy naive (mean CD4: 256 cells/mm), 21 patients on anti-retroviral therapy (mean CD4: 342 cells/mm) and 14 healthy controls were immunophenotyped for maturation markers at baseline and after 5 hour ex vivo stimulation with TLR-4 ligand, LPS, by flowcytometry. Subsequently, the expression of SOCS-1 gene and the cytokine levels were assessed in monocytederived DCs (Mo-DC) of healthy donors exposed to LPS and HIV-1 gp120 by real time PCR and flowcytometry respectively.

Preclinical Evaluation of Local JAK1 and JAK2 Inhibition in Cutaneous Inflammation

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

Tim-3 Negatively Regulates IL-12 Expression by Monocytes in HCV Infection

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.

Growth Hormone (GH)-Induced Insulin Resistance Is Rapidly Reversible: An Experimental Study in GH-Deficient Adults

It is clinically relevant and of physiological interest to investigate whether GH-induced insulin resistance depends on the timing of GH exposure relative to when insulin sensitivity is assessed. GH-induced insulin resistance is rapidly reversible. Eight male GH-deficient patients underwent a 6-h euglycemic-hyperinsulinemic glucose clamp thrice in a randomized crossover design receiving either no GH (study 0), a 7-h GH infusion (0.2-0.3 mg in total) that terminated 5 h before the clamp (study 1), or a similar GH infusion timed to continue during the first hour of the clamp (study 2). A muscle biopsy was obtained 30 min into the clamp. The patients were compared with eight healthy untreated control subjects (study c). The glucose infusion rate, indirect calorimetry, and free fatty acid metabolism were assessed. In muscle biopsies, protein phosphorylation of signal transducer and activator of transcription 5, Akt, and Akt substrate 160 (phospho-Akt substrate signal) and gene expression of IGF-I and SOCS1-3 were assessed. Insulin sensitivity differed significantly between the GH-deficiency studies (P = 0.005) with distinct insulin resistance in study 2 and increased insulin sensitivity in study 0 [area under the glucose infusion rate curve (mg/kg · min): 1663 ± 151 (study 0) vs. 1482 ± 166 (study 1) vs. 1123 ± 136 (study 2) vs. 1492 ± 229 (control group)]. Free fatty acid levels and lipid oxidation were elevated in response to GH exposure but became suppressed during the clamp. IGF-I and SOCS3 gene expression was increased in study 2. Very-low-dose GH exposure evokes acute insulin resistance that subsides after 5 h. This time-dependent reversibility should be considered when assessing the impact of GH on glucose homeostasis.

Lactobacillus rhamnosus GGandStreptococcus thermophilusinduce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.

Heme Oxygenase-1 Couples Activation of Mitochondrial Biogenesis to Anti-inflammatory Cytokine Expression

The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2(-/-) mice. Nfe2l2(-/-) mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2(-/-) mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-x(L) gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis.

Expression of suppressor of cytokine signaling 1 and 3 in ligature-induced periodontitis in rats

Objective: Evaluate expression of inducible negative regulators of JAK/STAT pathway and their target proteins during the course of ligature-induced experimental periodontal disease in rats. Design: Rats were sacrificed 07, 15 and 30days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western blot. Results: Ligature-induced model presented significant progressive bone loss from 7 to 30days. Inflammation was evident and similar for 07 and 15days; however, a decrease on severity at the end of the experimental period was observed. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control group at 15 and 30days. The SOCS1 and SOCS3 protein expression and activation of STAT1 and STAT3 were increased in earlier periods in the ligature model. Conclusion: This study suggests that SOCS1 and SOCS3 were directly correlated with regulatory mechanism of the inflammatory process responsible for the periodontal disease destruction.