Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-β and IFN-γ) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-β suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-γ reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-β preferentially induced SOCS1 rather than SOCS3, whereas IFN-γ strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-β-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-γ-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-β-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-γ with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-γ-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes.     

Inhibitor of κB kinase epsilon (IKK(epsilon)), STAT1, and IFIT2 proteins define novel innate immune effector pathway against West Nile virus infection

West Nile virus is an emerging virus whose virulence is dependent upon viral evasion of IFN and innate immune defenses. The actions of IFN-stimulated genes (ISGs) impart control of virus infection, but the specific ISGs and regulatory pathways that restrict West Nile virus (WNV) are not defined. Here we show that inhibitor of κB kinase ε (IKKε) phosphorylation of STAT1 at serine 708 (Ser-708) drives IFIT2 expression to mediate anti-WNV effector function of IFN. WNV infection was enhanced in cells from IKKε(-/-) or IFIT2(-/-) mice. In IKKε(-/-) cells, the loss of IFN-induced IFIT2 expression was linked to lack of STAT1 phosphorylation on Ser-708 but not Tyr-701 nor Ser-727. STAT1 Ser-708 phosphorylation occurs independently of IRF-3 but requires signaling through the IFN-α/β receptor as a late event in the IFN-induced innate immune response that coincides with IKKε-responsive ISGs expression. Biochemical analyses show that STAT1 tyrosine dephosphorylation and CRM1-mediated STAT1 nuclear-cytoplasmic shuttling are required for STAT1 Ser-708 phosphorylation. When compared with WT mice, WNV-infected IKKε(-/-) mice exhibit enhanced kinetics of virus dissemination and increased pathogenesis concomitant with loss of STAT1 Ser-708 phosphorylation and IFIT2 expression. Our results define an IFN-induced IKKε signaling pathway of specific STAT1 phosphorylation and IFIT2 expression that imparts innate antiviral immunity to restrict WNV infection and control viral pathogenesis.     

Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

Background

Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes.

Methodology/Principal Findings

Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta.

Conclusions/Significance

We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV.     

Analysis of interferon signaling by infectious hepatitis C virus clones with substitutions of core amino acids 70 and 91

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.     

Identification and partial characterization of FRAG-6, a novel interferon-stimulated gene that is expressed in an IRF-1-INDEPENDENT manner.

In order to identify new interferon-stimulated genes that could help in the better understanding of the mechanism of action of interferons (IFNs), we decided to compare, by differential display RT-PCR (DDRT-PCR), the pattern of gene expression between IFN-alpha treated and untreated mouse embryonic fibroblasts (MEFs). Here we describe the initial characterization of a new cDNA fragment, named FRAG-6, that is expressed only upon IFN stimulation. The IFN-induced expression of this new gene can be observed in both wild-type and IRF-1-deficient MEF. FRAG-6 cDNA hybridizes with an mRNA of 6-9 kb that is induced by IFNs in a time-dependent manner. Analysis of the cloned nucleotide sequence revealed a 174 amino acid (aa) open reading frame (ORF) contained within the 576 bp. No significant homology with known nucleotide or protein sequences was observed. FRAG-6 is induced in vitro upon treatment of wild type or IRF-1-null cells with IFN-alpha or -gamma, but not with TNF or IL-1. Treatment of mice with imiquimod, a potent inducer of IFN, led to induced expression of FRAG-6 mRNA in various organs from wild type or IRF-1-deficient mice, but not from STAT-1 or type I IFN receptor deficient animals. Our results demonstrate that FRAG-6 mRNA induction by interferons is IRF-1-independent and it is likely to be activated by the JAK/STAT pathway. Further characterization of FRAG-6 will help us in the understanding of the mechanism of action of IFNs.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Is sprint exercise a leptin signaling mimetic in human skeletal muscle?

This study was designed to determine whether sprint exercise activates signaling cascades linked to leptin actions in human skeletal muscle and how this pattern of activation may be interfered by glucose ingestion. Muscle biopsies were obtained in 15 young healthy men in response to a 30-s sprint exercise (Wingate test) randomly distributed into two groups: the fasting (n = 7, C) and the glucose group (n = 8, G), who ingested 75 g of glucose 1 h before the Wingate test. Exercise elicited different patterns of JAK2, STAT3, STAT5, ERK1/2, p38 MAPK phosphorylation, and SOCS3 protein expression during the recovery period after glucose ingestion. Thirty minutes after the control sprint, STAT3 and ERK1/2 phosphorylation levels were augmented (both, P < 0.05). SOCS3 protein expression was increased 120 min after the control sprint but PTP1B protein expression was unaffected. Thirty and 120 min after the control sprint, STAT5 phosphorylation was augmented (P < 0.05). Glucose abolished the 30 min STAT3 and ERK1/2 phosphorylation and the 120 min SOCS3 protein expression increase while retarding the STAT5 phosphorylation response to sprint. Activation of these signaling cascades occurred despite a reduction of circulating leptin concentration after the sprint. Basal JAK2 and p38 MAPK phosphorylation levels were reduced and increased (both P < 0.05), respectively, by glucose ingestion prior to exercise. During recovery, JAK2 phosphorylation was unchanged and p38 MAPK phosphorylation was transiently reduced when the exercise was preceded by glucose ingestion. In conclusion, sprint exercise performed under fasting conditions is a leptin signaling mimetic in human skeletal muscle.