IFN-? synergistically enhances LPS signalling in alveolar macrophages from COPD patients and controls by corticosteroid-resistant STAT1 activation.
ABSTRACT: BACKGROUND AND PURPOSE: IFN-? levels are increased in chronic obstructive airway disease (COPD) patients compared with healthy subjects and are further elevated during viral exacerbations. IFN-? can 'prime' macrophages to enhance the response to toll-like receptor (TLR) ligands, such as LPS. The aim of this study was to examine the effect IFN-? on corticosteroid sensitivity in alveolar macrophages (AM). EXPERIMENTAL APPROACH: AM from non-smokers, smokers and COPD patients were stimulated with IFN-? and/or LPS with or without dexamethasone. IL-6, TNF-? and IFN-?-induced protein 10?kDa (IP-10) levels were measured by elisa, and Western blots were used to investigate the IFN-?-stimulated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway. Real-time PCR and flow cytometry were used to investigate TLR levels following IFN-? treatment. KEY RESULTS: In all three subject groups, IFN-? alone had no effect on IL-6 and TNF-? production but enhanced the effects of LPS on these cytokines. In contrast, IFN-? alone increased the production of IP-10. IFN-? increased TLR2 and TLR4 expression in AM. Cytokine induction and STAT1 activation by IFN-? were insensitive to dexamethasone for all groups. The inhibition of JAK and STAT1 repressed all these IFN-? effects. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that IFN-?-induced STAT-1 signalling is corticosteroid resistant in AMs, and that targeting IFN-? signalling by JAK inhibitors is a potentially novel anti-inflammatory strategy in COPD.
Project description:CD8+ T-cells are located in the small airways of COPD patients and may contribute to pathophysiology. CD8+ cells express the chemokine receptor, CXCR3 that binds CXCL9, CXCL10 and CXCL11, which are elevated in the airways of COPD patients. These chemokines are released from airway epithelial cells via activation of receptor associated Janus kinases (JAK). This study compared the efficacy of two structurally dissimilar pan-JAK inhibitors, PF956980 and PF1367550, and the glucocorticosteroid dexamethasone, in BEAS-2B and human primary airway epithelial cells from COPD patients and control subjects.Cells were stimulated with either IFN? alone or with TNF?, and release of CXCL9, CXCL10 and CXCL11 measured by ELISA and expression of CXCL9, CXCL10 and CXCL11 by qPCR. Activation of JAK signalling was assessed by STAT1 phosphorylation and DNA binding.There were no differences in the levels of release of CXCL9, CXCL10 and CXCL11 from primary airway epithelial cells from any of the subjects or following stimulation with either IFN? alone or with TNF?. Dexamethasone did not inhibit CXCR3 chemokine release from stimulated BEAS-2B or primary airway epithelial cells. However, both JAK inhibitors suppressed this response with PF1367550 being ~50-65-fold more potent than PF956980. The response of cells from COPD patients did not differ from controls with similar responses regardless of whether inhibitors were added prophylactically or concomitant with stimuli. These effects were mediated by JAK inhibition as both compounds suppressed STAT1 phosphorylation and DNA-binding of STAT1 and gene transcription.These data suggest that the novel JAK inhibitor, PF1367550, is more potent than PF956980 and that JAK pathway inhibition in airway epithelium could provide an alternative anti-inflammatory approach for glucocorticosteroid-resistant diseases including COPD.
Project description:Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-?/?) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.
Project description:The recently discovered IFN-?4 has been found to have antiviral activity against several viruses. However, it's unknown whether IFN-?4 can inhibit HIV infection. Here, we show that IFN-?4 could suppress HIV infection of macrophages. This IFN-?4-mediated HIV inhibition was compromised by the antibodies against IFN-? receptor complex, IFN-?R1/IL-10R2. IFN-?4 enhanced the phosphorylation of STAT1, and induced antiviral interferon-stimulated genes. These findings indicated that IFN-?4 can inhibit HIV via JAK/STAT signalling pathway.
Project description:Hepatitis E virus (HEV) is responsible for large waterborne epidemics of hepatitis in endemic countries and is an emerging zoonotic pathogen worldwide. In endemic regions, HEV-1 or HEV-2 genotypes are frequently associated with fulminant hepatitis in pregnant women, while with zoonotic HEV (HEV-3 and HEV-4), chronic cases of hepatitis and severe neurological disorders are reported. Hence, it is important to characterize the interactions between HEV and its host. Here, we investigated the ability of the nonstructural polyprotein encoded by the first open reading frame (ORF1) of HEV to modulate the host early antiviral response and, in particular, the type I interferon (IFN-I) system. We found that the amino-terminal region of HEV-3 ORF1 (MetYPCP), containing a putative methyltransferase (Met) and a papain-like cysteine protease (PCP) functional domain, inhibited IFN-stimulated response element (ISRE) promoter activation and the expression of several IFN-stimulated genes (ISGs) in response to IFN-I. We showed that the MetYPCP domain interfered with the Janus kinase (JAK)/signal transducer and activator of the transcription protein (STAT) signalling pathway by inhibiting STAT1 nuclear translocation and phosphorylation after IFN-I treatment. In contrast, MetYPCP had no effect on STAT2 phosphorylation and a limited impact on the activation of the JAK/STAT pathway after IFN-II stimulation. This inhibitory function seemed to be genotype-dependent, as MetYPCP from HEV-1 had no significant effect on the JAK/STAT pathway. Overall, this study provides evidence that the predicted MetYPCP domain of HEV ORF1 antagonises STAT1 activation to modulate the IFN response.
Project description:Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes severe human disease, has been shown to block the interferon (IFN)-induced Janus kinase signal transducer and activation of transcription (Jak-Stat) signaling cascade by preventing Tyk2 tyrosine phosphorylation and Stat activation. In this study, we demonstrate that expression of the JEV nonstructural protein NS5 readily blocked IFN-stimulated Jak-Stat signaling events such as Stat1 nuclear translocation and tyrosine phosphorylation of Tyk2 and Stat1. The region of JEV NS5 responsible for Stat1 suppression was identified using various deletion clones. Deletion of 83 N-terminal residues of JEV NS5, but not the 143 C-terminal residues, abolished its ability to block IFN-stimulated Stat1 activation. The role of JEV NS5 as an IFN antagonist was further demonstrated by its ability to block the induction of interferon-stimulated genes and the antiviral effect of IFN-alpha against the IFN-sensitive encephalomyocarditis virus, which appears to replicate and kill cells that express NS5 even with alpha IFN treatment. Furthermore, the molecular mechanism responsible for IFN antagonism by NS5 probably involves protein tyrosine phosphatases (PTPs), as the IFN-blocking events in both JEV-infected and NS5-expressing cells were reversed by sodium orthovanadate, a broad-spectrum inhibitor of PTPs. We suggest that JEV NS5 is an IFN antagonist and that it may play a role in blocking IFN-stimulated Jak-Stat signaling via activation of PTPs during JEV infection.
Project description:BACKGROUND:We previously reported that JAK-STAT-pathway mediated regulation of IFN-regulatory factor genes could play an important role in SLE pathogenesis. Here, we evaluated the efficacy of the JAK inhibitor tofacitinib (TOFA) for controlling IFN signalling via the JAK-STAT pathway and as a therapeutic for SLE. RESULTS:We treated NZB/NZW F1 mice with TOFA and assessed alterations in their disease, pathological, and immunological conditions. Gene-expression results obtained from CD4+ T cells (SLE mice) and CD3+ T cells (human SLE patients) were measured by DNA microarray and qRT-PCR. TOFA treatment resulted in reduced levels of anti-dsDNA antibodies, decreased proteinuria, and amelioration of nephritis as compared with those observed in control animals. Moreover, we observed the rebalance in the populations of naïve CD4+ T cells and effector/memory cells in TOFA-treated mice; however, treatment with a combination of TOFA and dexamethasone (DEXA) elicited a stronger inhibitory effect toward the effector/memory cells than did TOFA or DEXA monotherapy. We also detected decreased expression of several IFN-signature genes Ifit3 and Isg15 in CD4+ from SLE-prone mice following TOFA and DEXA treatment, and IFIT3 in CD3+ T cells from human patients following immunosuppressant therapy including steroid, respectively. CONCLUSION:Modulation of type I IFN signalling via JAK-STAT inhibition may exert a beneficial effect in SLE patients, and our results suggest that TOFA could be utilised for the development of new SLE-specific therapeutic strategies.
Project description:Stringent regulation of the interferon (IFN) signalling pathway is essential for maintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. In resting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumour escape.
Project description:Interferons (IFNs) have been used in the treatment of viral hepatitis. However, their effectiveness is much reduced (<10%) in alcoholics. The mechanism underlying this resistance remains unknown. Here, we report that IFN-alpha/beta and IFN-gamma rapidly activate the JAK-STAT1 (Janus kinase-signal transducer and activator transcription factor 1) and p42/44 mitogen-activated protein kinase (p42/44 MAPK) in freshly isolated rat hepatocytes. Treatment of hepatocytes with 25-100 mM ethanol for 30 min inhibited IFN-beta- or IFN-gamma-induced STAT1 activation and tyrosine phosphorylation. The inhibitory effect of ethanol was not reversed by pretreatment with either sodium vanadate, a non-selective tyrosine phosphatase inhibitor, or with MG132, a specific proteasome inhibitor. This suggests that protein tyrosine phosphatases or the ubiquitin-proteasome pathway are not involved in the inhibitory action of ethanol. In contrast with the JAK-STAT signalling pathway, acute ethanol exposure significantly potentiated IFN-beta or IFN-gamma-induced activation of p42/44 MAPK, and caused marked activation of protein kinase C (PKC). Inhibition of PKC partially antagonized ethanol attenuation of IFN-induced STAT1 activation, suggesting that PKC may be involved. Taken together, these findings suggest that the ability of biologically relevant concentrations of ethanol (less than 100 mM) to markedly inhibit IFN-activated STAT1 is one of the cellular mechanisms responsible for the observed resistance of IFN therapy in alcoholics.
Project description:IFN responses to viral infection are necessary to establish intrinsic antiviral state, but if unchecked can lead to heightened inflammation. Recently, we showed that TLR2 activation contributes to limitation of rhinovirus (RV)-induced IFN response in the airway epithelial cells. We also demonstrated that compared with normal airway epithelial cells, those from patients with chronic obstructive pulmonary disease (COPD) show higher IFN responses to RV, but the underlying mechanisms are not known. Initially, RV-induced IFN responses depend on dsRNA receptor activation and then are amplified via IFN-stimulated activation of JAK/STAT signaling. In this study, we show that in normal cells, TLR2 limits RV-induced IFN responses by attenuating STAT1 and STAT2 phosphorylation and this was associated with TLR2-dependent SIRT-1 expression. Further, inhibition of SIRT-1 enhanced RV-induced IFN responses, and this was accompanied by increased STAT1/STAT2 phosphorylation, indicating that TLR2 may limit RV-induced IFN responses via SIRT-1. COPD airway epithelial cells showed attenuated IL-8 responses to TLR2 agonist despite expressing TLR2 similar to normal, indicating dysregulation in TLR2 signaling pathway. Unlike normal, COPD cells failed to show RV-induced TLR2-dependent SIRT-1 expression. Pretreatment with quercetin, which increases SIRT-1 expression, normalized RV-induced IFN levels in COPD airway epithelial cells. Inhibition of SIRT-1 in quercetin-pretreated COPD cells abolished the normalizing effects of quercetin on RV-induced IFN expression in these cells, confirming that quercetin exerts its effect via SIRT-1. In summary, we show that TLR2 is required for limiting RV-induced IFNs, and this pathway is dysregulated in COPD airway epithelial cells, leading to exaggerated IFN production.
Project description:Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-? only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-? signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-?-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-?-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIB?Vif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-?-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-? pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIB?Vif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be essential for Vif-mediated proteasomal degradation of STAT1 and STAT3. These results reveal a target for HIV-1-Vif and demonstrate how HIV-1 impairs the anti-viral activity of Type 1 IFNs, possibly explaining why both endogenous and therapeutic IFN-? fail to activate more effective control over HIV infection.