Search

Type I (IFNα/β) interferon signaling represents a critical transduction pathway involved in recognition and destruction of nascent tumor cells. Downregulation of this pathway to promote a more immunosuppressed microenvironment contributes to the ability of tumor cells to evade the immune system, a known Hallmark of Cancer. The present study investigates the progesterone receptor (PR), which is expressed in the vast majority of breast cancers, and its ability to inhibit efficient interferon signaling in tumor cells. We have shown that PR can block the interferon signaling cascade by promoting ubiquitination and degradation of STAT2. Targeting STAT2 is critical, as we show that it is an essential protein in inducing transcription of interferon-stimulated genes (ISG); shRNA-mediated knockdown of STAT2 severely abrogates the interferon response in vitro. Importantly, we were able to reverse this inhibition by treating with onapristone, an anti-progestin currently being investigated in breast cancer clinical trials. Additionally, we have found that an interferon-related gene signature (composed of ISGs) is inversely correlated with PR expression in human tumors. We speculate that PR inhibition of interferon signaling may contribute to creating an immunosuppressed microenvironment and reversal of this through anti-progestins may present a novel therapeutic target to promote immune activity within the tumor.

Type I interferons (IFN-I) protect us from viral infections. Signal transducer and activator of transcription 2 (STAT2) is a key component of interferon-stimulated gene factor 3 (ISGF3), which drives gene expression in response to IFN-I. Using electron microscopy, we found that, in naive cells, U-STAT2, lacking the activating tyrosine phosphorylation, forms a heterodimer with U-STAT1 in an inactive, anti-parallel conformation. A novel phosphorylation of STAT2 on T404 promotes IFN-I signaling by disrupting the U-STAT1-U-STAT2 dimer, facilitating the tyrosine phosphorylation of STATs 1 and 2 and enhancing the DNA-binding ability of ISGF3. IKK-ε, activated by virus infection, phosphorylates T404 directly. Mice with a T-A mutation at the corresponding residue (T403) are highly susceptible to virus infections. We conclude that T404 phosphorylation drives a critical conformational switch that, by boosting the response to IFN-I in infected cells, enables a swift and efficient antiviral defense.

OBJECTIVE: To investigate the expression of signal transduction and activator of transcription 2 (STAT2) in ovarian cancer and its correlation with the prognosis of ovarian cancer patients and explore the role of STAT2 inregulating metastasis of ovarian cancer cells. METHODS: RT-qPCR was performed to detect the expression of STAT2 mRNA in 62 fresh frozen ovarian cancer tissues and 62 normal ovarian tissues; immunohistochemistry was used to detect STAT2 protein expressions in 95 paraffin-embedded ovarian cancer samples and 33 normal ovarian tissues. Kaplan-Meier method was used to analyze the correlation between the expression of STAT2 and the prognosis of the patients. We also examined the relationship between STAT2 and the patients' prognosis by analyzing the data in Kaplan-Meier Plotter database. Western blotting was performed to detect the expression of STAT2 in different ovarian cancer cell lines. In A2780 cells with the highest STAT2 expression, we examined the effects of STAT2 interference on cell migration and invasiveness using Transwell migration assay and on the expressions of the downstream molecule epidermal growth factor receptor (EGFR). RESULTS: Ovarian cancer tissues expressed significantly higher levels of STAT2 mRNA than normal ovarian tissue. A high STAT2 mRNA expression was correlated with an advanced FIGO stage. Immunohistochemistry showed that 67.4% of the ovarian cancer samples, as compared with 28.3% of normal ovarian tissues, showed high STAT2 expressions. In ovarian cancer patients, a high expression of STAT2 protein was associated with ascites volume, distant metastasis and FIGO stage (P < 0.05). Survival analysis showed that ovarian cancer patients with a high expression of STAT2 protein had poor overall survival (P=0.021) and progression-free survival (P=0.018). STAT2 was overexpressed in all the ovarian cancer cell lines tested, and A2780 cell lines showed the highest expression. Interference of STAT2 significantly suppressed the migration and invasiveness (P < 0.01) and lowered the expression level of EGFR in A2780 cells. CONCLUSION: STAT2 is overexpressed in ovarian cancer. A high expression of STAT2 is associated with a poor prognosis of ovarian cancer patients. STAT2 may promote the metastasis of ovarian cancer by enhancing the expression of EGFR.

Virus infection of eukaryotes triggers cellular innate immune response, a major arm of which is the type I interferon (IFN) family of cytokines. Binding of IFN to cell surface receptors triggers a signaling cascade in which the signal transducer and activator of transcription 2 (STAT2) plays a key role, ultimately leading to an antiviral state of the cell. In retaliation, many viruses counteract the immune response, often by the destruction and/or inactivation of STAT2, promoted by specific viral proteins that do not possess protease activities of their own. This review offers a summary of viral mechanisms of STAT2 subversion with emphasis on degradation. Some viruses also destroy STAT1, another major member of the STAT family, but most viruses are selective in targeting either STAT2 or STAT1. Interestingly, degradation of STAT2 by a few viruses requires the presence of both STAT proteins. Available evidence suggests a mechanism in which multiple sites and domains of STAT2 are required for engagement and degradation by a multi-subunit degradative complex, comprising viral and cellular proteins, including the ubiquitin–proteasomal system. However, the exact molecular nature of this complex and the alternative degradation mechanisms remain largely unknown, as critically presented here with prospective directions of future study.

Endometrial gene expression is primarily regulated by the ovarian steroids and pregnancy recognition factors. This study was aimed to characterize differential expression genes (DEGs) in bovine endometrium together with the analysis of their promoter region. Bovine uteri at follicular stage (FS), luteal stage (LS), and implantation stage (IS) at Day 18 of pregnancy were collected. Total RNA extracted and prepared cDNA were then subjected to high-throughput sequencing. For promoter analysis, 1 kb upstream promoter region of each DEG was analyzed. The numbers of highly expressed DEGs were 496 and 597 at FS and LS, respectively. When compared the gene expression of IS with LS, 383 and 346 DEGs showed higher and lower expression at IS, respectively. It was also observed that 20-30 transcription factors (TFs) were included in each DEGs. In addition, promoter analyses estimated 150-160 TFs for each stage. DLX4 and interferon regulatory factor 4 (IRF4) at FS, and IRF5, IRF9, STAT1, and STAT2 at IS were in common to DEGs and estimated TFs, respectively. This study highlighted potential molecular mechanisms controlling endometrial function during estrus cycle and IS, which will further guide to better understand the endometrial functions in future studies.

Gruber et al. describe a gain-of-function mutation in STAT2 that leads to a lethal autoinflammatory disease with autosomal recessive inheritance. This syndrome defines a novel form of type I interferonopathy characterized by impaired regulation of the type I IFN response., Type I interferonopathies are monogenic disorders characterized by enhanced type I interferon (IFN-I) cytokine activity. Inherited USP18 and ISG15 deficiencies underlie type I interferonopathies by preventing the regulation of late responses to IFN-I. Specifically, USP18, being stabilized by ISG15, sterically hinders JAK1 from binding to the IFNAR2 subunit of the IFN-I receptor. We report an infant who died of autoinflammation due to a homozygous missense mutation (R148Q) in STAT2. The variant is a gain of function (GOF) for induction of the late, but not early, response to IFN-I. Surprisingly, the mutation does not enhance the intrinsic activity of the STAT2-containing transcriptional complex responsible for IFN-I–stimulated gene induction. Rather, the STAT2 R148Q variant is a GOF because it fails to appropriately traffic USP18 to IFNAR2, thereby preventing USP18 from negatively regulating responses to IFN-I. Homozygosity for STAT2 R148Q represents a novel molecular and clinical phenocopy of inherited USP18 deficiency, which, together with inherited ISG15 deficiency, defines a group of type I interferonopathies characterized by an impaired regulation of late cellular responses to IFN-I., Graphical Abstract

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I: C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish. (C) 2018 Elsevier Ltd. All rights reserved.

The nonstructural protein NSs of severe fever with thrombocytopenia syndrome phlebovirus blocks type I interferon (IFN)-stimulated JAK-STAT signaling. However, there is continuing controversy as to whether NSs targets STAT1 or STAT2 or both for this blockade. The present study was designed to gain a further understanding of the blockade mechanism. Immunoprecipitation experiments revealed a stronger interaction of NSs with STAT2 than with any other component constituting the JAK-STAT pathway. Expression of NSs resulted in the formation of cytoplasmic inclusion bodies (IBs), and affected cytoplasmic distribution of STAT2. STAT2 was relocated to NSs-induced IBs. Consequently, NSs inhibited IFN-α-stimulated tyrosine phosphorylation and nuclear translocation of STAT2. These inhibitory effects as well as the signaling blockade activity were not observed in NSs mutant proteins lacking the STAT2-binding ability. In contrast, NSs affected neither subcellular distribution nor phosphorylation of STAT1 in response to IFN-α and IFN-γ, demonstrating that NSs has little physical and functional interactions with STAT1. Taken together, these results suggest that NSs sequesters STAT2 into NSs-induced IBs, thereby blocking type I IFN JAK-STAT signaling.

Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG‐I‐like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG‐I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

Significance We describe unexpected cooperation between two cytokines that are important in regulating the growth of cancers, namely, type I interferons (IFNs) and interleukin 6 (IL6). It is well established that IL6 is vital for the ability of many tumor types to prosper, and the work in the current paper reveals that the signaling pathway driven by IFN, which is also evident in many cancers, increases the expression of IL6 through a direct effect on the IL6 gene. The findings may help to identify new antitumor targets for therapy., In response to IFNβ, the IL6 gene is activated, modestly at early times by ISGF3 (IRF9 plus tyrosine-phosphorylated STATs 1 and 2), and strongly at late times by U-ISGF3 (IRF9 plus U-STATs 1 and 2, lacking tyrosine phosphorylation). A classical IFN-stimulated response element (ISRE) at −1,513 to −1,526 in the human IL6 promoter is required. Pretreating cells with IFNβ or increasing the expression of U-STAT2 and IRF9 exogenously greatly enhances IL6 expression in response to the classical NF-κB activators IL1, TNF, and LPS. U-STAT2 binds tightly to IRF9, the DNA binding subunit of ISGF3, and also to the p65 subunit of NF-κB. Therefore, as shown by ChIP analyses, U-STAT2 can bridge the ISRE and κB elements in the IL6 promoter. In some cancer cells, the protumorigenic activation of STAT3 will be enhanced by the increased synthesis of IL6 that is facilitated by high expression of U-STAT2 and IRF9.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30–50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

Influenza viruses, the main cause of respiratory tract diseases, cause high morbidity and mortality in humans. Excessive inflammation in the lungs is proposed to be a hallmark for the severe influenza virus infection, especially influenza A virus infection. Strategies against inflammation induced by influenza A virus infection could be a potential anti-influenza therapy. Here, lethal dose of mouse-adapted H1N1 strain PR8A/PR/8/34 was inoculated C57BL/6 mice to detect the anti-influenza activity of andrographolide, the active component of traditional Chinese medicinal herb Andrographis paniculata, with or without influenza virus entry inhibitor CL-385319. Treatment was initiated on 4 days after infection. The survival rate, body weight, lung pathology, viral loads, cytokine expression were monitored in 14 days post inoculation. The combination group had the highest survival rate. Andrographolide treatment could increase the survival rate, diminish lung pathology, decrease the virus loads and the inflammatory cytokines expression induced by infection. Mechanism studies showed the NF-κB and JAK-STAT signaling pathway were involved in the activity of andrographolide. In conclusion, combination of virus entry inhibitor with immunomodulator might be a promising therapeutic approach for influenza.

Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity. We previously demonstrated by using protein crystallography that two molecules of Y3 (the C-terminal half of the C protein) can bind to the homodimer of the N-terminal domain of STAT1 (STAT1ND), elucidating the mechanism of inhibition of IFN-γ signal transduction. SeV C protein also blocks the signal transduction of IFN-α/β by inhibiting the phosphorylation of STAT1 and STAT2, although the mechanism for the inhibition is unclear. Therefore, we sought to elucidate the mechanism of inhibition of the IFN signal transduction via STAT1 and STAT2. Small angle X-ray scattering analysis indicated that STAT1ND associates with the N-terminal domain of STAT2 (STAT2ND) with the help of a Gly-rich linker. We generated a linker-less recombinant protein possessing a STAT1ND:STAT2ND heterodimeric structure via an artificial disulfide bond. Analytical size-exclusion chromatography and surface plasmon resonance revealed that one molecule of Y3 can associate with a linker-less recombinant protein. We propose that one molecule of C protein associates with the STAT1:STAT2 heterodimer, inducing a conformational change to an antiparallel form, which is easily dephosphorylated. This suggests that association of C protein with the STAT1ND:STAT2ND heterodimer is an important factor to block the IFN-α/β signal transduction.

The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-β) and type II interferon (IFN-ɣ) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-ɣ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-ɣ-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-ɣ-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.

Dengue fever is a significant mosquito-borne viral disease that affects millions of people every year. As a co-existing mechanism, DENV has evolved to evade elimination by the host antiviral immune system. DENV is reported to modulate host interferon response either by attenuating the factors that mediate interferon response like STAT1 and STAT2 or inhibiting the activation of STAT1 or by STAT2 degradation. Through this study we aim to understand how DENV modulates STAT3 mediated interferon response to its own advantage. We employed various techniques like Western blot, Confocal microscopy, RT-PCR to show that STAT3 acts as a pro-viral factor for DV-2 propagation. As per result of the present study STAT3 is upregulated as well as activated by phosphorylation in DV-2 infected A549 cells. Additionally, STAT3 knockdown led to a significant decrease in expression of viral proteins as well as viral replication. We show that DV-2 strategically tweaks STAT3 which is a negative regulator of Type I IFN signaling, in order to evade host Type I and Type III interferon response by upregulating its expression and activation. Our results demonstrate the proviral role of STAT3 for DV-2 propagation which is correlated to activation by tyrosine phosphorylation. Furthermore, since STAT3 is critical factor for DV-2 propagation, its modulation can facilitate targeted development of antivirals against Dengue.

Heartland virus (HRTV) is an emerging tick-borne virus (Bunyaviridae, Phlebovirus) that has caused sporadic cases of human disease in several central and mid-eastern states of America. Animal models of HRTV disease are needed to gain insights into viral pathogenesis and advancing antiviral drug development. Presence of clinical disease following HRTV challenge in hamsters deficient in STAT2 function underscores the important role played by type I interferon-induced antiviral responses. However, the recovery of most of the infected animals suggests that other mechanisms to control infection and limit disease offer substantial protection. The most prominent disease sign with HRTV infection in STAT2 knockout hamsters was dramatic weight loss with clinical laboratory and histopathology demonstrating acute inflammation in the spleen, lymph node, liver and lung. Finally, we show that HRTV disease in hamsters can be prevented by the use of favipiravir, a promising broad-spectrum antiviral in clinical development for the treatment of influenza.

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

Influenza A affects a large population worldwide. Influenza virus evades immune responses via various mechanisms, including through the modification of the immune microenvironment. Influenza virus non-structural protein 1 (NS1) encoded by the virus genome inhibits type I interferon (IFN) signaling pathways, which is essential for viral clearance. However, the precise mechanisms of NS1-mediated immune suppression remain poorly understood. The results of the present study demonstrated that mice infected with NS1-expressing influenza A H1N1 virus had lower expression levels of IFNβ in the lung. In addition, it was revealed that the human alveolar epithelial A549 cell line infected with influenza virus A H1N1 produced antiviral IFNβ. The production of IFNβ during infection was demonstrated to be a self-dependent autocrine process. A549 cells transfected with H1N1 NS1 lost the ability to produce IFNβ upon H1N1 infection or IFNβ stimulation. NS1 inhibited the expression of IFN receptors. Furthermore, NS1 inhibited the activation of signal transducers and activators of transcription (STAT)1 and STAT2, as well as the consequent IFNβ production. These data indicate that NS1 serves an important role during virus evasion by affecting the production of IFNβ via multiple mechanisms.

Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together, these data reveal a new mechanism for immune evasion of PKV.

Enterovirus 71 (EV71) has emerged as one of the most important enteroviruses since the eradication of poliovirus, and it causes severe neurological symptoms for which no effective antiviral drugs are available. Type I interferons (IFN) α/β have been used clinically as antiviral therapy as the first line of defense against virus infections successfully for decades. However, treatment with type I interferons has not been effective in patients with EV71 infection. In this study, we found that in cells pretreated with IFN-β, EV71 infection could still lead to a cytopathic effect, and the viral replication was not affected. The mechanism by which EV71 antagonizes interferon signaling, however, has been controversial. Our study indicated that EV71 infection did not inhibit phosphorylation of STAT1/2 induced by IFN-β stimulation, but p-STAT1/2 transport into the nucleus was significantly blocked. We showed that EV71 infection reduced the formation of STAT/karyopherin-α1 (KPNA1) complex upon interferon stimulation and that the virus down-regulated the expression of KPNA1, a nuclear localization signal receptor for p-STAT1. Using specific caspase inhibitors and siRNA for caspase-3, we demonstrated that EV71 infection induced degradation of cellular KPNA1 in a caspase-3-dependent manner, which led to decreased induction of interferon-inducible genes and IFN response. Viral 2A and 3C proteases did not degrade KPNA1, inhibit the activity of ISRE or suppress the transcription of interferon-inducible genes induced by IFN-β. Our study demonstrates a novel mechanism by which antiviral signaling is suppressed through degradation of KPNA1 by activated caspase-3 induced in an enteroviral infection.

IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

Background/Aims: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts. Methods: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes. Results: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL. Conclusion: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.

In fish, as well as vertebrates, type I interferons (IFNs) are important cytokines that help to provide innate, antiviral immunity. Although low amounts of IFN are constitutively secreted under normal physiological conditions, long-term and excessive IFN stimulation leads to reduced sensitivity to the IFN signal. This provides a negative feedback mechanism that prevents inappropriate responses and autoimmunity. At present, however, neither IFN desensitization nor the normal physiological role of constitutive IFN are well characterized in fish. The objective here was therefore to produce and characterize a transgenic medaka fish (Oryzias latipes), designated IFNd-Tg, that constitutively overexpressed the IFNd gene. A dual promoter expression vector was constructed for overexpression of IFNd under an EF1α promoter and a DsRed reporter gene under control of a γF-crystaline promoter. The phenotype of the IFNd-Tg fish had a lower response to poly(I:C) and increased susceptibility to nervous necrosis virus (NNV) infection compared to wild-type (WT). Furthermore, transduction of IFN signals for STAT1b, STAT2 and IRF9 were down-regulated in the IFNd-Tg fish, and expression levels of RLR signal molecules (MDA5, MITA, IRF1 and IRF3) were lower than in WT. The constitutive overexpression of IFNd resulted in desensitization of IFN-stimulation, apparently due to downregulation of IFN signal transduction, and this caused increased susceptibility to NNV.

An improved understanding of the mechanism of interferon (IFN)α activation in systemic lupus erythematosus (SLE) is likely to aid the identification of effective therapeutic targets. Increasing evidence has indicated that the activity of IFNα is mediated by the interplay of ubiquitylation/deubiquitylation enzyme regulators. The present study identified the deubiquitylation enzyme ubiquitin‑specific‑processing protease 7 (USP7) as a critical regulator of the human IFNα‑2 receptor (IFNAR1) protein levels. A co‑immunoprecipitation assay was used to demonstrate that USP7 was physically associated with IFNAR1 in vivo. A glutathione S‑transferase pull down assay revealed that USP7 interacted with IFNAR1 directly in vitro. Furthermore, USP7 may disassemble IFNAR1 dependent poly‑ubiquitin chains and stabilize IFNAR1 in vivo. The activation effects of USP7 on the IFNα pathway were confirmed by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Knockdown of USP7 expression consistently reduced the expression levels of signal transducer and activator of transcription (STAT)‑1, STAT‑2 and selected IFN‑inducible genes, including IFN‑induced protein with tetratricopeptide repeats 3, MX dynamin like GTPase 1 and 2'‑5'‑oligoadenylate synthetase 1. The present study demonstrated that USP7 was significantly overexpressed in 210 SLE patients compared with healthy controls. Furthermore, the association between USP7 levels, IFN scores, SLE disease activity index scores and anti‑double stranded DNA were analyzed and, as expected, positive correlations were demonstrated, indicating that USP7 may be associated with SLE disease activity through the stabilization of IFNAR1.

International audience; Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

A family of eleven proteins comprises the Janus kinases (JAK) and signal transducers and activators of transcription (STAT) signaling pathway, which enables transduction of signal from cytokine receptor to the nucleus and activation of transcription of target genes. Irregular functioning of the cascade may contribute to pathogenesis of autoimmune diseases; however, there are no reports concerning autoimmune bullous diseases yet to be published. The aim of this study was to evaluate the expression of proteins constituting the JAK/STAT signaling pathway in skin lesions and perilesional area in dermatitis herpetiformis (DH) and bullous pemphigoid (BP), as well as in the control group. Skin biopsies were collected from 21 DH patients, from 20 BP patients, and from 10 healthy volunteers. The localization and expression of selected STAT and JAK proteins were examined by immunohistochemistry and immunoblotting. We found significantly higher expression of JAK/STAT proteins in skin lesions in patients with BP and DH, in comparison to perilesional skin and the control group, which may be related to proinflammatory cytokine network and induction of inflammatory infiltrate in tissues. Our findings suggest that differences in the JAK and STAT expression may be related to distinct cytokines activating them and mediating neutrophilic and/or eosinophilic infiltrate.

Equid herpesvirus 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the beta interferon (IFN-β) response in vitro in equine endothelial cells (EECs) at 12 h postinfection (hpi). In the present study, we explored the molecular correlates of this inhibition as clues toward an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both Toll-like receptor 3 (TLR3) and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IFN regulatory factor 7 (IRF7) mRNA only at 12 hpi. EHV-1 did not alter the cellular level of Janus-activated kinase 1 (JAK1) at any time point. However, EHV-1 reduced the cellular level of expression of tyrosine kinase 2 (TYK2) at 12 hpi. Downstream of JAK1-TYK2 signaling, EHV-1 blocked the phosphorylation and activation of signal transducer and activator of transcription 2 (STAT2) when coincubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules, confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type I IFN sensitization and induction. IMPORTANCE To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by equid herpesvirus 1 (EHV-1), especially the neurologic form. The interferon (IFN) system, of which type I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type I IFN system has been exploited successfully to treat other viral infections, such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type I IFN has indicated transient induction of type I IFN production followed by a rapid shutdown in vitro in equine endothelial cells (EECs). The significance of our study is the identification of certain steps in the type I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1.

Severity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However its role in sterile inflammation is unknown. We hypothesised that Stat2 determines severity of non-infective inflammation in the pancreas.Wild type (WT) and Stat2−/− mice were injected intraperitoneally with cerulein or L-arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1h and 24h after the final dose of cerulein and up to 96h post L-arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2−/− bone-marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1(Pdx1)-expressing cells.Stat2−/− mice were protected from cerulein- and L-arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2−/− mice had lower cytokine levels including TNFα and IL-10 and reduced NF-kB nuclear localisation in pancreatic tissue compared to WT. Inhibition of TNFα improved (inhibition of IL-10 worsened) cerulein-induced pancreatitis in WT but not Stat2−/− mice. Phosphoproteomics showed down-regulation of mitogen-activated protein kinase (MAPK) mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2flox/Pdx1-cre) reduced pancreatic TNFα expression, but not histological injury or serum amylase. WT/Stat2−/− bone-marrow chimera were protected from pancreatitis irrespective of host or recipient genotype.Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow derived cells.

Type I/III interferons (IFN) induce expression of hundreds of interferon stimulated genes (ISGs) through the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway to combat viral infections. Although JAK/STAT signaling is seemingly straightforward, it is nevertheless subjected to complex cellular regulation. Herein we show that an ubiquitination regulatory X (UBX) domain containing protein, UBXN6, positively regulates JAK-STAT1/2 signaling. Overexpression of UBXN6 enhanced type I/III IFNs-induced ISGs expression, while deletion of UBXN6 inhibited their expression. RNA viral replication was increased in human UBXN6-deficient cell lines, accompanied by a reduction in both type I/III IFN expression, when compared to UBXN6-sufficient cell lines. Mechanistically, UBXN6 interacted with tyrosine kinase 2 (TYK2) and inhibited IFN-β-induced degradation of both TYK2 and type I IFN receptor. These results suggest that UBXN6 maintains normal JAK-STAT1/2 signaling by stabilizing key signaling components during viral infection.

Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever (BHF), is a New World arenavirus that was first isolated in Bolivia from a human spleen in 1963. Due to the lack of a specific vaccine or therapy, this virus is considered a major risk to public health and is classified as a category A priority pathogen by the U.S. National Institutes of Health. In this study, we used DNA vaccination against the MACV glycoprotein precursor complex (GPC) and murine hybridoma technology to generate 25 mouse monoclonal antibodies (MAbs) against the GPC of MACV. Out of 25 MAbs, five were found to have potent neutralization activity in vitro against a recombinant vesicular stomatitis virus expressing MACV GPC (VSV-MACV) as well as against authentic MACV. Furthermore, the five neutralizing MAbs exhibited strong antibody-dependent cellular cytotoxicity (ADCC) activity in a reporter assay. When tested in vivo using VSV-MACV in a Stat2(−/−) mouse model, three MAbs significantly lowered viral loads in the spleen. Our work provides valuable insights into epitopes targeted by neutralizing antibodies that could be potent targets for vaccines and therapeutics and shed light on the importance of effector functions in immunity against MACV. IMPORTANCE MACV infections are a significant public health concern and lead to high case fatality rates. No specific treatment or vaccine for MACV infections exist. However, cases of Junin virus infection, a related virus, can be treated with convalescent-phase serum. This indicates that a MAb-based therapy for MACV could be effective. Here, we describe several MAbs that neutralize MACV and could be used for this purpose.

The functions of the IL-36 cytokines remain poorly understood. We report a previously unrecognized mechanism whereby IL-36 promotes innate antiviral immunity in mouse and human models of herpes simplex virus-1 (HSV-1) infections. HSV-1 actively suppresses production of type I interferon (IFN); our data reveal that IL-36 overcomes this immune evasion strategy by increasing cellular sensitivity to IFN. IL-36β deficient mice display impaired IFN responses and poorly restrict viral replication in skin keratinocytes. In mouse and human keratinocytes IL-36 elicits an antiviral state driven by STAT1 and STAT2 via enhanced expression of IFNAR1 and IFNAR2 subunits of the type I IFN receptor. The degree of IFN regulatory factor 1 (IRF1) involvement is species dependent, with IRF1 playing a more prominent role in human cells. Similar mechanisms are activated by IL-1. Overall, IL-36 acts as an antiviral cytokine by potentiating type I IFN signaling and thereby upholds immune responses to viruses that limit the production of IFNs., Herpes simplex virus-1 (HSV-1) suppresses the induction of interferon to evade antiviral immunity. Here, Wang et al. show that during HSV-1 infection, IL-36 increases cellular sensitivity to interferon through induction of IRF1 and the interferon receptor.

Interferon (IFN) &beta, and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFN&beta, is reprogrammed by crosstalk with TNF. IFN&beta, mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFN&beta, and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFN&beta, and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFN&beta, and TNF.

Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5 h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity. IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Cardiac fibroblasts (CF) play a key role in the homeostasis of the extracellular matrix in cardiac tissue and are newly recognized as inflammatory supporter cells. Besides, CF-to-Cardiac myofibroblast differentiation is commanded by TGF-b, through SMAD signaling pathways, and these last cells are strongly implicated in cardiac fibrosis. In the heart IFN-β is produced by CF; however, the role of IFN-β, STAT proteins, and STAT-homo or heterodimers in the regulation of CF function with or without a fibrotic environment is unknown. CF were isolated from hearts of adult rats, and by western blot analysis we studied STAT1, STAT2, and STAT3 phosphorylation and through specific siRNA against these proteins we analyzed their role in CF functions such as differentiation (α-SMA expression); and pro-collagen type-I synthesis and secretion expression levels; collagen gels contraction and CF migration. In cultured adult rats CF, IFN-β increases phosphorylation of STAT1, STAT2, and STAT3. Both STAT1 and STAT2 were involved in decreasing α-SMA and CF migration induced by TGF-β1. Also, IFN-β through STAT1 regulated pro-collagen type-I protein expression levels, and collagen gels contraction induced by TGF-β1. STAT3 was not involved in any effects of IFN-β studied. In conclusion, IFN-β through STAT1 and STAT2 shows antifibrotic effects on CF TGF-β1-treated, whereas STAT3 did not participate in such effect.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA., A rapid cellular response to interferons (IFNs) is critical for establishing antimicrobial immunity, but how cells switch from from homeostasis to IFN signaling is not fully understood. Here, the authors provide evidence that IFNs induce gene expression by alternating subunits of transcription factor ISGF3.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

Type I interferon (IFN-I) is a family of multifunctional cytokines that modulate the innate and adaptive immunity and are used to treat mastocytosis. Although IFN-I is known to suppress mast cell function, including histamine release, the mechanisms behind its effects on mast cells have been poorly understood. We here investigated IFN-I’s action on mast cells using interferon-α/β receptor subunit 1 (Ifnar1)-deficient mice, which lack a functional IFN-I receptor complex, and revealed that IFN-I in the steady state is critical for mast cell homeostasis, the disruption of which is centrally involved in systemic anaphylaxis. Ifnar1-deficient mice showed exacerbated systemic anaphylaxis after sensitization, which was associated with increased histamine in the circulation, even though the mast cell numbers and high affinity immunoglobulin E receptor (FcεRI) expression levels were similar between Ifnar1-deficient and wild-type (WT) mice. Ifnar1-deficient mast cells showed increased secretory granule synthesis and exocytosis, which probably involved the increased transcription of Tfeb. Signal transducer and activator of transcription 1(Stat1) and Stat2 were unexpectedly insufficient to mediate these IFN-I functions, and instead, Stat3 played a critical role in a redundant manner with Stat1. Our findings revealed a novel regulation mechanism of mast cell homeostasis, in which IFN-I controls lysosome-related organelle biogenesis., This study reveals a novel role for type I interferon in mast cell homeostasis; spontaneous type I interferon signaling regulates the biogenesis of secretory granules and maturation of mast cells via STAT1 and STAT3, and limits the onset of systemic anaphylaxis.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel emerging virus that has been identified in China, South Korea, and Japan, and it induces thrombocytopenia and leukocytopenia in humans with a high case fatality rate. SFTSV is pathogenic to humans, while immunocompetent adult mice and golden Syrian hamsters infected with SFTSV never show apparent symptoms. However, mice deficient for the gene encoding the α chain of the alpha- and beta-interferon receptor (Ifnar1 -/- mice) and golden Syrian hamsters deficient for the gene encoding signal transducer and activator of transcription 2 (Stat2 -/- hamsters) are highly susceptible to SFTSV infection,with infection resulting in death. The nonstructural protein (NSs) of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT proteins.Here, we demonstrated that SFTSV induces lethal acute disease in STAT2-deficient mice but not in STAT1-deficient mice. Furthermore, we discovered that NSs cannot inhibit type I IFN signaling in murine cells due to an inability to bind to murine STAT2. Taken together, our results imply that the dysfunction of NSs in antagonizing murine STAT2 can lead to inefficient replication and the loss of pathogenesis of SFTSV in mice. IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTSV, which has been reported in China,South Korea, and Japan. Here, we revealed that mice lacking STAT2, which is an important factor for antiviral innate immunity, are highly susceptible to SFTSV infection. We also show that SFTSV NSs cannot exert its anti-innate immunity activity in mice due to the inability of the protein to bind to murine STAT2. Our findings suggest that the dysfunction of SFTSV NSs as an IFN antagonist in murine cells confers a loss of pathogenicity of SFTSV in mice., Journal of Virology, 93(10), art.no.e02226-18; 2019

Tightly interlinked feedback regulators control the dynamics of intracellular responses elicited by the activation of signal transduction pathways. Interferon alpha (IFNα) orchestrates antiviral responses in hepatocytes, yet mechanisms that define pathway sensitization in response to prestimulation with different IFNα doses remained unresolved. We establish, based on quantitative measurements obtained for the hepatoma cell line Huh7.5, an ordinary differential equation model for IFNα signal transduction that comprises the feedback regulators STAT1, STAT2, IRF9, USP18, SOCS1, SOCS3, and IRF2. The model‐based analysis shows that, mediated by the signaling proteins STAT2 and IRF9, prestimulation with a low IFNα dose hypersensitizes the pathway. In contrast, prestimulation with a high dose of IFNα leads to a dose‐dependent desensitization, mediated by the negative regulators USP18 and SOCS1 that act at the receptor. The analysis of basal protein abundance in primary human hepatocytes reveals high heterogeneity in patient‐specific amounts of STAT1, STAT2, IRF9, and USP18. The mathematical modeling approach shows that the basal amount of USP18 determines patient‐specific pathway desensitization, while the abundance of STAT2 predicts the patient‐specific IFNα signal response., Mathematical modeling based on quantitative data reveals the molecular mechanisms that cause hypersensitization of interferon alpha (IFNα) signaling by pre‐exposure with a low dose of IFNα and desensitization with a high dose of IFNα.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium., Author summary The spread of invading microbes is frequently contained by an inflammatory response. Yet, some pathogenic microbes have evolved to utilize inflammation for niche generation and to support their metabolism. Here, we demonstrate that S. Typhimurium exploits type I IFN signaling, a prototypical anti-viral response, to foster its own growth in the inflamed gut. In the absence of STAT2-dependent type I IFN, production of neutrophil reactive oxygen species was impaired, and epithelial metabolism returned to homeostatic hypoxia. Consequently, S. Typhimurium was unable to respire in the absence of type I IFN, and failed to expand in the gut lumen. Furthermore, perturbation of the gut microbiota was dependent on type I IFN signaling. Taken together, our work suggests that S. Typhimurium utilizes STAT2-dependent type I IFN signaling to generate a niche in the inflamed intestinal tract and outcompete the gut microbiota.

Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN α and β receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2-IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1-STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways.

The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to enhance viral replication and dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFN-α/β) for its engagement with human signal transducer and activator of transcription 2 (hSTAT2). In this manuscript, we report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFN-α/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses. IMPORTANCE Flaviviruses such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV) are important human pathogens. A common flavivirus trait is the antagonism of interferon (IFN) signaling to enhance viral replication and spread. We report that like ZIKV NS5 and DENV NS5, YFV NS5 binds human STAT2 (hSTAT2) but not mouse STAT2 (mSTAT2), a type I IFN (IFN-α/β) pathway component. Additionally, we show that contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, YFV NS5 is unable to interact with hSTAT2 in murine cells. We demonstrate that mSTAT2 restricts YFV replication in mice and that this correlates with a lack of IFN-α/β-induced YFV NS5 ubiquitination in murine cells. The lack of suitable animal models limits flavivirus pathogenesis, vaccine, and drug research. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.

Background and objectiveSystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes.MethodsChromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression.Results1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression.ConclusionSSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

Background Interferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells. Methods Gene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression. Results Microarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells. Conclusions JAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression. Electronic supplementary material The online version of this article (10.1186/s13045-019-0722-9) contains supplementary material, which is available to authorized users.

Key Points A stat2−/− Oncorhynchus tshawytscha cell line was isolated. STAT2 is required for type I but not type II signaling in salmonid cells., IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.