IFN-independent Manner Research Articles

The Evil DExH/D: Chikungunya virus runs but cannot hide from DDX39A

In this issue, Tapescu etal.1 identify DDX39A as a novel antiviral protein that acts on conserved features of alphavirus RNA to limit infection in an IFN-independent manner.

Antiviral response and immunopathogenesis of interleukin 27 in COVID-19

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a high mortality rate. The clinical course is attributed to the severity of pneumonia and systemic complications. In COVID-19 patients and murine models of SARS-CoV-2 infection, the disease may be accompanied by excessive production of cytokines, leading to an accumulation of immune cells in affected organs such as lungs. Previous reports have shown that SARS-CoV-2 infection antagonizes interferon (IFN)-dependent antiviral response, thereby preventing the expression of IFN-stimulated genes (ISGs). Lower IFN levels have been linked to more-severe COVID-19. Interleukin 27 (IL27) is a heterodimeric cytokine composed of IL27p28 and EBI3 subunits, which induce both pro- and anti-inflammatory responses. Recently, we and others have reported that IL27 also induces a strong antiviral response in an IFN-independent manner. Here, we investigated transcription levels of both IL27 subunits in COVID-19 patients. The results show that SARS-CoV-2 infection modulates TLR1/2-MyD88 signaling in PBMCs and monocytes and induces NF-κB activation and expression of NF-κB-target genes that are dependent on a robust pro-inflammatory response, including EBI3; and activates IRF1 signaling which induces IL27p28 mRNA expression. The results suggest that IL27 induces a robust STAT1-dependent pro-inflammatory and antiviral response in an IFN-independent manner in COVID-derived PBMCs and monocytes as a function of a severe clinical course of COVID-19. Similar results were observed in macrophages stimulated with the SARS-CoV-2 spike protein. Thus, IL27 can trigger an antiviral response in the host, suggesting the possibility of novel therapeutics against SARS-CoV-2 infection in humans.

IL-27 suppresses spring viremia of carp virus replication in zebrafish

Interleukin (IL) 27 is a member of the IL-12 family and is a heterodimeric cytokine composed of IL-27A and Epstein-Barr virus-induced 3 (EBI3). It plays an important role in regulating inflammation and cancer progression. IL-27A not only functions by dimerizing with EBI3 but also acts alone. Here, we report that IL-27A and EBI3 suppress spring viremia of carp virus (SVCV) replication in zebrafish. Expression analysis reveals that il-27a and ebi3 were significantly upregulated in the ZF4 cells by SVCV and poly(I:C), and in the zebrafish caudal fin (ZFIN) cells overexpressed with SVCV genes. Interestingly, il-27a and ebi3 were not modulated by IFNφ1, indicating that they are not IFN stimulated genes (ISGs). Furthermore, overexpression of IL-27A and EBI3 alone inhibited SVCV replication in the EPC cells, but less potent than co-expression of IL-27A and EBI3. Intriguingly, IL-27A could not induce the expression of irf3, ifn, isg15 and mx1. Taken together, our results demonstrate that IL-27A and EBI3 activate innate antiviral response in an IFN independent manner in zebrafish.

American-Asian- and African lineages of Zika virus induce differential pro-inflammatory and Interleukin 27-dependent antiviral responses in human monocytes

Zika virus (ZIKV) is an arbovirus that belongs to the Flaviviridae family and inflammatory responses play a critical role in ZIKV pathogenesis. As a first-line defense, monocytes are key components of innate immunity and host response to viruses. Monocytes are considered the earliest blood cell type to be infected by ZIKV and have been shown to be associated with ZIKV pathogenesis. The first ZIKV epidemic was reported in Africa and Asia although, it is less well known whether African- and Asian- lineages of ZIKV have different impacts on host immune response. We studied the pro-inflammatory and antiviral response of ZIKV-infected monocytes using publicly available RNA-seq analysis (GSE103114). We compared the transcriptomic profiles of human monocytes infected with ZIKV Puerto Rico strain (PRVABC59), American-Asian lineage, and ZIKV Nigeria strain (IBH30656), African lineage. We validated RNA-seq results by ELISA or RT-qPCR, in human monocytes infected with a clinical isolate of ZIKV from Colombia (American-Asian lineage), or with ZIKV from Dakar (African lineage). The transcriptomic analysis showed that ZIKV Puerto Rico strain promotes a higher pro-inflammatory response through TLR2 signaling and NF-kB activation and induces a strong IL27-dependent antiviral activity than ZIKV Nigeria strain. Furthermore, human monocytes are more susceptible to infection with ZIKV from Colombia than ZIKV from Dakar. Likewise, Colombian ZIKV isolate activated IL27 signaling and induced a robust antiviral response in an IFN-independent manner. Moreover, we show that treatment of monocytes with IL27 results in decreased release of ZIKV particles in a dose-dependent manner with an EC50 =2.870 ng/mL for ZIKV from Colombia and EC50 =10.23 ng/mL to ZIKV from Dakar. These findings highlight the differential inflammatory response and antiviral activity of monocytes infected with different lineages of ZIKV and may help better management of ZIKV-infected patients.

Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host’s innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-β mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host’s immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

SeV C Protein Plays a Role in Restricting Macrophage Phagocytosis by Limiting the Generation of Intracellular Double-Stranded RNA.

Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-β stimulation and neutralizing antibodies against IFN-β suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-β-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

Synergistic Effects of Toll-Like Receptor 1/2 and Toll-Like Receptor 3 Signaling Triggering Interleukin 27 Gene Expression in Chikungunya Virus-Infected Macrophages.

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and severe acute or chronic arthralgia. CHIKF is associated with immunopathology and high levels of pro-inflammatory factors. CHIKV is known to have a wide range of tropism in human cell types, including keratinocytes, fibroblasts, endothelial cells, monocytes, and macrophages. Previously, we reported that CHIKV-infected monocytes-derived macrophages (MDMs) express high levels of interleukin 27 (IL27), a heterodimeric cytokine consisting of IL27p28 and EBI3 subunits, that triggers JAK-STAT signaling and promotes pro-inflammatory and antiviral response, in interferon (IFN)-independent manner. Based on the transcriptomic analysis, we now report that induction of IL27-dependent pro-inflammatory and antiviral response in CHIKV-infected MDMs relies on two signaling pathways: an early signal dependent on recognition of CHIKV-PAMPs by TLR1/2-MyD88 to activate NF-κB-complex that induces the expression of EBI3 mRNA; and second signaling dependent on the recognition of intermediates of CHIKV replication (such as dsRNA) by TLR3-TRIF, to activate IRF1 and the induction of IL27p28 mRNA expression. Both signaling pathways were required to produce a functional IL27 protein involved in the induction of ISGs, including antiviral proteins, cytokines, CC- and CXC- chemokines in an IFN-independent manner in MDMs. Furthermore, we reported that activation of TLR4 by LPS, both in human MDMs and murine BMDM, results in the induction of both subunits of IL27 that trigger strong IL27-dependent pro-inflammatory and antiviral response independent of IFNs signaling. Our findings are a significant contribution to the understanding of molecular and cellular mechanisms of CHIKV infection.

CGAS exacerbates Schistosoma japonicum infection in a STING-type I IFN-dependent and independent manner.

Schistosomiasis, which is caused by infection with Schistosoma spp., is characterized by granuloma and fibrosis in response to egg deposition. Pattern recognition receptors are important to sense invading Schistosoma, triggering an innate immune response, and subsequently shaping adaptive immunity. Cyclic GMP-AMP synthase (cGAS) was identified as a major cytosolic DNA sensor, which catalyzes the formation of cyclic GMP-AMP (cGAMP), a critical second messenger for the activation of the adaptor protein stimulator of interferon genes (STING). The engagement of STING by cGAMP leads to the activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and the subsequent type I interferon (IFN) response. cGAS is suggested to regulate infectious diseases, autoimmune diseases, and cancer. However, the function of cGAS in helminth infection is unclear. In this study, we found that Cgas deficiency enhanced the survival of mice infected with S. japonicum markedly, without affecting the egg load in the liver. Consistently, Cgas deletion alleviated liver pathological impairment, reduced egg granuloma formation, and decreased fibrosis severity. In contrast, Sting deletion reduced the formation of egg granulomas markedly, but not liver fibrosis. Notably, Cgas or Sting deficiency reduced the production of IFNβ drastically in mice infected with S. japonicum. Intriguingly, intravenous administration of recombinant IFNβ exacerbated liver damage and promoted egg granuloma formation, without affecting liver fibrosis. Clodronate liposome-mediated depletion of macrophages indicated that macrophages are the major type of cells contributing to the induction of the type I IFN response during schistosome infection. Moreover, cGAS is important for type I IFN production and phosphorylation of TBK1 and IRF3 in response to stimulation with S. japonicum egg- or adult worm-derived DNA in macrophages. Our results clarified the immunomodulatory effect of cGAS in the regulation of liver granuloma formation during S. japonicum infection, involving sensing schistosome-derived DNA and producing type I IFN. Additionally, we showed that cGAS regulates liver fibrosis in a STING-type I–IFN-independent manner.

Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission.

ABSTRACTRotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1 day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells. We demonstrate that viperin upregulation by HSV-1 infection is cell-type-specific, with mouse cells typically showing greater increases compared with those of human origin. Further, overexpression and knockout of mouse, but not human viperin significantly impedes and increases HSV-1 replication, respectively. In primary mouse fibroblasts, viperin upregulation by infection requires viral gene transcription and occurs in a predominantly IFN-independent manner. Further we identify the N-terminal domain of viperin as being required for the anti-HSV-1 activity. Interestingly, this is the region of viperin that differs most between mouse and human, which may explain the apparent species-specific activity against HSV-1. Finally, we show that HSV-1 virion host shutoff (vhs) protein is a key viral factor that antagonises viperin in mouse cells. We conclude that viperin can be upregulated by HSV-1 in mouse and human cells, and that mouse viperin has anti-HSV-1 activity.

Interleukin 27 as an inducer of antiviral response against chikungunya virus infection in human macrophages

Chikungunya virus (CHIKV) is known to have a wide range of tropism in human cell types throughout infection, including keratinocytes, fibroblasts, endothelial cells, monocytes, and macrophages. We reported that human monocytes-derived macrophages (MDMs) are permissive to CHIKV infection in vitro. We found that the peak of CHIKV replication was at 24 hpi; however, at 48 hpi, a significant reduction in viral titer was observed that correlated with high expression levels of genes encoding antiviral proteins (AVPs) in an IFN-independent manner. To explore the molecular mechanisms involved in the induction of antiviral response in CHIKV-infected MDMs, we performed transcriptomic analysis by RNA-sequencing.Differential expression of genes at 24 hpi showed that CHIKV infection abrogated the expression of all types of IFNs in MDMs. However, we observed that CHIKV-infected MDMs activated the JAK-STAT signaling and induced a robust antiviral response associated with control of CHIKV replication. We identified that the IL27 pathway is activated in CHIKV-infected MDMs and that kinetics of IL27p28 mRNA expression and IL27 protein production correlated with the expression of AVPs in CHIKV-infected MDMs. Furthermore, we showed that stimulation of THP-1-derived macrophages with recombinant-human IL27 induced the activation of the JAK-STAT signaling and induced a robust pro-inflammatory and antiviral response, comparable to CHIKV-infected MDMs. Furthermore, pre-treatment of MDMs with recombinant-human IL27 inhibits CHIKV replication in a dose-dependently manner (IC50 = 1.83 ng/mL). Altogether, results show that IL27 is highly expressed in CHIKV-infected MDMs, leading to activation of JAK-STAT signaling and stimulation of pro-inflammatory and antiviral response to control CHIKV replication in an IFN-independent manner.

Conventional type 2 dendritic cells and natural killer cells mediate control of early metastatic seeding

Abstract The multistep progression of transformed primary tumors to metastatic dissemination and seeding of distant site is a lethal determinant of disease. Although the role of immune cells and immune sensors in promoting or restricting metastatic growth is well described, whether immune cells are capable of detecting and controlling metastatic cells during peripheral tissue seeding is unknown. Here we examine if local immune cells can confer early anti-metastatic control in the lung using intracardiac injection to mimic metastatic spread. Using a syngeneic mouse model of YUMMER1.7 melanoma expressing GFP and luciferase (YMR1.7-G/L), we demonstrate that lung resident conventional type 2 dendritic cells (cDC2) and extravascular NK cells confer host anti-tumor immunity during the first three days following intracardiac injection. Rapid production of IFN-γ by NK cells serves an essential role to limit metastatic burden. Using mice deficient in innate sensors and signaling pathways, we demonstrate that the transcription factors IRF3 and IRF7 are required for early metastatic control in a type I IFN-independent manner. In contrast, ablation of adaptive immune cells had no effect to initial metastatic burden. Collectively our results highlight a novel cDC2 - NK cell axis that orchestrates a non-redundant innate immune response program to limit initial metastatic burden in the lung.

Rhinovirus replication and innate immunity in highly differentiated human airway epithelial cells

BackgroundHuman rhinovirus (HRV) infections are the primary cause of the common cold and are a major trigger for exacerbations of lower airway diseases, such as asthma and chronic obstructive pulmonary diseases. Although human bronchial epithelial cells (HBE) are the natural host for HRV infections, much of our understanding of how HRV replicates and induces host antiviral responses is based on studies using non-airway cell lines (e.g. HeLa cells). The current study examines the replication cycle of HRV, and host cell responses, in highly differentiated cultures of HBE.MethodsHighly differentiated cultures of HBE were exposed to initial infectious doses ranging from 104 to 101 50% tissue culture-infective dose (TCID50) of purified HRV-16, and responses were monitored up to 144 h after infection. Viral genomic RNA and negative strand RNA template levels were monitored, along with levels of type I and II interferons and selected antivirals.ResultsRegardless of initial infectious dose, relatively constant levels of both genomic and negative strand RNA are generated during replication, with negative strand copy numbers being10,000-fold lower than those of genomic strands. Infections were limited to a small percentage of ciliated cells and did not result in any overt signs of epithelial death. Importantly, regardless of infectious dose, HRV-16 infections were cleared by HBE in the absence of immune cells. Levels of type I and type III interferons (IFNs) varied with initial infectious dose, implying that factors other than levels of double-stranded RNA regulate IFN induction, but the time-course of HRV-16 clearance HBE was the same regardless of levels of IFNs produced. Patterns of antiviral viperin and ISG15 expression suggest they may be generated in an IFN-independent manner during HRV-16 infections.ConclusionsThese data challenge a number of aspects of dogma generated from studies in HeLa cells and emphasize the importance of appropriate cell context when studying HRV infections.

Cell-type specificity and functional redundancy of RIG-I-like receptors in innate immune sensing of Coxsackievirus B3 and encephalomyocarditis virus

The contributions of RIG-I and MDA5 receptors to sensing viruses of the Picornaviridae family were investigated. The picornaviruses encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3) are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFNβ synthesis is reduced in the absence of each sensor, while IFNα production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate functional redundancy within the innate immune response to picornaviruses.

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses.

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα‐chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll‐like receptor (TLR) ligand activation of TCR‐transgenic murine dNKT cells. IFN‐γ production by dNKT cells required dendritic cells (DC), cell‐to‐cell contact and presence of TLR ligands. TLR‐stimulated DC activated dNKT cells to secrete IFN‐γ in a CD1d‐, CD80/86‐ and type I IFN‐independent manner. In contrast, a requirement for IL‐12p40, and a TLR ligand‐selective dependence on IL‐18 or IL‐15 was observed. TLR ligand/DC stimulation provoked early secretion of pro‐inflammatory cytokines by both CD62L+ and CD62L− dNKT cells. However, proliferation was limited. In contrast, TCR/co‐receptor‐mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L− dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co‐receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.

T time for ADAR: ADAR1 is required for T cell self-tolerance.

ADAR1, an RNA‐editing enzyme, plays a key role in preventing self‐RNAs from triggering autoinflammatory responses. In this issue of EMBO reports , Nakahama and colleagues uncover a novel role for ADAR1 in T cells [1]. The authors report that in T cells, ADAR1‐mediated suppression of type I interferon‐stimulated gene (ISG) expression is required for thymic T cell self‐tolerance and prevention of colitis. These findings establish a novel function of ADAR1 in T cells and suggest that autoreactive T cells may contribute to disease symptoms in autoinflammatory disorders.

IL-27 induces autophagy during monocyte-to-macrophage differentiation through a novel LC3-independent pathway

Abstract IL-27, a member of the IL-12 cytokine family composed of IL-12, IL-23 and IL-35, has previously been shown to be an antiviral cytokine inhibiting infection of several viruses, among which HIV-1, HIV-2, HSV, HCV, HBV and Influenza virus. We have already reported that IL-27 induces HIV-1 inhibition in macrophages and dendritic cells in an IFN-independent manner, and that the cytokine enhances potential reactive oxygen species induction from cells. In this study, we demonstrated for the first time that IL-27 triggers autophagy induction in human AB serum-induced macrophages, while other IL-12 cytokine family members did not have such effect on these cells; IL-27 was able to induce autophagy both in terminally differentiated macrophages, causing a 2 to 5-fold increase in autophagosome accumulation, as well as during monocyte-to-macrophage differentiation, where IL-27 effect was even stronger (generating a 20-fold increase in autophagosome formation). Furthermore, we also determined that two key autophagy proteins, mTOR and LC3, were not involved in this phenomenon: while we were able to assess autophagosome accumulation in IL-27-treated cells via electron microscopy and immunofluorescence, this was not linked to either mTOR dephosphorylation nor LC3 lipidation, implying that IL-27 induces autophagy through a novel non-canonical pathway. Finally, we established that blocking IL-27 signaling during macrophage differentiation using both Ruxoliotinib or Tofacitinib, respectively JAK1/2 and JAK3 inhibitors, successfully inhibited autophagy triggering together with STAT3 phosphorylation, which suggests that the JAK-STAT pathway is responsible for regulating IL-27-induced autophagy.

Repression of SUMOylation pathway by grass carp reovirus contributes to the upregulation of PKR in an IFN-independent manner

SUMOylation, a post-translational modification, is involved in interaction between hosts and viruses, and participates in diverse cellular processes including inflammatory responses and innate immunity. Here, we investigated the interaction between reovirus infection and the cellular SUMOylation machinery using grass carp reovirus (GCRV) as a model. Full-length cDNAs of grass carp SUMO-1 and SUMO-2 were obtained and phylogenetic analysis indicated that they shared high homology with those of higher vertebrates. The two modifiers and SUMO conjugating enzyme 9 (Ubc9) were ubiquitously expressed in all tested tissues of grass carp. During GCRV infection in CIK cells, transcriptional expressions of SUMO1/2 and Ubc9 were significantly inhibited; while UV-inactivated GCRV failed to inhibit the expression of the three molecules, which suggested that SUMOylation system was suppressed during viral replication. In CIK cells treated with inhibitor 2-D08 for SUMOylation, GCRV replication was not interfered; however, transcriptional analysis of immune genes involved in anti-viral interferon (IFN) response indicated that IRF2 and PKR were significantly up-regulated in CIK cells treated with inhibitor in contrast to IRF1, IRF7 and IFNI. Furthermore, 2-D08 treatment coupled with GCRV challenge resulted in higher IRF2 and PKR level during infection in comparison to those of CIK cells infected with GCRV only. These results indicated that inhibition of SUMOylation should result in the induction of PKR via IFN-independent manner, and both IFN-signaling and IFN-independent signaling seemed to involve in the upregulation of PKR during the process of GCRV infection. Repression of SUMOylation by GCRV might represent a cellular antiviral mechanism.

CXCL10 is produced in hepatitis A virus-infected cells in an IRF3-dependent but IFN-independent manner

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

STAT1 regulates marginal zone B cell differentiation in response to inflammation and infection with blood-borne bacteria.

Marginal zone B (MZ B) cells can rapidly produce antibody in response to infection with blood-borne encapsulated pathogens. Although TLR-mediated activation of MZ B is known to trigger humoral immune response, the signal cascade directing this response remains undefined. Here, we demonstrate that STAT1 plays an essential role in TLR-mediated antibody response of MZ B cells. Further, the TLR-induced IgM response is impaired in a type I and type II IFN-independent manner. Although activation, proliferation, and apoptosis are not affected, both differentiation into plasma cells and IgM production are impaired in Stat1-/- MZ B cells. Interestingly, STAT1 directly regulates the expression of Prdm1 (encodes BLIMP-1) by binding to its promoter, and Prdm1 expression is reduced in Stat1-/- MZ B cells. Restoration of BLIMP-1 to cells rescues TLR-induced IgM response. Moreover, Stat1-/- mice are more susceptible to S. pneumoniae infection, which can be rescued by the serum of bacteria-primed WT mice. The increased susceptibility to S. pneumoniae infection in Stat1-/- mice is also intrinsic to STAT1 requirement in MZ B cells. Collectively, these results define a differential regulation of TLR-mediated activation and differentiation of MZ B cells by STAT1 and reveal a STAT1-dependent, but IFN-independent, antibody response during infection and inflammation.