Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.     

Siglec1 suppresses antiviral innate immune response by inducing TBK1 degradation via the ubiquitin ligase TRIM27

Type I interferon (IFN) production plays pivotal roles in host antiviral innate immune responses, but an excessive production of type I IFN leads to the development of immunopathological conditions. Investigations on the regulatory mechanisms underlying host type I IFN production are currently of great interest. Here, we found that the expression of lectin family member Siglec1 was upregulated by viral infection in macrophages, which was dependent on the IFN/JAK/STAT1 signaling pathway. Siglec1 was found to negatively regulate viral infection-triggered type I IFN production. Mechanistically, Siglec1 associates with DAP12 to recruit and activate the scaffolding function of SHP2; SHP2 then recruits E3 ubiquitin ligase TRIM27, which induces TBK1 degradation via K48-linked ubiquitination at Lys251 and Lys372. Therefore, viral infection-induced upregulation of Siglec1 feedback loop inhibits type I IFN production and suppresses antiviral innate immune responses. Our study outlines a novel mechanism of negative regulation of type I IFN production, which may help virus to escape immune elimination.     

PD‐1/PD‐L1 expression on CD4+ T cells and myeloid DCs correlates with the immune pathogenesis of atrial fibrillation

Although immuno‐inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death‐1 (PD‐1)/PD‐1 ligand (PD‐L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD‐1 and PD‐L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD‐1 on CD⁴⁺ T cells and PD‐L1 on myeloid dendritic cells (mDCs) in AF patients is significantly down‐regulated compared with healthy volunteers. In addition, the extent of PD‐1/PD‐L1 down‐regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD‐L1 down‐regulation is associated with increased T cell (CD⁴⁺ and CD⁸⁺) proliferation, increased type 1 effector cytokines (IL‐2 and IFN‐γ) secretion, and decreased type 2 effector cytokine (IL‐10) secretion. Then, PD‐L1 up‐regulation by the stimulation of IFN‐α can significantly convert this representation. Collectively, our report suggest that T(CD⁴⁺)/mDCs‐associated PD‐1/PD‐L1 pathway plays a key role in AF immune regulation. PD‐1/PD‐L1 down‐regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.     

IL‐12 stimulates the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) gene expression in CD4+ T cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (?1 to ?1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009. © 2009 Wiley‐Liss, Inc.     

SARI suppresses colitis‐associated cancer development by maintaining MCP‐1‐mediated tumour‐associated macrophage recruitment

SARI (suppressor of AP‐1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour‐associated inflammation microenvironment is still elusive. In our study, the colitis‐dependent and ‐independent primary model were established in SARI deficiency mice and immuno‐reconstructive mice to investigate the functional role of SARI in regulating tumour‐associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis‐associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour‐associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down‐regulates p‐STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP‐1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP‐1 expression and p‐STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.     

miR‐181a and miR‐150 regulate dendritic cell immune inflammatory responses and cardiomyocyte apoptosis via targeting JAK1–STAT1/c‐Fos pathway

The immune inflammatory response plays a crucial role in many cardiac pathophysiological processes, including ischaemic cardiac injury and the post‐infarction repair process. MicroRNAs (miRNAs) regulate the development and function of dendritic cells (DCs), which are key players in the initiation and regulation of immune responses; however, the underlying regulatory mechanisms remain unclear. Here, we used the supernatants of necrotic primary cardiomyocytes (Necrotic‐S) to mimic the myocardial infarction (MI) microenvironment to investigate the role of miRNAs in the regulation of DC‐mediated inflammatory responses. Our results showed that Necrotic‐S up‐regulated the DC maturation markers CD40, CD83 and CD86 and increased the production of inflammatory cytokines, concomitant with the up‐regulation of miR‐181a and down‐regulation of miR‐150. Necrotic‐S stimulation activated the JAK/STAT pathway and promoted the nuclear translocation of c‐Fos and NF‐κB p65, and silencing of STAT1 or c‐Fos suppressed Necrotic‐S‐induced DC maturation and inflammatory cytokine production. The effects of Necrotic‐S on DC maturation and inflammatory responses, its activation of the JAK/STAT pathway and the induction of cardiomyocyte apoptosis under conditions of hypoxia were suppressed by miR‐181a or miR‐150 overexpression. Taken together, these data indicate that miR‐181a and miR‐150 attenuate DC immune inflammatory responses via JAK1–STAT1/c‐Fos signalling and protect cardiomyocytes from cell death under conditions of hypoxia.     

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

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

Hepatitis C Virus Core Protein Inhibits Interferon Production by a Human Plasmacytoid Dendritic Cell Line and Dysregulates Interferon Regulatory Factor-7 and Signal Transducer and Activator of Transcription (STAT) 1 Protein Expression

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.     

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

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

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi–Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR–Cas9 gene KO or lentiviral viral protein X–mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2–infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.     

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ‐induced responses, for example, expression of IFN‐stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress‐induced‐phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca²⁺ levels while inhibiting nuclear translocalization of STAT1 and NFAT‐1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca²⁺/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5‐mediated inhibition of IFNβ‐induced responses, for example, IFN‐sensitive response element driven luciferase, STAT1‐dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5‐expressing cells in response to IFNβ. Results prove Ca²⁺, calreticulin, and calcineurin involvement in STAT1‐mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.