"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-κB-dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-κB induction in STAT1(-/-) mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.

Diabetes is a proinflammatory state. The pattern recognition receptors, Toll-like receptors (TLRs), are increased in diabetic patients and have been suggested to play a role in diabetic nephropathy (DN). Progression of DN involves altered mesangial cell (MC) function with an expansion of the mesangial matrix. There is a paucity of data examining the role of TLR and its expression in MC. We hypothesize the expression of TLRs in the mesangium might be an important factor contributing to mesangium expansion and nephropathy. Thus we evaluated the effect of high glucose on TLR2 and TLR4 expression in mouse mesangial cells (MMC) in vitro. Exposure of MMC to 25 mM glucose for 24 h resulted in increased TLR4 mRNA and cell surface receptor expression compared with 5.5 mM glucose (P < 0.05). Interestingly, we were not able to detect expression of TLR2 in MMC. Furthermore, expression of a TLR4 downstream signaling cascade including myeloid differentiation factor 88 (MyD88), interferon regulatory factor 3 (IRF3), and Toll interleukin receptor domain containing adaptor inducing interferon-β (TRIF)-related adaptor molecule (TRAM) were significantly increased in cells exposed to 25 mM glucose (P < 0.05). There was also a significant increase in NF-κB activation along with increased secretion of inflammatory cytokines IL-6 and monocyte chemotactic protein-1. Levels of transforming growth factor-β were also significantly increased in the presence of 25 mM glucose (P < 0.05). Collectively, these data suggest that hyperglycemia activates TLR4 expression and activity in MC and could contribute to DN.

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally and play critical roles in intricate networks of host-pathogen interactions and innate immunity. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases affecting swine industry worldwide. Here, we demonstrated that miR-23, miR-378, and miR-505 were antiviral host factors against PRRS virus (PRRSV). Over-expression of the three miRNAs inhibited PRRSV infection in a dose-dependent manner, respectively. Blockage of the three endogenously expressed miRNAs significantly enhanced PRRSV replication. Different type 2 PRRSV strains harbored conserved miR-23, miR-378, and miR-505 target sites (TSs) that were sufficient to confer miRNA-mediated repression of PRRSV replication. Interestingly, miR-23 was capable of inducing type I interferon expression during PRRSV infection through IRF3/IRF7 activation, which might further lead to the inhibition of virus infection. These results suggest that miR-23, miR-378, and miR-505, especially miR-23, may have the potential to be used as antiviral therapy against PRRSV infection.

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis; some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, IGF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TAC1. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung.

The RNA helicases RIG-I and MDA5 detect virus infection of dendritic cells (DCs) leading to cytokine induction. Maximal sensitivity for virus detection by these helicases is obtained after their upregulation, which is thought to occur primarily through type I interferon (IFN) signaling. Here we demonstrate that in response to paramyxovirus infection, RIG-I upregulation requires type I IFN whereas MDA5 expression is increased by Sendai virus infection independently of signaling mediated by type I IFN, STAT1, tumor necrosis factor alpha, or NF-kappaB. This MDA5 upregulation is largely lost in IRF3 knockout DCs and is achieved in type I IFN-deficient cells expressing constitutively active IRF3.

A member of the IL-1 receptor (IL-1R)-associated kinase (IRAK) family, IRAK4, has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. IRAK4 kinase-inactive knockin mice have been shown to be completely resistant to LPS- and CpG-induced shock, due to impaired TLR-mediated induction of pro-inflammatory cytokines and chemokines. A reduction of LPS-, R848- and IL-1-mediated mRNA stability contributes to the reduced cytokine and chemokine production in bone marrow (BM)-derived macrophages from IRAK4 kinase-inactive knockin mice: however, not all of the TLR/IL-1R signaling events are ablated in IRAK4 kinase-inactive knockin mice. A paper in this issue of the European Journal of Immunology shows that, while JNK activation is significantly impaired, NF-kappaB and IRF3 activation are retained in the absence of IRAK4 kinase activity. These residual TLR/IL-1R-induced signaling events allow the production of some cytokines and chemokines (including TNFalpha and CXCL1); at early times after the stimulation and induction of a group of TLR-mediated MyD88/IRAK4-independent genes in IRAK4 kinase-inactive knockin cells. Therefore, pharmacological blocking of IRAK4 kinase activity will retain some levels of host defence, while reducing the levels and duration of inflammatory responses, which should provide beneficial therapies for sepsis and chronic inflammatory diseases.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection has claimed hundreds of lives and has become a global threat since its emergence in Saudi Arabia in 2012. The ability of MERS-CoV to evade the host innate antiviral response may contribute to its severe pathogenesis. Many MERS-CoV-encoded proteins were identified to have interferon (IFN)-antagonizing properties, which correlates well with the reduced IFN levels observed in infected patients and ex vivo models. In this study, we fully characterized the IFN-antagonizing property of the MERS-CoV M protein. Expression of MERS-CoV M protein suppressed type I IFN expression in response to Sendai virus infection or poly(I:C) induction. This suppressive effect was found to be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-κB. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3-TBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response.

BACKGROUND

Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown.

RESULTS

Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection.

CONCLUSIONS

Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection.

Viral infection causes activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. The mitochondrial outer membrane protein VISA acts as a critical adapter for assembling a virus-induced complex that signals NF-kappaB and IRF3 activation. Using a biochemical purification approach, we identified the WD repeat protein WDR5 as a VISA-associated protein. WDR5 was recruited to VISA in a viral infection dependent manner. Viral infection also caused translocation of WDR5 from the nucleus to mitochondria. Knockdown of WDR5 impaired the formation of virus-induced VISA-associated complex. Consistently, knockdown of WDR5 inhibited virus-triggered activation of IRF3 and NF-kappaB as well as transcription of the IFNB1 gene. These findings suggest that WDR5 is essential in assembling a virus-induced VISA-associated complex and plays an important role in virus-triggered induction of type I IFNs.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double-stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNbeta) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kappaB and IRF3 (interferon regulatory factor 3) and induced IFNbeta (interferon beta) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNbeta expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies.

Members of the Bcl-2 family of apoptosis-regulating proteins contain at least one of the four evolutionarily conserved domains, termed BH1, BH2, BH3, or BH4. Here, we report the identification, cloning, physical mapping, and expression pattern of BCL2L12, a novel gene that encodes a BCL2-like proline-rich protein. Proline-rich sites have been shown to interact with Src homology region 3 (SH3) domains of several tyrosine kinases, mediating their oncogenic potential. This new gene maps to chromosome 19q13.3 and is located between the IRF3 and the PRMT1/HRMT1L2 genes, close to the RRAS gene. BCL2L12 is composed of seven coding exons and six intervening introns, spanning a genomic area of 8.8 kb. All of the exon-intron splice sites conform to the consensus sequence for eukaryotic splice sites. The BCL2L12 protein is composed of 334 amino acids, with a calculated molecular mass of 36.8 kDa and an isoelectric point of 9.45. The BCL2L12 protein contains one BH2 homology domain, one proline-rich region similar to the TC21 protein and, five consensus PXXP tetrapeptide sequences. BCL2L12 is expressed mainly in breast, thymus, prostate, fetal liver, colon, placenta, pancreas, small intestine, spinal cord, kidney, and bone marrow and to a lesser extent in many other tissues. We also identified one splice variant of BCL2L12 that is primarily expressed in skeletal muscle.

This study investigated the immunomodulating effect of melatonin on toll-like receptor (TLR)-stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high-mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF-κB) and phosphorylated c-Jun, a component of activator protein 1. The increased level of toll-receptor-associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN-β was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase-1 (HO-1) was augmented. Our results suggest that melatonin ameliorates I/R-induced liver damage by modulation of TLR-mediated inflammatory responses.

Several innate sensing pathways contribute to the control of early cytomegalovirus (CMV) infection, leading to a multiphasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells; however, the initial burst of IFN-I that occurs within the first few hours in vivo is TLR independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3) signaling. Disruption of STING expression in endothelial cells by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 revealed that it is essential for the induction of IFN-I and restriction of CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in nonhematopoietic cells and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo.

Cytomegalovirus (CMV) is one of the most common viral pathogens, with the majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immunocompromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPRs revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice, STING is necessary for the first phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection.

Borrelia burgdorferi elicits a potent cytokine response through activation of multiple signaling receptors on innate immune cells. Spirochetal lipoproteins initiate expression of NF-κB-dependent cytokines primarily via TLR2, whereas type I interferon (IFN) production is induced through the endosomal receptors TLR7 and TLR9 in human dendritic cells and TLR8 in monocytes. We demonstrate that DNA and RNA are the B. burgdorferi components that initiate a type I IFN response by human peripheral blood mononuclear cells (PBMCs). IFN-α protein and transcripts for IRF7, MX1, and OAS1 were induced by endosomal delivery of B. burgdorferi DNA, RNA, or whole-cell lysate, but not by lysate that had been treated with DNase and RNase. Induction of IFN-α and IFN-λ1, a type III IFN, by B. burgdorferi RNA or live spirochetes required TLR7-dependent signaling and correlated with significantly enhanced transcription and expression of IRF7 but not IRF3. Induction of type I and type III IFNs by B. burgdorferi RNA could be completely abrogated by a TLR7 inhibitor, IRS661. In addition to type I and type III IFNs, B. burgdorferi RNA contributed to the production of the NF-κB-dependent cytokines, IFN-γ, interleukin-10 (IL-10), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α), by human PBMCs. Collectively, these data indicate that TLR7-dependent recognition of RNA is pivotal for IFN-α and IFN-λ1 production by human PBMCs, and that RNA-initiated signaling contributes to full potentiation of the cytokine response generated during B. burgdorferi infection.

Toll-like receptors (TLRs) are major receptors that enable inflammatory cells to recognize invading microbial pathogens. MicroRNAs are small non-coding RNAs that play important regulatory roles in a variety of biological processes. In this study, we found that a microRNA, miR-147, was induced upon stimulation of multiple TLRs and functioned as a negative regulator of TLR-associated signaling events in murine macrophages. We first demonstrated that the NMES1 transcript was a functional primary miR-147. miR-147 was induced in LPS-stimulated mouse macrophages and under in vivo conditions in the lungs of LPS-treated mice. Expression of miR-147 was greater after cellular activation by TLR4 than after engagement of either TLR2 or TLR3, suggesting that maximal induction of miR-147 required activation of both NF-kappaB and IRF3. TLR4-induced miR-147 expression was both MyD88- and TRIF-dependent. The miR-147 promoter was responsive to TLR4 stimulation and both NF-kappaB and STAT1alpha bound to the miR-147 promoter. miR-147 mimics or induced expression of miR-147 decreased, whereas miR-147 knockdown increased inflammatory cytokine expression in macrophages stimulated with ligands to TLR2, TLR3, and TLR4. These data demonstrate a negative-feedback loop in which TLR stimulation induces miR-147 to prevent excessive inflammatory responses.

The innate immune response provides the first line of defense against foreign pathogens by responding to molecules that are a signature of a pathogenic infection. Certain RNA viruses, such as influenza virus, produce double-stranded RNA as an intermediate during the replication life cycle, which activates pathogen recognition receptors capable of inducing interferon production. By engaging interferon receptors, interferon activates the JAK-STAT pathway and results in the positive feedback of interferon production, amplifying the response to viral infection. To examine how deficiencies in interferon signaling affect the cellular response to infection, we performed influenza virus infections of mouse embryonic fibroblasts lacking the alpha/beta interferon receptor, the gamma interferon receptor, or both. In the absence of the alpha/beta interferon receptor, we observed increased viral replication but decreased activation of PKR, Stat1, and NF-kappaB; the presence or absence of the gamma interferon receptor did not exhibit discernible differences in these readouts. Analysis of gene expression profiles showed that while cells lacking the alpha/beta interferon receptor exhibited decreased levels of transcription of antiviral genes, genes related to inflammatory and apoptotic responses were transcribed to levels similar to those of cells containing the receptor. These results indicate that while the alpha/beta interferon receptor is needed to curb viral replication, it is dispensable for the induction of certain inflammatory and apoptotic genes. We have identified potential pathways, via interferon regulatory factor 3 (IRF3) activation or Hoxa13, Polr2a, Nr4a1, or Ing1 induction, that contribute to this redundancy. This study illustrates another way in which the host has evolved to establish several overlapping mechanisms to respond to viral infections.

BACKGROUND

Japanese encephalitis virus (JEV) infection leads to Japanese encephalitis (JE) in humans. JEV is transmitted through mosquitoes and maintained in a zoonotic cycle. This cycle involves pigs as the major reservoir, water birds as carriers and mosquitoes as vectors. JEV invasion into the central nervous system (CNS) may occur via antipodal transport of virions or through the vascular endothelial cells. Microglial cells get activated in response to pathogenic insults. JEV infection induces the innate immune response and triggers the production of type I interferons. The signaling pathway of type I interferon production is regulated by a number of molecules. TRIM proteins are known to regulate the expression of interferons; however, the involvement of TRIM genes and their underlying mechanism during JEV infection are not known.

METHODS

Human microglial cells (CHME3) were infected with JEV to understand the role of TRIM21 in JEV infection and its effect on type I interferon (IFN-β) production. Cells were infected in presence and absence of exogenous TRIM21 as well as after knocking down the TRIM21 mRNA. Levels of activated IRF3 expression were measured through Western blot analyses of anti-p-IRF3 antibody, and IFN-β production was measured by using IFN-β real-time PCR and luciferase activity analyses.

RESULTS

JEV infection increased expression of TRIM21 in CHME3 cells. JEV induced an innate immune response by increasing production of IFN-β via IRF3 activation and phosphorylation. Overexpression of TRIM21 resulted in downregulation of p-IRF3 and IFN-β, while silencing led to increased production of p-IRF3 and IFN-β in JEV-infected CHME3 cells.

CONCLUSIONS

This report demonstrates TRIM21 as a negative regulator of interferon-β (IFN-β) production mediated by IRF-3 during JEV infection in human microglial cells.

Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-kappaBp65. T-cell receptor delta (TCRdelta)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing gammadeltaT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection.

Interferon regulatory factor (IRF) 3, a member of the highly conserved IRF family transcription factors, plays a pivotal role in innate immune response, apoptosis, and oncogenesis. Recent studies have implicated IRF3 in a wide range of host defense. However, whether IRF3 induces defensive responses to hypertrophic stresses such as biomechanical stress and neurohumoral factors remains unclear. Herein, we employed an IRF3-deficient mouse model, cardiac-specific IRF3-overexpression mouse model and isolated cardiomyocytes to investigate the role of IRF3 in cardiac hypertrophy induced by aortic banding (AB) or isoproterenol (ISO). The extent of cardiac hypertrophy was quantitated by echocardiography as well as by pathological and molecular analysis. Our results demonstrate that IRF3 deficiency profoundly exacerbated cardiac hypertrophy, whereas overexpression of IRF3 in the heart significantly blunted pathological cardiac remodeling induced by pressure overload. Similar results were also observed in cultured cardiomyocytes upon the treatment with ISO. Mechanistically, we discovered that IRF3 interacted with ERK2 and thereby inhibited the ERK1/2 signaling. Furthermore, inactivation of ERK1/2 by U0126 offset the IRF3-deficient-mediated hypertrophic response induced by aortic banding. Altogether, these data demonstrate that IRF3 plays a protective role in AB-induced hypertrophic response by inactivating ERK1/2 in the heart. Therefore, IRF3 could be a new target for the prevention and therapy of cardiac hypertrophy and failure.

MDA5 plays a critical role in antiviral innate immunity by functioning as a cytoplasmic double-stranded RNA sensor that can activate type I interferon signaling pathways, but the mechanism for the activation of MDA5 is poorly understood. Here, we show that TRIM65 specifically interacts with MDA5 and promotes K63-linked ubiquitination of MDA5 at lysine 743, which is critical for MDA5 oligomerization and activation. Trim65 deficiency abolishes MDA5 agonist or encephalomyocarditis virus (EMCV)-induced interferon regulatory factor 3 (IRF3) activation and type I interferon production but has no effect on retinoic acid-inducible I (RIG-I), Toll-like receptor 3 (TLR3), or cyclic GMP-AMP synthase signaling pathways. Importantly, Trim65-/- mice are more susceptible to EMCV infection than controls and cannot produce type I interferon in vivo. Collectively, our results identify TRIM65 as an essential component for the MDA5 signaling pathway and provide physiological evidence showing that ubiquitination is important for MDA5 oligomerization and activation.

Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain-containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868-880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 "pocket," completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.

Induction of type I interferon is a central event of innate immunity, essential for host defense. Here we report that the transcription factor ELF4 is induced by type I interferon and upregulates interferon expression in a feed-forward loop. ELF4 deficiency leads to reduced interferon production, resulting in enhanced susceptibility to West Nile virus encephalitis in mice. After viral infection, ELF4 is recruited by STING, interacts with and is activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind interferon promoters. Cooperative binding with ELF4 increases the binding affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by EICE elements. Thus, in addition to identifying a regulator of innate immune signaling, we uncovered a role for EICE elements in interferon transactivation.