Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.

The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.

Complement factors B, D, C3bBbP and risk of future venous thromboembolism.

The complement system appears to be involved in the pathogenesis of venous thromboembolism (VTE). We investigated the association of complement factors (CF) B, D, and the alternative pathway convertase, C3bBbP, measured at inclusion, with the risk of future VTE in a nested case-control study; 380 VTE patients and 804 age- and sex-matched controls derived from the Tromsø study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE across tertiles of CF concentrations were estimated using logistic regression. There was no association between CFB or CFD and risk of future VTE. Higher levels of C3bBbP gave an increased risk of provoked VTE; subjects in Q4 had a 1.68-fold higher OR compared with Q1 in the age-, sex- and BMI-adjusted model (OR 1.68; 95% CI 1.08-2.64). There was no increased risk of future VTE in individuals with higher levels of complement factors B or D of the alternative pathway. Increased levels of the alternative pathway activation product, C3bBbP, showed an association with future risk of provoked VTE.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

IFI16 is an innate immune sensor for intracellular DNA.

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-ß (IFN-ß). IFI16 directly associated with IFN-ß-inducing viral DNA motifs. STING, a critical mediator of IFN-ß responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-ß induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Sensing the hybrid--a novel PAMP for TLR9.

The host immune response is initiated by pattern recognition receptors (PRR) when triggered by specific ligands known as pathogen-associated molecular patterns (PAMP). Toll-like receptor 9 is highly expressed in plasmacytoid dendritic cells and is well described as a PRR for CpG DNA which induce the production of cytokines and type I interferons. In this issue of The EMBO Journal, Rigby et al identify RNA-DNA hybrids as a novel PAMP specifically recognized by TLR9 in dendritic cells.

Listeria monocytogenes induces IFNß expression through an IFI16-, cGAS- and STING-dependent pathway.

Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon ß (IFNß) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNß expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNß expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.

IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication.

Replication of lentiviruses generates different DNA forms, including RNA:DNA hybrids, ssDNA, and dsDNA. Nucleic acids stimulate innate immune responses, and pattern recognition receptors detecting dsDNA have been identified. However, sensors for ssDNA have not been reported, and the ability of RNA:DNA hybrids to stimulate innate immune responses is controversial. Using ssDNAs derived from HIV-1 proviral DNA, we report that this DNA form potently induces the expression of IFNs in primary human macrophages. This response was stimulated by stem regions in the DNA structure and was dependent on IFN-inducible protein 16 (IFI16), which bound immunostimulatory DNA directly and activated the stimulator of IFN genes -TANK-binding kinase 1 - IFN regulatory factors 3/7 (STING-TBK1-IRF3/7) pathway. Importantly, IFI16 colocalized and associated with lentiviral DNA in the cytoplasm in macrophages, and IFI16 knockdown in this cell type augmented lentiviral transduction and also HIV-1 replication. Thus, IFI16 is a sensor for DNA forms produced during the lentiviral replication cycle and regulates HIV-1 replication in macrophages.

Interferon γ-inducible protein (IFI) 16 transcriptionally regulates type i interferons and other interferon-stimulated genes and controls the interferon response to both DNA and RNA viruses.

The interferon γ-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-κB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-κB-regulated cytokines IL-6 and IL-1ß was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-α and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.

Activation of autophagy by α-herpesviruses in myeloid cells is mediated by cytoplasmic viral DNA through a mechanism dependent on stimulator of IFN genes.

Autophagy has been established as a player in host defense against viruses. The mechanisms by which the host induces autophagy during infection are diverse. In the case of HSV type 1 (HSV-1), dsRNA-dependent protein kinase is essential for induction of autophagy in fibroblasts through phosphorylation of eukaryotic initiation factor 2α (eIF2α). HSV-1 counteracts autophagy via ICP34.5, which dephosphorylates eIF2α and inhibits Beclin 1. Investigation of autophagy during HSV-1 infection has largely been conducted in permissive cells, but recent work suggests the existence of a eIF2α-independent autophagy-inducing pathway in nonpermissive cells. To clarify and further characterize the existence of a novel autophagy-inducing pathway in nonpermissive cells, we examined different HSV and cellular components in murine myeloid cells for their role in autophagy. We demonstrate that HSV-1-induced autophagy does not correlate with phosphorylation of eIF2α, is independent of functional dsRNA-dependent protein kinase, and is not antagonized by ICP34.5. Autophagy was activated independent of viral gene expression, but required viral entry. Importantly, we found that the presence of genomic DNA in the virion was essential for induction of autophagy and, conversely, that transfection of HSV-derived DNA induced microtubule-associated protein 1 L chain II formation, a marker of autophagy. This occurred through a mechanism dependent on stimulator of IFN genes, an essential component for the IFN response to intracellular DNA. Finally, we observed that HSV-1 DNA was present in the cytosol devoid of capsid material following HSV-1 infection of dendritic cells. Thus, our data suggest that HSV-1 genomic DNA induces autophagy in nonpermissive cells in a stimulator of IFN gene-dependent manner.

High plasma levels of C1-inhibitor are associated with lower risk of future venous thromboembolism.

BACKGROUND: C1-inhibitor (C1INH) is a broad-acting serine protease inhibitor with anticoagulant activity. The impact of C1INH plasma levels within the normal physiological range on risk of venous thromboembolism (VTE) is unknown. We assessed the association of plasma C1INH levels and VTE risk and evaluated the impact of C1INH on thrombin and plasmin generation in ex vivo assays. METHODS: A nested case-control study with 405 patients with VTE and 829 age- and sex-matched controls was derived from the Tromsø Study. Odds ratios (ORs) with 95% confidence intervals (95% CI) for VTE were estimated across plasma C1INH quartiles. Genetic regulation of C1INH was explored using quantitative trait loci analysis of whole exome sequencing data. The effect of plasma C1INH levels on coagulation was evaluated ex vivo by calibrated automated thrombography. RESULTS: Individuals with C1INH levels in the highest quartile had a lower risk of VTE (OR 0.68, 95% CI: 0.49-0.96) compared with those with C1INH in the lowest quartile. In subgroup analysis, the corresponding ORs were 0.60 (95% CI: 0.39-0.89) for deep vein thrombosis and 0.85 (95% CI: 0.52-1.38) for pulmonary embolism, respectively. No significant genetic determinants of plasma C1INH levels were identified. Addition of exogenous C1INH to normal human plasma reduced thrombin generation triggered by an activator of the intrinsic coagulation pathway, but not when triggered by an activator of the extrinsic coagulation pathway. CONCLUSIONS: High plasma levels of C1INH were associated with lower risk of VTE, and C1INH inhibited thrombin generation initiated by the intrinsic coagulation pathway ex vivo.

Elevated Systemic Levels of Markers Reflecting Intestinal Barrier Dysfunction and Inflammasome Activation Are Correlated in Severe Mental Illness.

BACKGROUND AND HYPOTHESIS: Gut microbiota alterations have been reported in severe mental illness (SMI) but fewer studies have probed for signs of gut barrier disruption and inflammation. We hypothesized that gut leakage of microbial products due to intestinal inflammation could contribute to systemic inflammasome activation in SMI. STUDY DESIGN: We measured plasma levels of the chemokine CCL25 and soluble mucosal vascular addressin cell adhesion molecule-1 (sMAdCAM-1) as markers of T cell homing, adhesion and inflammation in the gut, lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP) as markers of bacterial translocation and gut barrier dysfunction, in a large SMI cohort (n = 567) including schizophrenia (SCZ, n = 389) and affective disorder (AFF, n = 178), relative to healthy controls (HC, n = 418). We assessed associations with plasma IL-18 and IL-18BPa and leukocyte mRNA expression of NLRP3 and NLRC4 as markers of inflammasome activation. STUDY RESULTS: Our main findings were: (1) higher levels of sMAdCAM-1 (P = .002), I-FABP (P = 7.6E-11), CCL25 (P = 9.6E-05) and LBP (P = 2.6E-04) in SMI compared to HC in age, sex, BMI, CRP and freezer storage time adjusted analysis; (2) the highest levels of sMAdCAM-1 and CCL25 (both P = 2.6E-04) were observed in SCZ and I-FABP (P = 2.5E-10) and LBP (3) in AFF; and (3), I-FABP correlated with IL-18BPa levels and LBP correlated with NLRC4. CONCLUSIONS: Our findings support that intestinal barrier inflammation and dysfunction in SMI could contribute to systemic inflammation through inflammasome activation.

Cellular Requirements for Sensing and Elimination of Incoming HSV-1 DNA and Capsids.

Incoming viruses challenge the cell with diverse foreign molecules, which need to be sensed quickly to initiate immune responses and to remove the viral components. In this study, we investigate the cellular requirements for sensing and degradation of incoming viral DNA and capsids during herpes simplex virus type 1 (HSV-1) infections. Using click chemistry labeling of the viral genome, we found that HSV-1 DNA was released from a subset of capsids into the cytosol early in infection. By next-generation sequencing of cyclic GMP-AMP (cGAMP) synthase (cGAS)-bound DNA from HSV-1-infected cells, we show that HSV-1 DNA was bound by the cytosolic DNA sensor cGAS. Activation of cGAS enzymatic activity by viral DNA did not require proteasomal activity, indicating that viral DNA release into the cytosol is not proteasome-dependent. However, induction of interferon (IFN)-ß expression was blocked by inhibition of the proteasome, suggesting a contribution of the proteasome to IFN-ß induction through the cGAS-stimulator of interferon genes pathway. Viral DNA was cleared from the cytosol within few hours, in a manner dependent on TREX1 and a cGAS-dependent process. Capsid material in the cytoplasm was also degraded rapidly. This was partially blocked by treatment with a proteasome inhibitor. This treatment led to accumulation of DNA-containing viral capsids near the nucleus and reduced nuclear entry of viral DNA. Thus, cells infected with HSV-1 use a panel of mechanisms to eliminate viral DNA and capsids. This represents a barrier for establishment of infection and potentially enables the host to gear the IFN-ß response to a level required for antiviral defense without causing immunopathology.

Two-wave mixing in a broad-area semiconductor amplifier.

The two-wave mixing in the broad-area semiconductor amplifier was investigated, both theoretically and experimentally. In detail we investigated how the optical gain is affected by the presence of the two-wave mixing interference grating. In the experimental setup we are able to turn on and off the interference pattern in the semiconductor amplifier. This arrangement allows us to determine the two-wave mixing gain. The coupled-wave equations of two-wave mixing were derived based on the Maxwell's wave equation and rate equation of the carrier density. The analytical solutions of the coupled-wave equations were obtained in the condition of small signal and the total intensity is far below the saturation intensity of the amplifier. The results show that when the amplifier is operated below transparency we obtain an increase in the optical gain, and when the amplifier is operated above transparency we obtain a decrease in the optical gain. The experimental results obtained in an 810 nm, 200 microm wide GaAlAs amplifier show good agreement with the theory. A diffusion length of 2.0 microm is determined from the experiment.

Herpes simplex virus infection is sensed by both Toll-like receptors and retinoic acid-inducible gene- like receptors, which synergize to induce type I interferon production.

The innate antiviral response is initiated by pattern recognition receptors, which recognize viral pathogen-associated molecular patterns. Here we show that retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) in cooperation with Toll-like receptor (TLR) 9 is required for expression of type I interferons (IFNs) after infection with herpes simplex virus (HSV). Our work also identified RNase L as a critical component in IFN induction. Moreover, we found that TLR9 and RLRs activate distinct, as well as overlapping, intracellular signalling pathways. Thus, RLRs are important for recognition of HSV infection, and cooperate with the Toll pathway to induce an antiviral response.

RIG-I-mediated activation of p38 MAPK is essential for viral induction of interferon and activation of dendritic cells: dependence on TRAF2 and TAK1.

The innate immune system provides an initial defense system against microbial infections and contributes to the development of adaptive immune response. Type I interferons play a pivotal role for the first line of defense against virus infections, and dendritic cells (DCs) are important sensors of pathogens responsible for priming of adaptive immune responses in lymphoid organs. Here we have investigated the role and mechanisms of activation of the MAPK pathway in innate immune responses induced by Sendai virus, a negative sense single-stranded RNA virus. Both p38 and JNK were activated in fibroblasts and DCs after infection with Sendai virus in a manner dependent on virus replication and RIG-I. Virus replication was also required for stimulation of interferon production in both cell types and interleukin-12 production in DCs. Blocking of p38 MAPK activation by the specific inhibitor SB202190 abolished the expression of these cytokines. p38 MAPK exerted its function independent of the MAPK-activated protein kinases MK2, MNK, and MSK1/2. We also observed that TRAF2 and TAK1 were essential for RIG-I-mediated activation of p38 MAPK. Interestingly, the kinase activity of p38 MAPK was required for its own phosphorylation, which was kinetically associated with TAB1 interaction. By contrast, the canonical p38 upstream kinase MKK3 was not involved in the p38-dependent response. Thus, activation of p38 MAPK by RIG-I proceeds via a TRAF2-TAK1-dependent pathway, where the enzymatic activity of the kinase plays an essential role. The p38 MAPK in turn stimulates important processes in the innate antiviral response.

Activation of innate defense against a paramyxovirus is mediated by RIG-I and TLR7 and TLR8 in a cell-type-specific manner.

Recognition of pathogens by the innate immune system is mediated by pattern recognition receptors (PRRs), which recognize specific molecular structures of the infectious agents and subsequently trigger expression of genes involved in host defense. Toll-like receptors (TLRs) represent a well-characterized class of membrane-bound PRRs, and the RNA helicase retinoic acid inducible gene I (RIG-I) has recently been described as a novel cytoplasmic PRR recognizing double-stranded RNA (dsRNA). Here we show that activation of signal transduction and induction of cytokine expression by the paramyxovirus Sendai virus is dependent on virus replication and involves PRRs in a cell-type-dependent manner. While nonimmune cells relied entirely on recognition of dsRNA through RIG-I for activation of an antiviral response, myeloid cells utilized both the single-stranded RNA sensing TLR7 and TLR8 and dsRNA-dependent mechanisms independent of RIG-I, TLR3, and dsRNA-activated protein kinase R to trigger this response. Therefore, there appears to be a large degree of cell-type specificity in the mechanisms used by the host to recognize infecting viruses.