Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.     

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.     

Antiviral signaling through pattern recognition receptors

Viral infection is detected by the host innate immune system. Innate immune cells such as dendritic cells and macrophages detect nucleic acids derived from viruses through pattern recognition receptors (PRRs). Viral recognition by PRRs initiates the activation of signaling pathways that lead to production of type I interferon and inflammatory cytokines, which are important for the elimination of viruses. Two types of PRRs that recognize viral nucleic acids, Toll-like receptors (TLR) and RIG-I-like RNA helicases (RLH), have been identified. Of the TLRs, TLR3 recognizes viral double-stranded (ds) RNA, TLR7 and human TLR8 identify viral single-stranded (ss) RNA and TLR9 detects viral DNA. TLRs are located in endosomal compartments, whereas RLH are present in the cytoplasm where they detect viral dsRNA or ssRNA. Here we review the role of TLRs and RLHs in the antiviral innate immune response.     

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1‐caspase‐1 axis

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.     

Toll-like receptors that sense viral infection

Anti-viral host defense harbors a variety of strategies to coup with viral infection. Recent findings suggested that Toll-like receptors (TLRs) and their signaling pathways involve type I IFN induction in response to virus-specific molecular patterns. TLR 3 and TLR 4 in myeloid dendritic cells (mDCs) recognize viral dsRNA and putative viral products, respectively, to induce IFN-beta via IRF-3 activation. On the other hand, TLR 7 and TLR 9 in plasmacytoid DCs (pDCs) induce IFN-alpha in response to their ligands, U/G-rich ssRNA and non-methylated CpG DNA. We identified TICAM-1 which is recruited to the cytoplasmic domain (designated TIR) of TLR 3 and allows to select the pathway to activation of IRF-3. We also identified TICAM-2 which binds TLR 4 and together with TICAM-1 activates IRF-3. TICAM-1 knockdown by RNAi supported the key role of TICAM-1 in IFN-beta induction. Hence, the IFN-beta induction in mDCs appears in part due to the function of TICAM-1. Viruses are known to activate kinases that directly activate IRF-3 inside the cells, and this pathway may merge with the TLR 3-TICAM-1 pathway. Here we review the relationship between the TLR 3-TICAM-1 pathway and viral infection.     

Toll样受体信号转导对树突状细胞活化的影响

树突状细胞（dendriticcells,DCs）是目前已知功能最强的抗原提呈细胞（antigenpresentingcell,APC）,是介导固有免疫和适应性免疫的桥梁,在机体抗感染、抗肿瘤等方面发挥重要作用。Toll样受体（toll．1ikereceptor,TLRs）是一类重要的模式识别受体（paRemrecognitionreceptors,PRRs）,可识别入侵的病原体相关分子模式（pathogen-associatedmoleculepatterns,PAMPs）,通过招募接头蛋白、活化蛋白激酶和激活转录因子进行信号传导,从而引起效应细胞的活化和促炎因子的释放。不同亚型的DCs分布有不同的TLRs,多种TLRs可识别外来入侵的病原体成分,发挥重要的免疫学作用：诱导DCs分化成熟,摄取递呈抗原,促进DCs分泌多种细胞因子发挥作用。在炎症、病毒感染、自身免疫性疾病和肿瘤等疾病状态下,DCs表面TLRs的表达上调或下调,并且存在功能障碍,可影响DCs的分化成熟,导致其功能低下,这与疾病的发生和发展密切相关。本文综述了TLRs及其信号通路对树突状细胞的活化及功能的影响。     

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Human TLR3 recognizes dengue virus and modulates viral replication in vitro

The elicitation of large amount inflammatory cytokine in serum has been developed as the cause of the plasma leakage in dengue fever (DF)/dengue haemorrhagic fever (DHF) infection. Virus recognition in innate immunity is the key. The Toll-like receptors (TLRs) play an important role in pathogen recognition towards cytokine induction among several viruses; however, the role of TLRs on innate immune recognition against DENV remains unclear. This study aims at the interaction between dengue virus (DENV) and human TLRs at the incipient stage of infection in vitro . Our experiment reveals that stably expression of TLR3, 7, 8 on HEK293 enables IL-8 secretion after DENV recognition. By the model of human monocytic cells U937, we demonstrated the trigger of IL-8 after viral recognition of human monocytic cell is primary through TLR3 following endosomal acidification. Silencing of TLR3 in U937 cells significantly blocks the DENV-induced IL-8 production. Besides, the interaction is further corroborated by colocalization of TLR3 and DENV RNA upon DENV internalization. Furthermore, in this study we found the expression of TLR3 can mediate strong IFN-α/β release and inhibit DENV viral replication significantly, thus limit the cytopathic effect.     

Ebolavirus VP35 suppresses IFN production from conventional but not plasmacytoid dendritic cells

Ebolaviruses naturally infect a wide variety of cells including macrophages and dendritic cells (DCs), and the resulting cytokine and interferon-α/β (IFN) responses of infected cells are thought to influence viral pathogenesis. The VP35 protein impairs RIG-I-like receptor-dependent signaling to inhibit IFN production, and this function has been suggested to promote the ineffective host immune response characteristic of ebolavirus infection. To assess the impact of VP35 on innate immunity in biologically relevant primary cells, we used a recombinant Newcastle disease virus encoding VP35 (NDV/VP35) to infect macrophages and conventional DCs, which primarily respond to RNA virus infection via RIG-I-like pathways. VP35 suppressed not only IFN but also tumor necrosis factor (TNF)-α secretion, which are normally produced from these cells upon NDV infection. Additionally, in cells susceptible to the activity of VP35, IRF7 activation is impaired. In contrast, NDV/VP35 infection of plasmacytoid DCs, which activate IRF7 and produce IFN through TLR-dependent signaling, leads to robust IFN production. When plasmacytoid DCs deficient for TLR signaling were infected, NDV/VP35 was able to inhibit IFN production. Consistent with this, VP35 was less able to inhibit TLR-dependent versus RIG-I-dependent signaling in vitro. These data demonstrate that ebolavirus VP35 suppresses both IFN and cytokine production in multiple primary human cell types. However, cells that utilize the TLR pathway can circumvent this inhibition, suggesting that the presence of multiple viral sensors enables the host to overcome viral immune evasion mechanisms.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

MIM through MOM: the awakening of Bax and Bak pores

The mechanism whereby mitochondrial DNA (mtDNA) is released into the cytosol and activates the cGAS/STING inflammatory pathway during Bax/Bax‐mediated apoptosis is unknown. In this issue, Riley et al ( 2018 ) report that widening of Bax and Bak pores on the mitochondrial outer membrane (MOM) during apoptosis allows the extrusion of the mitochondrial inner membrane (MIM) into the cytosol and its permeabilization to release mtDNA independently of caspases. In this scenario, Bax and Bak emerge as key modulators of the apoptotic immunogenic response.     

Increased plasma levels of extracellular mitochondrial DNA during HIV infection: a new role for mitochondrial damage-associated molecular patterns during inflammation

HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV⁺ patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4⁺ T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.     

TLR9-signaling pathways are involved in Kilham rat virus-induced autoimmune diabetes in the biobreeding diabetes-resistant rat

Viral infections are associated epidemiologically with the expression of type 1 diabetes in humans, but the mechanisms underlying this putative association are unknown. To investigate the role of viruses in diabetes, we used a model of viral induction of autoimmune diabetes in genetically susceptible biobreeding diabetes-resistant (BBDR) rats. BBDR rats do not develop diabetes in viral-Ab-free environments, but approximately 25% of animals infected with the parvovirus Kilham rat virus (KRV) develop autoimmune diabetes via a mechanism that does not involve beta cell infection. Using this model, we recently documented that TLR agonists synergize with KRV infection and increase disease penetrance. We now report that KRV itself activates innate immunity through TLR ligation. We show that KRV infection strongly stimulates BBDR splenocytes to produce the proinflammatory cytokines IL-6 and IL-12p40 but not TNF-alpha. KRV infection induces high levels of IL-12p40 by splenic B cells and Flt-3-ligand-induced bone marrow-derived dendritic cells (DCs) but only low levels of IL-12p40 production by thioglycolate-elicited peritoneal macrophages or GM-CSF plus IL-4-induced bone marrow-derived DCs. KRV-induced cytokine production is blocked by pharmacological inhibitors of protein kinase R and NF-kappaB. Genomic KRV DNA also induces BBDR splenocytes and Flt-3L-induced DCs from wild-type but not TLR9-deficient mice to produce IL-12p40; KRV-induced up-regulation of B lymphocytes can be blocked by TLR9 antagonists including inhibitory CpG and chloroquine. Administration of chloroquine to virus-infected BBDR rats decreases the incidence of diabetes and decreases blood levels of IL-12p40. Our data implicate the TLR9-signaling pathway in KRV-induced innate immune activation and autoimmune diabetes in the BBDR rat.     

Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

Malaria parasites require TLR9 signaling for immune evasion by activating regulatory T cells

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9(-/-) mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.     

Mitochondrial DNA in the regulation of innate immune responses

Mitochondrion is known as the energy factory of the cell, which is also a unique mammalian organelle and considered to be evolved from aerobic prokaryotes more than a billion years ago. Mitochondrial DNA, similar to that of its bacterial ancestor’s, consists of a circular loop and contains significant number of unmethylated DNA as CpG islands. The innate immune system plays an important role in the mammalian immune response. Recent research has demonstrated that mitochondrial DNA (mtDNA) activates several innate immune pathways involving TLR9, NLRP3 and STING signaling, which contributes to the signaling platforms and results in effector responses. In addition to facilitating antibacterial immunity and regulating antiviral signaling, mounting evidence suggests that mtDNA contributes to inflammatory diseases following cellular damage and stress. Therefore, in addition to its well-appreciated roles in cellular metabolism and energy production, mtDNA appears to function as a key member in the innate immune system. Here, we highlight the emerging roles of mtDNA in innate immunity.