RIG-I and Other RNA Sensors in Antiviral Immunity.

Pattern recognition receptors (PRRs) survey intra- and extracellular spaces for pathogen-associated molecular patterns (PAMPs) within microbial products of infection. Recognition and binding to cognate PAMP ligand by specific PRRs initiates signaling cascades that culminate in a coordinated intracellular innate immune response designed to control infection. In particular, our immune system has evolved specialized PRRs to discriminate viral nucleic acid from host. These are critical sensors of viral RNA to trigger innate immunity in the vertebrate host. Different families of PRRs of virus infection have been defined and reveal a diversity of PAMP specificity for wide viral pathogen coverage to recognize and extinguish virus infection. In this review, we discuss recent insights in pathogen recognition by the RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensor PRRs, to present emerging themes in innate immune signaling during virus infection.

A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Hematopoietic stem and progenitor cells integrate microbial signals to promote post-inflammation gut tissue repair.

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated following bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we show that acute gut inflammation expands HSPCs in the BM and directs them to inflamed mesenteric lymph nodes through GM-CSFR activation for further expansion and potential differentiation into Ly6C+ /G+ myeloid cells specialized in gut tissue repair. We identified this process to be mediated by Bacteroides, a commensal gram-negative bacteria that activates innate immune signaling. These findings establish cross-organ communication between the BM and distant inflamed sites, whereby a certain subset of multipotent progenitors is specified to respond to imminent hematopoietic demands and to alleviate inflammatory symptoms.

Targeting protein condensation in cGAS-STING signaling pathway.

The cGAS-STING signaling pathway plays a pivotal role in sensing cytosolic DNA and initiating innate immune responses against various threats, with disruptions in this pathway being associated with numerous immune-related disorders. Therefore, precise regulation of the cGAS-STING signaling is crucial to ensure appropriate immune responses. Recent research, including ours, underscores the importance of protein condensation in driving the activation and maintenance of innate immune signaling within the cGAS-STING pathway. Consequently, targeting condensation processes in this pathway presents a promising approach for modulating the cGAS-STING signaling and potentially managing associated disorders. In this review, we provide an overview of recent studies elucidating the role and regulatory mechanism of protein condensation in the cGAS-STING signaling pathway while emphasizing its pathological implications. Additionally, we explore the potential of understanding and manipulating condensation dynamics to develop novel strategies for mitigating cGAS-STING-related disorders in the future.

Microglia in antiviral immunity of the brain and spinal cord.

Viral infections of the central nervous system (CNS) are a significant cause of neurological impairment and mortality worldwide. As tissue resident macrophages, microglia are critical initial responders to CNS viral infection. Microglia seem to coordinate brain-wide antiviral responses of both brain resident cells and infiltrating immune cells. This review discusses how microglia may promote this antiviral response at a molecular level, from potential mechanisms of virus recognition to downstream cytokine responses and interaction with antiviral T cells. Recent advancements in genetic tools to specifically target microglia in vivo promise to further our understanding about the precise mechanistic role of microglia in CNS infection.

Long noncoding RNA CHROMR regulates antiviral immunity in humans.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their contribution to immune regulation in humans remains poorly understood. Here, we report that the primate-specific lncRNA CHROMR is induced by influenza A virus and SARS-CoV-2 infection and coordinates the expression of interferon-stimulated genes (ISGs) that execute antiviral responses. CHROMR depletion in human macrophages reduces histone acetylation at regulatory regions of ISG loci and attenuates ISG expression in response to microbial stimuli. Mechanistically, we show that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional corepressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Consequently, CHROMR expression is essential to restrict viral infection of macrophages. Our findings identify CHROMR as a key arbitrator of antiviral innate immune signaling in humans.

Mitochondrial double-stranded RNA triggers induction of the antiviral DNA deaminase APOBEC3A and nuclear DNA damage.

APOBEC3A is an antiviral DNA deaminase often induced by virus infection. APOBEC3A is also a source of cancer mutation in viral and nonviral tumor types. It is therefore critical to identify factors responsible for APOBEC3A upregulation. Here, we test the hypothesis that leaked mitochondrial (mt) double-stranded (ds)RNA is recognized as foreign nucleic acid, which triggers innate immune signaling, APOBEC3A upregulation, and DNA damage. Knockdown of an enzyme responsible for degrading mtdsRNA, the exoribonuclease polynucleotide phosphorylase, results in mtdsRNA leakage into the cytosol and induction of APOBEC3A expression. APOBEC3A upregulation by cytoplasmic mtdsRNA requires RIG-I, MAVS, and STAT2 and is likely part of a broader type I interferon response. Importantly, although mtdsRNA-induced APOBEC3A appears cytoplasmic by subcellular fractionation experiments, its induction triggers an overt DNA damage response characterized by elevated nuclear γ-H2AX staining. Thus, mtdsRNA dysregulation may induce APOBEC3A and contribute to observed genomic instability and mutation signatures in cancer.

USP38 Inhibits Type I Interferon Signaling by Editing TBK1 Ubiquitination through NLRP4 Signalosome.

TBK1 is a component of the type I interferon (IFN) signaling pathway, yet the mechanisms controlling its activity and degradation remain poorly understood. Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo. USP38 specifically cleaves K33-linked poly-ubiquitin chains from TBK1 at Lys670, and it allows for subsequent K48-linked ubiquitination at the same position mediated by DTX4 and TRIP. Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signaling. Our findings identify an essential role for USP38 in negatively regulating type I IFN signaling, and they provide insights into the mechanisms by which USP38 regulates TBK1 ubiquitination through the NLRP4 signalosome.

First Discovery of Beta-Sitosterol as a Novel Antiviral Agent against White Spot Syndrome Virus.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.

Viperin: An ancient radical SAM enzyme finds its place in modern cellular metabolism and innate immunity.

Viperin plays an important and multifaceted role in the innate immune response to viral infection. Viperin is also notable as one of very few radical SAM-dependent enzymes present in higher animals; however, the enzyme appears broadly conserved across all kingdoms of life, which suggests that it represents an ancient defense mechanism against viral infections. Although viperin was discovered some 20 years ago, only recently was the enzyme's structure determined and its catalytic activity elucidated. The enzyme converts CTP to 3'-deoxy-3',4'-didehydro-CTP, which functions as novel chain-terminating antiviral nucleotide when misincorporated by viral RNA-dependent RNA polymerases. Moreover, in higher animals, viperin interacts with numerous other host and viral proteins, and it is apparent that this complex network of interactions constitutes another important aspect of the protein's antiviral activity. An emerging theme is that viperin appears to facilitate ubiquitin-dependent proteasomal degradation of some of the proteins it interacts with. Viperin-targeted protein degradation contributes to the antiviral response either by down-regulating various metabolic pathways important for viral replication or by directly targeting viral proteins for degradation. Here, we review recent advances in our understanding of the structure and catalytic activity of viperin, together with studies investigating the interactions between viperin and its target proteins. These studies have provided detailed insights into the biochemical processes underpinning this unusual enzyme's wide-ranging antiviral activity. We also highlight recent intriguing reports that implicate a broader role for viperin in regulating nonpathological cellular processes, including thermogenesis and protein secretion.

Innate Immune Nod1/RIP2 Signaling Is Essential for Cardiac Hypertrophy but Requires Mitochondrial Antiviral Signaling Protein for Signal Transductions and Energy Balance.

BACKGROUND: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. METHODS: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1-/-, RIP2-/-, or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1-/- and RIP2-/- mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1-/- and RIP2-/- mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. CONCLUSIONS: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.

Neuroimmune mechanisms of cognitive impairment in a mouse model of Gulf War illness.

Gulf War Illness (GWI) is a chronic, multi-symptom disorder affecting approximately 30 percent of the nearly 700,000 Veterans of the 1991 Persian Gulf War. GWI-related chemical (GWIC) exposure promotes immune activation that correlates with cognitive impairment and other symptoms of GWI. However, the molecular mechanisms and signaling pathways linking GWIC to inflammation and neurological symptoms remain unclear. Here we show that acute exposure of murine macrophages to GWIC potentiates innate immune signaling and inflammatory cytokine production. Using an established mouse model of GWI, we report that neurobehavioral changes and neuroinflammation are attenuated in mice lacking the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) and NOD-, LRR- or pyrin domain-containing protein 3 (NLRP3) innate immune pathways. In addition, we report sex differences in response to GWIC, with female mice showing more pronounced cognitive impairment and hippocampal astrocyte hypertrophy. In contrast, male mice display a GWIC-dependent upregulation of proinflammatory cytokines in the plasma that is not present in female mice. Our results indicate that STING and NLRP3 are key mediators of the cognitive impairment and inflammation observed in GWI and provide important new information on sex differences in this model.

[Innate immune signal-mediated regulation and alterations in hematopoiesis].

Hematopoietic stem cells (HSCs) are maintained in steady state in the bone marrow (BM); these cells are capable of continuous self-renewal and have the potential for multilineage-differentiation into all blood cell lineages. The BM has long been considered as an immune-privilege organ with little immunological reactions. However, recent findings have revealed that immunological/hematopoietic challenges such as infection or inflammation induce broad spectrum of immune and inflammatory responses in BM. While these responses play a beneficial role to boost immune activation and blood production, chronic challenge might lead to BM pathology and dysregulation, including hematopoietic aplasia or neoplasia. We will introduce recent findings focused on hematopoietic activation induced by existing outside of our body or co-existing with us, and discuss to what degree and how function of HSCs and progenitors is regulated and altered by bacterial insult.

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective.

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.

Bacterial TIR domain-derived peptides inhibit innate immune signaling and catabolic responses in chondrocyte.

Osteoarthritis (OA) is a degenerative joint disease, in which low-grade inflammation plays an important role at the initiating step. Low-doses of LPS-induced inflammation in the plasma activate chondrocytes and promote the secretion proinflammatory cytokines, leading to secondary inflammation. Blocking OA-associated TLR activation is a promising strategy for the development of suitable therapies. Here, we want to find some bacteria-derived peptides that can block TLR signaling in chondrocytes more efficiently. Based on previous studies, we screened 12 TIR domain-derived peptides for their effects on NF-кB activation induced by LPS, IL-1ß or TNF-α in murine ATDC-5 cells. We evaluated their effects on LPS-induced cytokine expression and secretion. Among them, two bacteria-derived peptides, TcpC-DD and TcpB-DD, showed the most potent inhibitory activities. In comparison with TcpB-DD, TcpC-DD exhibited broader TLR-inhibitory specificity during inflammation in chondrocytes. Furthermore, both TcpC-DD and TcpB-DD displayed strong inhibition of LPS- and IL-1ß-induced catabolic reactions in chondrocytes. However, only TcpC-DD exhibited obvious suppression of TNF-α-induced catabolism. In conclusion, we identified two novel inhibitory peptides that modulate catabolism in chondrocytes and innate immune responses, and these peptides could be used to develop novel therapeutic strategies for OA.

Foot-and-mouth disease virus infection stimulates innate immune signaling in the mouse macrophage RAW 264.7 cells.

The innate immune system acts as the first line of defense against invasion by bacterial and viral pathogens. The role of macrophages in innate immune responses to foot-and-mouth disease virus (FMDV) is poorly understood. To determine the mechanism underlying activation of innate immunity after FMDV infection in macrophages, we performed FMDV infection in mouse macrophage RAW 264.7 cells and found that FMDV serotype O infection induced a cytopathic effect. We then evaluated the gene expression profile in macrophage RAW 264.7 cells after FMDV infection using systematic microarray analysis. Gene ontology annotation and enrichment analysis revealed that FMDV promoted expression in a group of genes that are enriched in innate immune response and inflammatory response processes. Further research demonstrated that FMDV serotype O infection enhanced NF-κB, Toll-like, and RIG-I-like receptor signaling pathways and proteins expression and increased transcription and expression of a series of cytokines and interferons, as proved by qRT-PCR, Western blot, ELISA, and dual-luciferase reporter assay. Our study concluded that FMDV infection triggers the innate immune response in macrophages after activation of multiple innate immune pathway receptors and proteins by FMDV serotype O, resulting in activation and secretion of a series of cytokines and interferons.

Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue.

Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.

Gcn5 and PCAF negatively regulate interferon-ß production through HAT-independent inhibition of TBK1.

Viral infection triggers innate immune signaling, which in turn induces interferon-ß (IFN-ß) production to establish innate antiviral immunity. Previous studies showed that Gcn5 (Kat2a), a histone acetyltransferase (HAT) with partial functional redundancy with PCAF (Kat2b), and Gcn5/PCAF-mediated histone H3K9 acetylation (H3K9ac) are enriched on the active IFNB gene promoter. However, whether Gcn5/PCAF and H3K9ac regulate IFN-ß production is unknown. Here, we show that Gcn5/PCAF-mediated H3K9ac correlates well with, but is surprisingly dispensable for, the expression of endogenous IFNB and the vast majority of active genes in fibroblasts. Instead, Gcn5/PCAF repress IFN-ß production and innate antiviral immunity in several cell types in a HAT-independent and non-transcriptional manner: by inhibiting the innate immune signaling kinase TBK1 in the cytoplasm. Our results thus identify Gcn5 and PCAF as negative regulators of IFN-ß production and innate immune signaling.

Identification and functional characterization of three TLR signaling pathway genes in Cyclina sinensis.

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play a critical role in initiating and activating the innate immune system. In this study, we identified two TLR genes (CsTLR4 and CsTLR13) and the MyD88 (CsMyD88) gene using a transcriptome library from Cyclina sinensis. The sequence features and mRNA expression profiles of the genes were characterized, and their functions in the immune response were investigated to validate the TLR signaling pathway and its potential role in immune defense. The expression patterns of CsTLR4, CsTLR13 and CsMyD88 were detected in all the tissues examined from healthy clams and were primarily expressed in the hemocytes (P < 0.05), as shown by real-time PCR. Upon challenge with Vibrio anguillarum and Micrococcus luteus, they were significantly increased in hemocytes (P < 0.01), whereas only CsTLR13 and CsMyD88 were up-regulated (P < 0.01) by poly (I:C) challenge. In addition, the mRNA expression level of CsC-LYZ and CsAMP was down-regulated at 72 h (P < 0.01) after injection with CsMyD88 RNAi. These findings might be valuable for understanding the innate immune signaling pathways of C. sinensis and enabling future studies on host-pathogen interactions.

TBK1-like transcript negatively regulates the production of IFN and IFN-stimulated genes through RLRs-MAVS-TBK1 pathway.

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN and host antiviral defense. In the present study, TBK1-like transcript, namely TBK1L, was cloned from zebrafish. Compared with TBK1, TBK1L contains an incomplete S_TKc domain, and lacks UBL_TBK1_like domain. Realtime PCR showed that TBK1L was constitutively produced in embryos, early larvae and ZF4 cells, and unchanged in ZF4 cells following SVCV infection. Overexpression of TBK1 but not TBK1L resulted in significant activation of zebrafish IFN1 and IFN3 promoters. Similarly, TBK1L had little impact on the antiviral state of the cells. However, the overexpression of TBK1L negatively regulated the induction of zebrafish IFN1 and/or IFN3 promoters mediated by the retinoic acid-inducible gene I-like receptors (RLRs), MAVS and TBK1. In addition, the overexpression of TBK1L in zebrafish embryos led to the decreased production of many IFN-stimulated genes induced by TBK1. Collectively, these data support that zebrafish TBK1L negatively regulates RLRs-MAVS-TBK1 pathway.