A Guideline Strategy for Identifying Genes/Proteins Regulating Antiviral Innate Immunity.

Antiviral innate immunity is a complicated system initiated by the induction of type I interferon (IFN-I) and downstream interferon-stimulated genes (ISGs) and is finely regulated by numerous positive and negative factors at different signaling adaptors. During this process, posttranslational modifications, especially ubiquitination, are the most common regulatory strategy used by the host to switch the antiviral innate signaling pathway and are mainly controlled by E3 ubiquitin ligases from different protein families. A comprehensive understanding of the regulatory mechanisms and a novel discovery of regulatory factors involved in the IFN-I signaling pathway are important for researchers to identify novel therapeutic targets against viral infectious diseases based on innate immunotherapy. In this section, we use the E3 ubiquitin ligase as an example to guide the identification of a protein belonging to the RING Finger (RNF) family that regulates the RIG-I-mediated IFN-I pathway through ubiquitination.

Identifying RNA Sensors in Antiviral Innate Immunity.

The innate immune system plays a pivotal role in pathogen recognition and the initiation of innate immune responses through its Pathogen Recognition Receptors (PRRs), which detect Pathogen-Associated Molecular Patterns (PAMPs). Nucleic acids, including RNA and DNA, are recognized as particularly significant PAMPs, especially in the context of viral pathogens. During RNA virus infections, specific sequences in the viral RNA mark it as non-self, enabling host recognition through interactions with RNA sensors, thereby triggering innate immunity. Given that some of the most lethal viruses are RNA viruses, they pose a severe threat to human and animal health. Therefore, understanding the immunobiology of RNA PRRs is crucial for controlling pathogen infections, particularly RNA virus infections. In this chapter, we will introduce a "pull-down" method for identifying RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensors.

Polymorphisms in the genes encoding RLR and TLR3 and CMV DNAemia in subjects coinfected with human immunodeficiency virus and cytomegalovirus.

Cytomegalovirus (CMV) is a pathogen that is common worldwide and is often present in individuals infected with human immunodeficiency virus (HIV). Pattern recognition receptors (PRRs) are host sensors that activate the immune response against infectious agents. However, it is unclear whether PRR single-nucleotide polymorphisms (SNPs) are associated with the occurrence of CMV DNAemia in subjects coinfected with HIV and CMV. HIV/CMV-coinfected patients with and without CMV DNAemia were recruited for this study. The DDX58 rs10813831 and IFIH1 (rs3747517 and rs1990760) polymorphisms were genotyped using the TaqMan Allelic Discrimination Assay, whereas the DDX58 rs12006123 and TLR3 (rs3775291 and rs3775296) SNPs were analyzed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. A mutation present in at least one allele of the DDX58 rs12006123 SNP occurred at least two times more frequently in HIV/CMV-coinfected patients with CMV DNAemia than in coinfected subjects without CMV DNAemia (OR, 2.50; 95% CI, 1.33-4.68; p = 0.004, in the dominant model). A higher level of CMV DNAemia was observed in subjects who had the heterozygous (GA) or homozygous recessive (AA) genotype for the DDX58 rs12006123 SNP compared with those who had the wild-type (GG) genotype (p = 0.0003). Moreover, in subjects with a mutation detected in at least one allele of the DDX58 rs12006123 SNP, a lower serum IFN-ß concentration was found compared with those who had a wild-type (GG) genotype for this polymorphism (p = 0.024). The DDX58 rs12006123 SNP is associated with CMV DNAemia in HIV/CMV-coinfected patients.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7.

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Release of mitochondrial dsRNA into the cytosol is a key driver of the inflammatory phenotype of senescent cells.

The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases.

Recent developments in understanding RIG-I's activation and oligomerization.

Insights into mechanisms driving either activation or inhibition of immune response are crucial in understanding the pathology of various diseases. The differentiation of viral from endogenous RNA in the cytoplasm by pattern-recognition receptors, such as retinoic acid-inducible gene I (RIG-I), is one of the essential paths for timely activation of an antiviral immune response through induction of type I interferons (IFN). In this mini-review, we describe the most recent developments centered around RIG-I's structure and mechanism of action. We summarize the paradigm-changing work over the past few years that helped us better understand RIG-I's monomeric and oligomerization states and their role in conveying immune response. We also discuss potential applications of the modulation of the RIG-I pathway in preventing autoimmune diseases or induction of immunity against viral infections. Overall, our review aims to summarize innovative research published in the past few years to help clarify questions that have long persisted around RIG-I.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

Histone deacetylase 8 promotes innate antiviral immunity through deacetylation of RIG-I.

Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.

Nsp10-interacting host protein SAP18 restricts PEDV replication in Marc-145 cells via enhancing dephosphorylation of RIG-I.

PEDV, a single-stranded RNA virus, causes significant economic losses in the pig industry. Sin3-associated protein 18 (SAP18) is known for its role in transcriptional inhibition and RNA splicing. However, research on SAP18's involvement in PEDV infection is limited. Here, we identified an interaction between SAP18 and PEDV nonstructural protein 10 (Nsp10) using immunoprecipitation-mass spectrometry (IP-MS) and confirmed it through immunoprecipitation and laser confocal microscopy. Additionally, PEDV Nsp10 reduced SAP18 protein levels and induced its cytoplasmic accumulation. Overexpressing SAP18 suppressed PEDV replication, meanwhile its knockdown via short interfering RNA (siRNA) enhanced replication. SAP18 overexpression boosted IRF3 and NF-κB P65 phosphorylation, nuclear translocation, and IFN-ß antiviral response. Furthermore, SAP18 upregulated RIG-I expression and facilitated its dephosphorylation, while SAP18 knockdown had the opposite effect. Finally, SAP18 interacted with phosphatase 1 (PP1) catalytic subunit alpha (PPP1CA), promoting PPP1CA-RIG-I interaction during PEDV infection. These findings highlight SAP18's role in activating the type I interferon pathway and inhibiting viral replication by promoting RIG-I dephosphorylation through its interaction with PPP1CA.

Glaesserella parasuis serotype 5 induces pyroptosis via the RIG-I/MAVS/NLRP3 pathway in swine tracheal epithelial cells.

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.

Transmissible gastroenteritis virus induces inflammatory responses via RIG-I/NF-κB/HIF-1α/glycolysis axis in intestinal organoids and in vivo.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

Nlrp6 protects from corticosterone-induced NSPC ferroptosis by modulating RIG-1/MAVS-mediated mitophagy.

Hippocampal neural stem/progenitor cells (NSPCs) are highly vulnerable to different stress stimuli, resulting in adult neurogenesis decline and eventual cognitive defects. Our previous study demonstrated that NOD-like receptor family pyrin domain-containing 6 (Nlrp6) highly expressed in NSPCs played a critical role in sustaining hippocampal neurogenesis to resist stress-induced depression, but the underlying mechnistms are still unclear. Here, we found that Nlrp6 depletion led to cognitive defects and hippocampal NSPC loss in mice. RNA-sequencing analysis of the primary NSPCs revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process. Upon siNlrp6 transfection, as well as corticosterone (CORT) exposure, downregulation of Nlrp6 suppressed retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS)-mediated autophagy, but drove NSPC ferroptotic death. More interesting, short chain fatty acids (SCFAs) upregulated Nlrp6 expression and promoted RIG-1/MAVS-mediated mitophagy, preventing CORT-induced NSPC ferroptosis. Our study further demonstrates that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy and play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs. These results suggests that Nlrp6 should be a potential drug target to combat neurodegenerative diseases relative with chronic stress.

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Mammalian orthoreovirus capsid protein σ3 antagonizes RLR-mediated antiviral responses by degrading MAVS.

Mammalian orthoreovirus (MRV) outer capsid protein σ3 is a multifunctional protein containing a double-stranded RNA-binding domain, which facilitates viral entry and assembly. We reasoned that σ3 has an innate immune evasion function. Here, we show that σ3 protein localizes in the mitochondria and interacts with mitochondrial antiviral signaling protein (MAVS) to activate the intrinsic mitochondria-mediated apoptotic pathway. Consequently, σ3 protein promotes the degradation of MAVS through the intrinsic caspase-9/caspase-3 apoptotic pathway. Moreover, σ3 protein can also inhibit the expression of the components of the RNA-sensing retinoic acid-inducible gene (RIG)-like receptor (RLR) signaling pathway to block antiviral type I interferon responses. Mechanistically, σ3 inhibits RIG-I and melanoma differentiation-associated gene 5 expression is independent of its inhibitory effect on MAVS. Overall, we demonstrate that the MRV σ3 protein plays a vital role in negatively regulating the RLR signaling pathway to inhibit antiviral responses. This enables MRV to evade host defenses to facilitate its own replication providing a target for the development of effective antiviral drugs against MRV. IMPORTANCE: Mammalian orthoreovirus (MRV) is an important zoonotic pathogen, but the regulatory role of its viral proteins in retinoic acid-inducible gene-like receptor (RLR)-mediated antiviral responses is still poorly understood. Herein, we show that MRV σ3 protein co-localizes with mitochondrial antiviral signaling protein (MAVS) in the mitochondria and promotes the mitochondria-mediated intrinsic apoptotic pathway to cleave and consequently degrade MAVS. Furthermore, tryptophan at position 133 of σ3 protein plays a key role in the degradation of MAVS. Importantly, we show that MRV outer capsid protein σ3 is a key factor in antagonizing RLR-mediated antiviral responses, providing evidence to better unravel the infection and transmission mechanisms of MRV.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas.

BACKGROUND: Current cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5'-C; antisense strand, 3'-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy. METHODS: IsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5'-C; antisense strand, 3'-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-ß and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection. RESULTS: IsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-ß and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors. CONCLUSIONS: This study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5'-C; antisense strand, 3'-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

Feruloylated Oligosaccharides Prevented Influenza-Induced Lung Inflammation via the RIG-I/MAVS/TRAF3 Pathway.

Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-ß, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.