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IKKε is an IκB kinase participating in the control of NF-κB and type I IFN signal pathways in mammals. However, the function of IKKε in regulating immune response is largely unknown in teleost. Herein, an IKKε homologue named AjIKKε was characterized in Japanese eel (Anguilla japonica). AjIKKε has an N-terminal kinase domain, a ubiquitin-like domain, and a coiled coil-containing domain (CC), which is conserved and similar to its counterpart in mammals. Expression analysis showed that AjIKKε could be up-regulated in kidney, spleen, and particularly in liver under the stimulation of poly I:C, LPS, and Aeromonas hydrophila infection. In vitro, the mRNA levels of AjIKKε were significantly provoked in eel liver cells stimulated by LPS and poly I:C, or the different concentrations of A. hydrophila. The overexpression of AjIKKε could not only induce a significantly higher level of promoter activity of human NF-κB, AP-1, and IFN-β in a dose-dependent manner but also upregulate the activation of promoters of Japanese eel cRel, AP1, IL6, IFN4, IRF3, and IRF7 in HEK293 cells. RNAi studies showed that after AjIKKε was knocked down, the expression levels of IL1, IL6, TNFα, c-Jun, IFN2, IFN3, MX1, MX2, and IRF3 genes were significantly down-regulated in liver, spleen, and kidney of Japanese eels. In addition, the mutants of AjIKKε-K39A, AjIKKε-S174A, and AjIKKε-ΔCC failed to activate Japanese eel IFN4, IRF3 and human IFN-β promoters in HEK293 cells. Collectively, these results suggest that AjIKKε may function as a positive regulator of NF-κB, MAPK, and IRF3-mediated type I IFN signaling pathways related to immune response evoked by bacterial and viral infection. [ABSTRACT FROM AUTHOR]

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tb) is a significant intracellular pathogen responsible for numerous infectious disease‐related deaths worldwide. It uses ESX‐1 T7SS to damage phagosomes and to enter the cytosol of host cells after phagocytosis. During infection, M. tb and host mitochondria release dsDNA, which activates the CGAS‐STING1 pathway. This pathway leads to the production of type I interferons and proinflammatory cytokines and activates autophagy, which targets and degrades bacteria within autophagosomes. However, the role of type I IFNs in immunity against M. tb is controversial. While previous research has suggested a protective role, recent findings from cgas‐sting1 knockout mouse studies have contradicted this. Additionally, a study using knockout mice and non‐human primate models uncovered a new mechanism by which neutrophils recruited to lung infections form neutrophil extracellular traps. Activating plasmacytoid dendritic cells causes them to produce type I IFNs, which interfere with the function of interstitial macrophages and increase the likelihood of tuberculosis. Notably, M. tb uses its virulence proteins to disrupt the CGAS‐STING1 signaling pathway leading to enhanced pathogenesis. Investigating the CGAS‐STING1 pathway can help develop new ways to fight tuberculosis. [ABSTRACT FROM AUTHOR]

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective. IMPORTANCE The innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses. [ABSTRACT FROM AUTHOR]

Porcine respiratory and reproductive syndrome (PRRS) is one of the most devastating infectious diseases of pigs, causing reproductive failures in sows and severe respiratory symptoms in piglets and growing pigs. MicroRNAs (miRNAs) are reported to play an essential role in virus–host interactions. In this study, we demonstrated that miR-451 enhanced type I interferon (IFN-I) production through targeting proteasome subunit β8 (PSMB8), therefore restricting PRRS virus (PRRSV) replication. We showed that the expression of PSMB8 was upregulated by PRRSV infection, and knockdown of PSMB8 inhibited PRRSV replication by promoting IFN-I production. Moreover, we demonstrated that PSMB8 interacted with the regulatory domain of IRF3 to mediate K48-linked polyubiquitination and degradation of IRF3. Also, importantly, we showed that PSMB8, as a target gene of miR-451, negatively regulated IFN-I production by promoting IRF3 degradation, which is a previously unknown mechanism for PSMB8 to modulate innate immune responses. IMPORTANCE Porcine respiratory and reproductive syndrome virus (PRRSV), as a huge threat to the swine industry, is a causative agent that urgently needs to be solved. The dissecting of PRRSV pathogenesis and understanding of the host–pathogen interaction will provide insights into developing effective anti-PRRSV strategies. In this study, we showed that miR-451 dramatically inhibited PRRSV replication by targeting proteasome subunit β8 (PSMB8), a subunit of the immunoproteasome. Mutation of PSMB8 is often related to autoinflammatory diseases due to the elevated IFN production. We revealed that PSMB8 downregulated IFN production by promoting IRF3 degradation. In addition, we showed that PRRSV infection upregulated PSMB8 expression. Taken together, our findings reveal that miR-451 is a negative regulator of PRRSV replication, and PSMB8, a target gene of miR-451, negatively regulates IFN-I production by promoting IRF3 degradation, which is a previously unknown mechanism for PSMB8 to regulate innate immune responses. [ABSTRACT FROM AUTHOR]

ImportanceInactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

Abstract The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D: IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer’s disease, notably apolipoprotein-e. Using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 (ZBP1) as a target of IRF3 that is relevant across various neuroinflammatory disorders. Lastly, we show IRF3 phosphorylation and IRF3-dependent ZBP1 induction in response to Aβ in primary microglia cultures. Together, our results identify IRF3 as an important regulator of LPS and Aβ -mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

Bile acids (BAs) are natural metabolites in mammals and have the potential to function as drugs against viral infection. However, the limited understanding of chenodeoxycholic acid (CDCA) receptors and downstream signaling, along with its lower suppression efficiency in inhibiting virus infection limits its clinical application. In this study, we demonstrate that farnesoid X receptor (FXR), the receptor of CDCA, negatively regulates interferon signaling, thereby contributing to the reduced effectiveness of CDCA against virus replication. FXR deficiency or pharmacological inhibition enhances interferon signaling activation to suppress virus infection. Mechanistically, FXR impairs the DNA binding and transcriptional abilities of activated interferon regulatory factor 3 (IRF3) through interaction. Reduced IRF3 transcriptional activity by FXR–IRF3 interaction significantly undermines the expression of Interferon Beta 1 (IFNB1) and the antiviral response of cells, especially upon the CDCA treatment. In FXR-deficient cells, or when combined with Z-guggulsterone (GUGG) treatment, CDCA exhibits a more potent ability to restrict virus infection. Thus, these findings suggest that FXR serves as a limiting factor for CDCA in inhibiting virus replication, which can be attributed to the “signaling-brake” roles of FXR in interferon signaling. Targeting FXR inhibition represents a promising pharmaceutical strategy for the clinical application of BAs metabolites as antiviral drugs.

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D: IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer's disease, notably apolipoprotein-e. Using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 (ZBP1) as a target of IRF3 that is relevant across various neuroinflammatory disorders. Lastly, we show IRF3 phosphorylation and IRF3-dependent ZBP1 induction in response to Aβ in primary microglia cultures. Together, our results identify IRF3 as an important regulator of LPS and Aβ -mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases. [ABSTRACT FROM AUTHOR]

Bile acids (BAs) are natural metabolites in mammals and have the potential to function as drugs against viral infection. However, the limited understanding of chenodeoxycholic acid (CDCA) receptors and downstream signaling, along with its lower suppression efficiency in inhibiting virus infection limits its clinical application. In this study, we demonstrate that farnesoid X receptor (FXR), the receptor of CDCA, negatively regulates interferon signaling, thereby contributing to the reduced effectiveness of CDCA against virus replication. FXR deficiency or pharmacological inhibition enhances interferon signaling activation to suppress virus infection. Mechanistically, FXR impairs the DNA binding and transcriptional abilities of activated interferon regulatory factor 3 (IRF3) through interaction. Reduced IRF3 transcriptional activity by FXR–IRF3 interaction significantly undermines the expression of Interferon Beta 1 (IFNB1) and the antiviral response of cells, especially upon the CDCA treatment. In FXR -deficient cells, or when combined with Z -guggulsterone (GUGG) treatment, CDCA exhibits a more potent ability to restrict virus infection. Thus, these findings suggest that FXR serves as a limiting factor for CDCA in inhibiting virus replication, which can be attributed to the "signaling-brake" roles of FXR in interferon signaling. Targeting FXR inhibition represents a promising pharmaceutical strategy for the clinical application of BAs metabolites as antiviral drugs. FXR inhibition enhances antiviral potency of chenodeoxycholic acid by relieving FXR interference with interferon signaling, indicating FXR as a promising target for augmenting bile acids' antiviral effects. [Display omitted] [ABSTRACT FROM AUTHOR]

Herpesviruses antagonize host antiviral responses through a myriad of molecular strategies culminating in the death of the host cells. Pseudorabies virus (PRV) is a significant veterinary pathogen in pigs, causing neurological sequalae that ultimately lead to the animal's demise. PRV is known to trigger apoptotic cell death during the late stages of infection. The virion host shutdown protein (VHS) encoded by UL41 plays a crucial role in the PRV infection process. In this study, we demonstrate that UL41 inhibits PRV-induced activation of inflammatory cytokine and negatively regulates the cGAS-STING-mediated antiviral activity by targeting IRF3, thereby inhibiting the translocation and phosphorylation of IRF3. Notably, mutating the conserved amino acid sites (E192, D194, and D195) in the RNase domain of UL41 or knocking down UL41 inhibits the immune evasion of PRV, suggesting that UL41 may play a crucial role in PRV's evasion of the host immune response during infection. These results enhance our understanding of how PRV structural proteins assist the virus in evading the host immune response. [ABSTRACT FROM AUTHOR]

Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous μ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV μ1 protein expression by shRNA could impair MRV proliferation. Specifically, μ1 protein inhibited MRV or poly(I:C)-induced IFN-β expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that μ1 protein significantly decreased IFN-β mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that μ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein μ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection. • MRV μ1 protein promotes the replication of MRV in vitro. • MRV µ1 protein blocks MRV infection or poly(I:C)-mediated type I IFN responses. • MRV μ1 protein attenuates RLRs-mediated IFN-β activation by directly interacting with the RIG-I/MDA5 signaling molecules. • MRV µ1 protein decreases the expression level of RLRs components through proteasome-lysosomal degradation pathway. • MRV μ1 protein is to hinder nuclear translocation of IRF3 resulting in dampened type I IFN responsesresponses. [ABSTRACT FROM AUTHOR]

Simple Summary: A238L, a non-structural protein of the African swine fever virus (ASFV), inhibits the activation of NF-κB by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. In contrast, A238L did not inhibit but rather increased TBK1 and IRF3 phosphorylation and enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals an unrecognized role for A238L in promoting antiviral immune responses by activating the TBK1-IRF3 pathway. African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation. [ABSTRACT FROM AUTHOR]

Abstract The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to over six million deaths worldwide. In human immune system, the type 1 interferon (IFN) pathway plays a crucial role in fighting viral infections. However, the ORF8 protein of the virus evade the immune system by interacting with IRF3, hindering its nuclear translocation and consequently downregulate the type I IFN signaling pathway. To block the binding of ORF8–IRF3 and inhibit viral pathogenesis a quick discovery of an inhibitor molecule is needed. Therefore, in the present study, the interface between the ORF8 and IRF3 was targeted on a high-affinity carbon nanotube by using computational tools. After analysis of 62 carbon nanotubes by multiple docking with the induced fit model, the top five compounds with high docking scores of − 7.94 kcal/mol, − 7.92 kcal/mol, − 7.28 kcal/mol, − 7.19 kcal/mol and − 7.09 kcal/mol (top hit1-5) were found to have inhibitory activity against the ORF8–IRF3 complex. Molecular dynamics analysis of the complexes revealed the high compactness of residues, stable binding, and strong hydrogen binding network among the ORF8-nanotubes complexes. Moreover, the total binding free energy for top hit1-5 was calculated to be − 43.21 ± 0.90 kcal/mol, − 41.17 ± 0.99 kcal/mol, − 48.85 ± 0.62 kcal/mol, − 43.49 ± 0.77 kcal/mol, and − 31.18 ± 0.78 kcal/mol respectively. These results strongly suggest that the identified top five nanotubes (hit1-5) possess significant potential for advancing and exploring innovative drug therapies. This underscores their suitability for subsequent in vivo and in vitro experiments, marking them as promising candidates worthy of further investigation.

Abstract Background Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS–STING in SAP-induced lung injury (LI) remains unknown. Methods Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS −/− and sting −/− mice. Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. Results SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS/STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA–cGAS–STING axis and the NLRP3–pyroptosis axis. Conclusions Negative regulation of any molecule in the mtDNA–cGAS–STING–IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to over six million deaths worldwide. In human immune system, the type 1 interferon (IFN) pathway plays a crucial role in fighting viral infections. However, the ORF8 protein of the virus evade the immune system by interacting with IRF3, hindering its nuclear translocation and consequently downregulate the type I IFN signaling pathway. To block the binding of ORF8–IRF3 and inhibit viral pathogenesis a quick discovery of an inhibitor molecule is needed. Therefore, in the present study, the interface between the ORF8 and IRF3 was targeted on a high-affinity carbon nanotube by using computational tools. After analysis of 62 carbon nanotubes by multiple docking with the induced fit model, the top five compounds with high docking scores of − 7.94 kcal/mol, − 7.92 kcal/mol, − 7.28 kcal/mol, − 7.19 kcal/mol and − 7.09 kcal/mol (top hit1-5) were found to have inhibitory activity against the ORF8–IRF3 complex. Molecular dynamics analysis of the complexes revealed the high compactness of residues, stable binding, and strong hydrogen binding network among the ORF8-nanotubes complexes. Moreover, the total binding free energy for top hit1-5 was calculated to be − 43.21 ± 0.90 kcal/mol, − 41.17 ± 0.99 kcal/mol, − 48.85 ± 0.62 kcal/mol, − 43.49 ± 0.77 kcal/mol, and − 31.18 ± 0.78 kcal/mol respectively. These results strongly suggest that the identified top five nanotubes (hit1-5) possess significant potential for advancing and exploring innovative drug therapies. This underscores their suitability for subsequent in vivo and in vitro experiments, marking them as promising candidates worthy of further investigation. [ABSTRACT FROM AUTHOR]

Background: Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS–STING in SAP-induced lung injury (LI) remains unknown. Methods: Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS −/− and sting −/− mice. Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. Results: SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS/STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA–cGAS–STING axis and the NLRP3–pyroptosis axis. Conclusions: Negative regulation of any molecule in the mtDNA–cGAS–STING–IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model. [ABSTRACT FROM AUTHOR]

The human gastrointestinal system is a complex ecosystem crucial for well-being. During sepsis-induced gut injury, the integrity of the intestinal barrier can be compromised. Lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, disrupts the intestinal barrier, contributing to inflammation and various dysfunctions. The current study explores the protective effects of limonene, a natural compound with diverse biological properties, against LPS-induced jejunal injury in mice. Oral administration of limonene at dosages of 100 and 200 mg/kg was used in the LPS mouse model. The Murine Sepsis Score (MSS) was utilized to evaluate the severity of sepsis, while serum levels of urea and creatinine served as indicators of renal function. Our results indicated that LPS injection induced renal function deterioration, evidenced by elevated serum urea and creatinine levels compared to control mice. However, pretreatment with limonene at doses of 100 and 200 mg/kg mitigated this decline in renal function, evidenced from the reduced levels of serum urea and creatinine. Limonene demonstrated anti-inflammatory effects by reducing pro-inflammatory cytokines (TNF-α, IL-1β, COX-2), suppressing the TLR4/NF-κB/AP-1 but not IRF3 signaling pathways, and modulating oxidative stress through Nrf2 activation. The results suggest that limonene holds promise as a potential therapeutic agent for mitigating intestinal inflammation and preserving gastrointestinal health. [ABSTRACT FROM AUTHOR]

Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

Abstract Background Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-Gα protein interaction. Results IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant Gα subunits and Phos-tag immunoblotting, Gαs was found out to strongly phosphorylate IRF3. Stimulation of Gαs by glucagon or epinephrine or by Gαs-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of Gαs. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of Gαs signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by α-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody. Conclusions Gαs-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease. Video Abstract

Abstract Background The cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease. Methods The effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice. Results In this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3. Conclusion Glab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases.

Objective Often, alternative splicing is used by cancer cells to produce or increase proteins that promote growth and survival through alternative splicing. Although RNA-binding proteins are known to regulate alternative splicing events associated with tumorigenesis, their role in oesophageal cancer (EC) has rarely been explored. Methods We analysed the expression pattern of several relatively well characterized splicing regulators on 183 samples from TCGA cohort of oesophageal cancer; the effectiveness of the knockdown of SRSF2 was subsequently verified by immunoblotting; we measured the ability of cells treated with lenti-sh-SRSF2/lenti-sh2-SRSF2 to invade through an extracellular matrix coating by transwell invasion assay; using RNA-seq data to identify its potential target genes; we performed qRT-PCR to detect the changes of exon 2 usage in lenti-sh-SRSF2 transduced KYSE30 cells to determine the possible effect of SRSF2 on splicing regulation of IRF3; RNA Electrophoretic mobility shift assay (RNA-EMSA) was performed by the incubation of purified SRSF2 protein and biotinylated RNA probes; we performed luciferase assay to confirm the effect of SRSF2 on IFN1 promoter activity. Results We found upregulation of SRSF2 is correlated with the development of EC; Knock-down of SRSF2 inhibits EC cell proliferation, migration, and invasion; SRSF2 regulates the splicing pattern of IRF3 in EC cells; SRSF2 interacts with exon 2 of IRF3 to regulate its exclusion; SRSF2 inhibits the transcription of IFN1 in EC cells. Conclusion This study identified a novel regulatory axis involved in EC from the various aspects of splicing regulation.

DP96R of African swine fever virus (ASFV), also known as uridine kinase (UK), encodes a virulence-associated protein. Previous studies have examined DP96R along with other genes in an effort to create live attenuated vaccines. While experiments in pigs have explored the impact of DP96R on the pathogenicity of ASFV, the precise molecular mechanism underlying this phenomenon remains unknown. Here, we describe a novel molecular mechanism by which DP96R suppresses interferon regulator factor-3 (IRF3)-mediated antiviral immune responses. DP96R interacts with a crucial karyopherin (KPNA) binding site within IRF3, disrupting the KPNA-IRF3 interaction and consequently impeding the translocation of IRF3 to the nucleus. Under this mechanistic basis, the ectopic expression of DP96R enhances the replication of DNA and RNA viruses by inhibiting the production of IFNs, whereas DP96R knock-down resulted in higher IFNs and IFN-stimulated gene (ISG) transcription during ASFV infection. Collectively, these findings underscore the pivotal role of DP96R in inhibiting IFN responses and increase our understanding of the relationship between DP96R and the virulence of ASFV. [ABSTRACT FROM AUTHOR]

Histone H1.2 is a member of the linker histone family, which plays extensive and crucial roles not only in the regulation of chromatin dynamics, cell cycle, and cell apoptosis, but also in viral diseases and innate immunity response. Recently, it was discovered that H1.2 regulates interferon-β and inhibits influenza virus replication, whereas its role in other viral infections is poorly reported. Here, we first found the up-regulation of H1.2 during Encephalomyocarditis virus (EMCV) infection, implying that H1.2 was involved in EMCV infection. Overexpression of H1.2 inhibited EMCV proliferation, whereas knockdown of H1.2 showed a significant promotion of virus infection in HEK293T cells. Moreover, we demonstrated that overexpression of H1.2 remarkably enhanced the production of EMCV-induced type I interferon, which may be the crucial factor for H1.2 proliferation–inhibitory effects. We further found that H1.2 up-regulated the expression of the proteins of the MDA5 signaling pathway and interacted with MDA5 and IRF3 in EMCV infection. Further, we demonstrated that H1.2 facilitated EMCV-induced phosphorylation and nuclear translocation of IRF3. Briefly, our research uncovers the mechanism of H1.2 negatively regulating EMCV replication and provides new insight into antiviral targets for EMCV. [ABSTRACT FROM AUTHOR]

Background and Aim: Previous reports have suggested IFI16 as a tumor suppressor in hepatocellular carcinoma (HC). Nonetheless, the biological significance of IFI16 and its mechanism concerning resistance to cisplatin (DDP) in HC requires further exploration. Methods: Samples of tumor and corresponding para-carcinoma tissues were acquired from patients with HC. Furthermore, DDP-resistant cell lines of HC, specifically HCC, Huh7 and Hepatoblastoma, HepG3, were generated by gradually increasing the concentration of DDP. Cell apoptosis and DNA damage were evaluated by utilizing flow cytometry assay and TUNEL staining. The interaction between IFI16 and interferon regulatory factor 3 (IRF3) proteins were analyzed using Co-Immunoprecipitation (Co-IP) assay. In vivo assays were conducted by establishing HC subcutaneous xenograft tumor models. Results: The study found a reduction in IFI16 expression in both HC tissues and DDP-resistant HC cell lines. The binding of IFI16 to IRF3 regulated DNA damage-associated markers in vitro. Overexpression of IFI16 heightened the susceptibility of DDP-induced apoptosis and DNA damage, which was counteracted by IRF3 knockdown, while strengthened by IRF3 overexpression. Moreover, overexpression of IFI16 diminished in vivo DDP-resistant HC tumorigenicity. Conclusion: In summary, our findings suggest that IFI16 serves as a tumor suppressor in HC by promoting DNA damage via its interaction with IRF3, thereby reversing DDP resistance. Cisplatin-induced damage in tumor cells, with the involvement of DNA repair, leads to survival of tumor cells, manifesting as drug resistance of the tumor. However, the binding of IRF3 and IFI16 inhibits DNA repair, resulting in the ultimate fate of cell death. [ABSTRACT FROM AUTHOR]

RNF5 is an E3 ubiquitin ligase involved in various physiological processes such as protein localization and cancer progression. Recent studies have shown that RNF5 significantly inhibits antiviral innate immunity by promoting the ubiquitination and degradation of STING and MAVS, which are essential adaptor proteins, as well as their downstream signal IRF3. The abundance of RNF5 is delicately regulated by both host factors and viruses. Host factors have been found to restrict RNF5-mediated ubiquitination, maintaining the stability of STING or MAVS through distinct mechanisms. Meanwhile, viruses have developed ingenious strategies to hijack RNF5 to ubiquitinate and degrade immune proteins. Moreover, recent studies have revealed the multifaceted roles of RNF5 in the life cycle of various viruses, including SARS-CoV-2 and KSHV. Based on these emerging discoveries, RNF5 represents a novel means of modulating antiviral immunity. In this review, we summarize the latest research on the roles of RNF5 in antiviral immunity and virus life cycle. This comprehensive understanding could offer valuable insights into exploring potential therapeutic applications focused on targeting RNF5 during viral infections. [ABSTRACT FROM AUTHOR]

Background: Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-Gα protein interaction. Results: IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant Gα subunits and Phos-tag immunoblotting, Gαs was found out to strongly phosphorylate IRF3. Stimulation of Gαs by glucagon or epinephrine or by Gαs-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of Gαs. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of Gαs signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by α-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody. Conclusions: Gαs-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease. 3Z-4y3zsWdGiZ_62kYezA1 Video Abstract [ABSTRACT FROM AUTHOR]

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK. [ABSTRACT FROM AUTHOR]

Background: The cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease. Methods: The effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice. Results: In this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3. Conclusion: Glab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. Methods: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. Results: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1–Interferon regulatory factor 3 (IRF3)–Stimulator of interferon genes (STING) cascade. Conclusions: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target. [ABSTRACT FROM AUTHOR]

To combat microbial infections, mammalian cells use a variety of innate immune response pathways to induce synthesis of anti‐microbial proteins. The cGAS/STING pathway recognizes cytoplasmic viral or cellular DNA to elicit signals that lead to type I interferon and other cytokine synthesis. cGAMP, synthesized by DNA‐activated cGAS, activates the ER‐associated protein, STING, which oligomerizes and translocates to other intracellular membrane compartments to trigger different branches of signaling. We have reported that, in the ER, EGFR‐mediated phosphorylation of Tyr245 of STING is required for its transit to the late endosomes, where it recruits and activates the transcription factor IRF3 required for IFN induction. In the current study, we inquired whether STING Tyr245 phosphorylation per se or STING's location in the late endosomes was critical for its ability to recruit IRF3 and induce IFN. Using pharmacological inhibitors or genetic ablation of proteins that are essential for specific steps of STING trafficking, we demonstrated that the presence of STING in the late endosomal membranes, even without Tyr245 phosphorylation, was sufficient for IRF3‐mediated IFN induction. [ABSTRACT FROM AUTHOR]

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Alzheimer's disease (AD) is one of the leading causes of death throughout the world. Z-DNA binding protein 1 (ZBP1), a DNA-related gene, is associated with inflammation, and its expression is altered in AD brain. We aimed to elucidate the exact role of ZBP1 in AD development and its potential regulatory mechanism. First, we constructed both in vivo and in vitro models of AD and investigated the ZBP1 expression profile. A loss-of-function assay was performed by transfecting lentivirus carrying ZBP1 short hairpin RNA (shRNA). By evaluating cell death, oxidative stress, inflammation response and pyroptosis, the function of ZBP1 was validated. Finally, the correlation between ZBP1 and interferon regulatory factor 3 (IRF3) was verified. We also performed rescue experiments to validate the crucial role of IRF3 in ZBP1-mediated AD progression. According to our results, ZBP1 was upregulated in AD rat tissue and AD neurons. Silencing ZBP1 dramatically decreased cell injury, oxidative stress and inflammation in AD neurons and improved the cognitive function of AD rats. Additionally, IRF3 expression and phosphorylation were significantly elevated during AD development and positively correlated with ZBP1. Taken together, silencing ZBP1 suppressed cell injury and pyroptosis of AD neurons and improved cognitive function of AD rats via inhibiting IRF3. These findings might provide a novel insight for AD target diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. Methods PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-β signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. Results Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-β signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. Conclusions These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-β and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation.

Abstract Background Metabolic reprogramming is a critical event for cell fate and function, making it an attractive target for clinical therapy. The function of metabolic reprogramming in Helicobacter pylori (H. pylori)-infected gastric intestinal metaplasia remained to be identified. Methods Xanthurenic acid (XA) was measured in gastric cancer cells treated with H. pylori or H. pylori virulence factor, respectively, and qPCR and WB were performed to detect CDX2 and key metabolic enzymes expression. A subcellular fractionation approach, luciferase and ChIP combined with immunofluorescence were applied to reveal the mechanism underlying H. pylori mediated kynurenine pathway in intestinal metaplasia in vivo and in vitro. Results Herein, we, for the first time, demonstrated that H. pylori contributed to gastric intestinal metaplasia characterized by enhanced Caudal-related homeobox transcription factor-2 (CDX2) and mucin2 (MUC2) expression, which was attributed to activation of kynurenine pathway. H. pylori promoted kynurenine aminotransferase II (KAT2)-mediated kynurenine pathway of tryptophan metabolism, leading to XA production, which further induced CDX2 expression in gastric epithelial cells. Mechanically, H. pylori activated cyclic guanylate adenylate synthase (cGAS)-interferon regulatory factor 3 (IRF3) pathway in gastric epithelial cells, leading to enhance IRF3 nuclear translocation and the binding of IRF3 to KAT2 promoter. Inhibition of KAT2 could significantly reverse the effect of H. pylori on CDX2 expression. Also, the rescue phenomenon was observed in gastric epithelial cells treated with H. pylori after IRF3 inhibition in vitro and in vivo. Most importantly, phospho-IRF3 was confirmed to be a clinical positive relationship with CDX2. Conclusion These finding suggested H. pylori contributed to gastric intestinal metaplasia through KAT2-mediated kynurenine pathway of tryptophan metabolism via cGAS-IRF3 signaling, targeting the kynurenine pathway could be a promising strategy to prevent gastric intestinal metaplasia caused by H. pylori infection. Video Abstract

Diabetes could directly induce cardiac injury, leading to cardiomyopathy. However, treatment strategies for diabetic cardiomyopathy remain limited. ZNF593-AS knockout and cardiomyocyte-specific transgenic mice were constructed. In addition, high-fat diet (HFD)-induced diabetic mouse model and db/db mice, another classic diabetic mouse model, were employed. ZNF593-AS was silenced using GapmeR, a modified antisense oligonucleotide, while overexpressed using a recombinant adeno-associated virus serotype 9-mediated gene delivery system. Transcriptome sequencing, RNA pull-down assays, and RNA immunoprecipitation assays were also performed to investigate the underlying mechanisms. ZNF593-AS expression was decreased in diabetic hearts. ZNF593-AS attenuated the palmitic acid-induced apoptosis of cardiomyocytes in vitro. In HFD-induced diabetic mice, ZNF593-AS deletion aggravated cardiac dysfunction and enhanced cardiac apoptosis and inflammation. In contrast, HFD-induced cardiac dysfunction was improved in ZNF593-AS transgenic mice. Consistently, ZNF593-AS exerted the same cardioprotective effects in db/db mice. Mechanistically, ZNF593-AS directly interacted with the functional domain of interferon regulatory factor 3 (IRF3), and suppressed fatty acid-induced phosphorylation and activation of IRF3, contributing to the amelioration of cardiac cell death and inflammation. In conclusion, our results identified the protective role of ZNF593-AS in diabetic cardiomyopathy, suggesting a novel potential therapeutic target.

ABSTRACTAlternative splicing is a process causing mRNA translation to produce different proteins, and it is crucial for the development of tumours. In this study, we constructed a prognostic model related to alternative splicing events in hepatocarcinoma using bioinformatics analysis, including the alternative splicing of CSAD, AFMID, ZDHHC16, and IRF3. The model is an independent prognostic factor and can accurately predict a patient’s prognosis. IRF3 is a transcription factor related to the immune response. Its alternative splicing can affect the expression of various genes related to prognosis and plays an essential role in the tumour microenvironment. We also verified the expression of IRF3 exon skipping isoform in hepatocarcinoma at the mRNA level. In conclusion, we discovered that the alternative splicing of IRF3 is essential for the development of hepatocarcinoma. This study provides new insight into the development of treatments for hepatocarcinoma.

ABSTRACTEnteroviruses (EVs)—positive-sense single-strand RNA viruses of Picornaviridae—are among the most common human viral pathogens. Unlike other cytoplasmic viral RNAs (vRNAs), which are detected by RIG-I, sensing of EV RNAs is mediated primarily by MDA5. EVs evolved with rapid, cytotoxic life cycles as a means of host cell interference, ostensibly to suppress MDA5-orchestrated antiviral type-I interferon (IFN) responses. At the core of this strategy are viral 2A protease (2Apro)-directed cleavage of the eukaryotic initiation factor (eIF)4G—the central translation initiation scaffold and ribosome adaptor—and degradation of nuclear pores. EV MDA5 agonism has an intriguing innate footprint, characterized by polar TBK1-IFN regulatory factor 3 (IRF3) signaling, which fuels sustained type-I IFN release to provide context for antitumor CD8+ T-cell priming after in situ cancer vaccination. Here we compared EV-host interactions between wild-type EVs and the highly attenuated recombinant polio:rhinovirus chimera and cancer immunotherapy agent (PVSRIPO) to test how targeted inhibition of discrete steps in the viral life cycle affects the innate antiviral response. Our investigations demonstrate that sustained MDA5-TBK1-IRF3 signaling elicited by PVSRIPO is the result of inefficient immediate 2Apro-directed eIF4G degradation, while wild-type EVs counter host innate type-I IFN defenses via rapid eIF4G cleavage. The MDA5-directed innate signature of profoundly attenuated EVs may provide efficient innate immune-stimulatory assets, for example, in in situ cancer vaccination strategies.IMPORTANCEMultiple pattern recognition receptors sense vRNAs and initiate downstream innate signaling: endosomal Toll-like receptors (TLRs) 3, 7, and 8 and cytoplasmic RIG-I-like receptors (RLRs) RIG-I, and MDA5. They engage distinct signaling scaffolds: mitochondrial antiviral signaling protein (RLR), MyD88, and TLR-adaptor interacting with SLC15A4 on the lysosome (TLR7 and TLR8) and toll/IL-1R domain-containing adaptor inducing IFN (TLR3). By virtue of their unusual vRNA structure and direct host cell entry path, the innate response to EVs uniquely is orchestrated by MDA5. We reported that PVSRIPO’s profound attenuation and loss of cytopathogenicity triggers MDA5-directed polar TBK1-IRF3 signaling that generates priming of polyfunctional antitumor CD8+ T-cell responses and durable antitumor surveillance in vivo. Here we unraveled EV-host relations that control suppression of host type-I IFN responses and show that PVSRIPO’s deficient immediate host eIF4G cleavage generates unopposed MDA5-directed downstream signaling cascades resulting in sustained type-I IFN release.

As a member of the inositol polyphosphate-5-phosphatase family, INPP5D (inositol polyphosphate-5-phosphatase D) is an important regulator of immune cell activation. To date, the mechanisms underlying anti-malarial immunity have not been elucidated. We recently identified INPP5D as a negative regulator of IFN-I (type I interferon) signaling by promoting autophagic degradation of IRF3 (interferon regulatory factor 3) during malaria infection. Mechanistically, INPP5D enhances the association between IRF3 and the autophagy receptor CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2), which promotes the K63-linked ubiquitination of IRF3 at K313 and serves as a signal for CALCOCO2-dependent selective macroautophagy (hereafter autophagy). Moreover, INPP5D is downregulated by IFN-I-induced miR-155-5p after Plasmodium yoelii (P. yoelii) nigeriensis N67 infection and plays a role as a feedback loop between IFN-I signaling and autophagy. Thus, our study reveals the key role of INPP5D in mediating the crosstalk between IFN-I response and autophagy during anti-malarial immune responses, and suggests that INPP5D may be a potential therapeutic target to control malaria. Abbreviations: ATG5; autophagy-related 5; CALCOCO2/NDP52, calcium binding and coiled-coil domain 2; CQ, chloroquine; INPP5D/SHIP1, inositol polyphosphate-5-phosphatase D; IRF3, interferon regulatory factor 3; IFN-I, type I interferon; 3-MA, 3-methyladenine.

Background: Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. Methods: PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-β signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. Results: Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-β signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. Conclusions: These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-β and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response. [ABSTRACT FROM AUTHOR]

A viral infection activates the transcription factors IRF3 and NF-κB, which synergistically induces type I interferons (IFNs). Here, we identify the E3 ubiquitin ligase RNF138 as an important negative regulator of virus-triggered IRF3 activation and IFN-β induction. The overexpression of RNF138 inhibited the virus-induced activation of IRF3 and the transcription of the IFNB1 gene, whereas the knockout of RNF138 promoted the virus-induced activation of IRF3 and transcription of the IFNB1 gene. We further found that RNF138 promotes the ubiquitination of PTEN and subsequently inhibits PTEN interactions with IRF3, which is essential for the PTEN-mediated nuclear translocation of IRF3, thereby inhibiting IRF3 import into the nucleus. Our findings suggest that RNF138 negatively regulates virus-triggered signaling by inhibiting the interaction of PTEN with IRF3, and these data provide new insights into the molecular mechanisms of cellular antiviral responses. [ABSTRACT FROM AUTHOR]

Transforming growth factor beta kinase 1 (TAK1, also known as MAP3K7) has been identified as an adaptor protein in the NF-κB and IRF3 signaling pathways in teleost fishes, and it plays a crucial role in both inflammatory responses and anti-viral actions. SET and MYND domain protein 3 (Smyd3) belongs to the Smyd3 lysine methylase family and possess significant functions in the methylation process of diverse histone and non-histone targets. However, the exact role of Smyd3 in innate immune regulation has not yet been fully elucidated. Siniperca chuatsi rhabdovirus (SCRV) is a negative single-stranded RNA virus that has the potential to cause widespread fish diseases. In this study, we discovered that overexpression of Smyd3 facilitates the replication of SCRV, enabling the virus to evade detection of the immune system through Smyd3. Furthermore, we observed that Smyd3 promotes the degradation of TAK1 by inducing K48-linked ubiquitination of TAK1. As a result, Smyd3 exerts a negative regulatory effect on the TAK1-mediated NF-κB signaling pathway and the IRF3 signaling pathway. Thus, our findings demonstrate that Smyd3 plays a role in promoting the degradation of the TAK1 protein. Moreover, we discovered that the degradation of TAK1 by Smyd3 occurs independently of its methyltransferase activity. This study not only enhances our understanding of anti-viral immunity in fish but also offers a novel perspective for investigating anti-viral immunity in mammals. [ABSTRACT FROM AUTHOR]

Innate immunity plays an essential role in defending the host against pathogenic infections. Appropriate controls are required to exert antiviral effects and avoid inflammatory disorders, but the negative regulation mechanisms are not fully understood. Here, Prohibitin1 (PHB1) was identified as a negative regulator of innate immune responses. We found that PHB1 protein and mRNA levels were promoted by virus-induced beta interferon (IFN-β) and subsequently suppressed the antiviral innate immune responses, thereby facilitating the replication of multiple RNA viruses. Further studies revealed that PHB1 interacted with IFN regulatory factor 3 (IRF3) to restrain the binding of IRF3 to nuclear import proteins, thereby suppressing the nuclear import of IRF3 and the downstream production of IFN-β. In summary, we elucidated the mechanism by which PHB1 regulates host antiviral innate immunity by inhibiting the nuclear translocation of IRF3, which contributed to the understanding of IRF3 regulation and revealed a novel role of PHB1 in host innate immunity. IMPORTANCE Type I interferon (IFN-I), produced by the innate immune system, plays an essential role in host antiviral responses. Proper regulation of IFN-I production is required for the host to balance immune responses and prevent superfluous inflammation. IFN regulatory factor 3 (IRF3) and subsequent sensors are activated by RNA virus infection to induce IFN-I production. Therefore, proper regulation of IRF3 serves as an important way to control innate immunity and viral replication. Here, we first identified Prohibitin1 (PHB1) as a negative regulator of host IFN-I innate immune responses. Mechanistically, PHB1 inhibited the nucleus import of IRF3 by impairing its binding with importin subunit alpha-1 and importin subunit alpha-5. Our study demonstrates the mechanism by which PHB1 facilitates the replication of multiple RNA viruses and provides insights into the negative regulation of host immune responses. [ABSTRACT FROM AUTHOR]

Cyclic GMP-AMP synthase (cGAS), which recognizes double-stranded DNA (dsDNA) and activates the innate immune system, is mainly localized in the cytosol, but also shows nuclear localization. Here, we sought to determine the role of nuclear cGAS by mutating known nuclear localization signal (NLS) motifs in cGAS and assessing its functionality by monitoring phosphorylation of the downstream target, interferon regulatory factor-3 (IRF3). Interestingly, NLS2-mutated cGAS failed to promote phosphorylation of IRF3, reflecting the loss of its ability to produce cyclic GMP-AMP (cGAMP). We further found that insertion of an NLS from SV40 large T antigen could not restore this loss of activity, indicating that this loss was attributable to the mutation of NLS2 itself, but not dependent on the inability of cGAS to enter the nucleus. NLS2-mutant cGAS protein also showed decreased stability dependent on polyubiquitination, an effect that was independent of both its loss of catalytic function and its inability to enter into the nucleus. Collectively, these findings indicate that the NLS2 motif of cGAS is not only involved in regulating the subcellular localization of cGAS protein but also influences its stability and enzymatic activity through independent mechanisms, highlighting the novel roles of NLS2 in regulating the intracellular functions of cGAS. • The cGAS protein senses DNA and initiates the cGAS/STING/IRF3 innate immune pathway. • NLS2 motif of cGAS affects both nuclear localization and enzyme activity. • NLS2 motif also affects protein stability of cGAS via changing polyubiquitination level. • These effects of NLS2 on cGAS do not appear to be interdependent. [ABSTRACT FROM AUTHOR]

Swine acute diarrhea syndrome (SADS) is first reported in January 2017 in Southern China. It subsequently causes widespread outbreaks in multiple pig farms, leading to economic losses. Therefore, it is an urgent to understand the molecular mechanisms underlying the pathogenesis and immune evasion of Swine acute diarrhea syndrome coronavirus (SADS‐CoV). Our research discovered that SADS‐CoV inhibited the production of interferon‐β (IFN‐β) during viral infection. The nonstructural protein 1 (nsp1) prevented the phosphorylation of TBK1 by obstructing the interaction between TBK1 and Ub protein. Moreover, nsp1 induced the degradation of CREB‐binding protein (CBP) through the proteasome‐dependent pathway, thereby disrupting the IFN‐β enhancer and inhibiting IFN transcription. Finally, we identified nsp1‐Phe39 as the critical amino acid that downregulated IFN production. In conclusion, our findings described two mechanisms in nsp1 that inhibited IFN production and provided new insights into the evasion strategy adopted by SADS‐CoV to evade host antiviral immunity. [ABSTRACT FROM AUTHOR]

Disrupting the balance of neuronal excitation and inhibition (E/I) is an important pathogenic mechanism of anxiety and depression. Interferon regulatory factor 3 (IRF3) plays a key role in the innate immune response, and activation of IRF3 triggers the expression of type I interferons and downstream interferon-stimulated genes, which are associated with anxiety and depression. However, whether IRF3 participates in the pathogenesis of anxiety/depression by regulating E/I balance remains poorly understood. Here, we reported that global knockout (KO) of IRF3 (IRF3−/−) significantly increased anxiety/depression-like behaviors, but did not affect normal spatial learning and memory. Compared with wild type (WT) control mice, the E/I balance was disrupted, as reflected by enhanced glutamatergic transmission and decreased GABAergic transmission in the neurons of hippocampal CA1 and medial prefrontal cortex (mPFC) in IRF3-KO mice. Importantly, genetic rescue of IRF3 expression by adeno-associated virus (AAV) was sufficient to alleviate anxiety/depression-like behaviors and restore the neuronal E/I balance in IRF3-KO mice. Taken together, our results indicate that IRF3 is critical in maintaining neuronal E/I balance, thereby playing an essential role in ensuring emotional stability.