Background Acute pancreatitis (AP) is a prevalent and serious condition within the digestive system, with approximately 20% to 30% of cases advancing to severe acute pancreatitis (SAP). During the initial phases of SAP, macrophages are activated in response to the substantial amounts of acinar cell contents and damage-associated molecular patterns (DAMPs) resulting from acinar cell destruction. Subsequently, activated macrophages release a significant array of pro-inflammatory factors that exacerbate the progression of SAP. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic protein that is intimately linked to immune regulation. While the role of CIP2A in T-cell-mediated specific immune responses has been reported, its function and mechanism in macrophages, a component of non-specific immunity, have not been widely studied. This research fills this knowledge gap by elucidating the critical role of CIP2A in regulating macrophage autophagy and inflammation. This finding not only expands our understanding of CIP2A in immune modulation but also provides a new scientific basis and potential application prospects for targeting CIP2A in the treatment of AP. Methods We established AP using a combination of palmitoleic acid with anhydrous ethanol or using caerulein alone. The effects of TD52 and Ethoxysanguinarine (Etho) on SAP were evaluated through serological, histopathological, and tissue inflammation observations. The effect of TD52 on macrophage activation in vitro was examined using primary macrophages (PMs) and RAW264.7 cells. Results We found that TD52 and Etho inhibit CIP2A expression while reducing the levels of serum amylase, lipase, and inflammatory cytokines, thereby alleviating the pathological symptoms of SAP. Additionally, TD52 could reduce the infiltration of macrophages into pancreatic tissue. Therefore, we established a model of macrophage inflammatory response mimicking the pathophysiological process of AP and detected changes in inflammation, apoptosis, and autophagy through pre-treatment of macrophages with TD52. The results show that inhibiting CIP2A expression decreases the release of inflammatory cytokines and reduces apoptosis in macrophages. Further exploration revealed that TD52 promoted macrophage autophagy regulation and inhibited the AKT-mTOR pathway to modulate macrophage activation. Conclusion In summary, our findings indicate that TD52 and Etho can alleviate the severity of SAP. TD52 can block the AKT-mTOR pathway to promote macrophage autophagy, thereby improving SAP. Thus, CIP2A may serve as one of the molecular targets in SAP, highlighting its potential as a therapeutic option.

Interferon regulatory factor 3 (IRF3) is a central hub transcription factor that controls host antiviral innate immunity. The expression and function of IRF3 are tightly regulated by the post-translational modifications. However, it is unknown whether unanchored ubiquitination and deubiquitination of IRF3 involve modulating antiviral innate immunity against RNA viruses. Here, we find that USP5, a deubiquitinase (DUB) regulating unanchored polyubiquitin, is downregulated during host anti-RNA viral innate immunity in a type I interferon (IFN-I) receptor (IFNAR)-dependent manner. USP5 is further identified to inhibit IRF3-triggered antiviral immune responses through its DUB enzyme activity. K48-linked unanchored ubiquitin promotes IRF3-driven transcription of IFN-β and induction of IFN-stimulated genes (ISGs) in a dose-dependent manner. USP5 simultaneously removes both K48-linked unanchored and K63-linked anchored polyubiquitin chains on IRF3. Our study not only provides evidence that unanchored ubiquitin regulates anti-RNA viral innate immunity but also proposes a novel mechanism for DUB-controlled IRF3 activation, suggesting that USP5 is a potential target for the treatment of RNA viral infectious diseases.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), a novel bunyavirus primarily transmitted by Haemaphysalis longicornis, induces severe disease with a high mortality rate. N6-methyladenosine (m6A) is a prevalent internal chemical modification in eukaryotic mRNA that has been reported to regulate viral infection. However, the role of m6A modification during SFTSV infection remains elusive. We here reported that SFTSV RNAs bear m6A modification during infection. Manipulating the expressions or activities of host m6A regulators significantly impacted SFTSV infection. Mechanistically, SFTSV recruited m6A regulators through the nucleoprotein to modulate the m6A modification of viral RNA, eventually resulting in enhanced infection by promoting viral mRNA translation efficiency and/or genome RNA stability. m6A mutations in the S genome diminished virus particle production, while m6A mutations in the G transcript impaired the replication of recombinant vesicular stomatitis virus (rVSV) expressing G protein in vitro and in vivo. Interestingly, m6A modification was evolutionarily conserved and facilitated SFTSV infection in primary tick cells. These findings may open an avenue for the development of m6A-targeted anti-SFTSV vaccines, drugs, and innovative strategies for the prevention and control of tick-borne disease.

U1 small nuclear RNA (snRNA) is an abundant and evolutionarily conserved 164 -nucleotide RNA species that functions in pre-mRNA splicing, and it is considered to be a housekeeping non -coding RNA. However, the role of U1 snRNA in regulating host antiviral immunity remains largely unexplored. Here, we find that RNVU1-18 , a U1 pseudogene, is significantly upregulated in the host infected with RNA viruses, including influenza and respiratory syncytial virus. Overexpression of U1 snRNA protects cells against RNA viruses, while knockdown of U1 snRNA leads to more viral burden in vitro and in vivo . Knockout of RNVU1-18 is sufficient to impair the type I interferon -dependent antiviral innate immunity. U1 snRNA is required to fully activate the retinoic acid -inducible gene I (RIG -I) -dependent antiviral signaling, since it interacts with tripartite motif 25 (TRIM25) and enhances the RIG-I-TRIM25 interaction to trigger K63 -linked ubiquitination of RIG -I. Our study reveals the important role of housekeeping U1 snRNA in regulating host antiviral innate immunity and restricting RNA virus infection.

Background and Aims: Hyperactivated inflammatory responses induced by cytokine release syndrome are the primary causes of tissue damage and even death. The translation process is precisely regulated to control the production of proinflammatory cytokines. However, it is largely unknown whether targeting translation can effectively limit the hyperactivated inflammatory responses during acute hepatitis and graft-versus-host disease. Approach and Results: By using in vitro translation and cellular overexpression systems, we have found that the nonstructural protein gene NS2A of Zika virus functions as RNA molecules to suppress the translation of both ectopic genes and endogenous proinflammatory cytokines. Mechanistically, results from RNA pulldown and co-immunoprecipitation assays have demonstrated that NS2A RNA interacts with the translation initiation factor eIF2α to disrupt the dynamic balance of the eIF2/eIF2B complex and translation initiation, which is the rate-limiting step of translation. In the acetaminophen-induced, lipopolysaccharide/D-galactosamine-induced, viral infection-induced acute hepatitis, and graft-versus-host disease mouse models, mice with myeloid cell-specific knock-in of NS2A show decreased levels of serum proinflammatory cytokines and reduced tissue damage. Conclusions: Zika virus NS2A dampens the production of proinflammatory cytokines and alleviates inflammatory injuries by interfering translation process as RNA molecules, which suggests that NS2A RNA is potentially used to treat numerous acute inflammatory diseases characterized by cytokine release syndrome.

BackgroundOver-activated osteoclast (OC) is a major cause of diseases related to bone loss and bone metabolism. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is an important interferon-stimulated and apoptotic gene. However, how XAF1 regulates bone formation and remodeling is unknown.MethodsWe generate global and chimeric Xaf1 knockout mouse models and utilize these models to explore the function and mechanism of XAF1 in regulating bone formation and remodeling in vivo and in vitro.ResultsWe show that XAF1 depletion enhances osteoclast generation in vitro. XAF1 knockout increases osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (a potent XIAP inhibitor) suppresses osteoclast formation. Mechanistically, XAF1 deletion decreases osteoclast apoptosis by facilitating the interaction between XIAP and caspase-3/7.ConclusionsOur data illustrates an essential role of XAF1 in controlling osteoclastogenesis in both osteoporosis and osteolysis mouse models and highlights its underlying mechanism, indicating a potential role in clinical treatment.The translational potential of this article: The translation potential of this article is that we first indicated that osteoclast apoptosis induced by XAF1 contribute to the progression of osteoporosis and osteolysis, which provides a novel strategy in the prevention of osteoporosis and osteolysis.

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2-mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin-proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild-type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265-type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs.

Spliceosome dysfunction and aberrant RNA splicing underline unresolved inflammation and immunopathogenesis. Here, we revealed the misregulation of mRNA splicing via the spliceosome in the pathogenesis of rheumatoid arthritis (RA). Among them, decreased expression of RNA binding motif protein 25 (RBM25) was identified as a major pathogenic factor in RA patients and experimental arthritis mice through increased proinflammatory mediator production and increased hyperinflammation in macrophages. Multiomics analyses of macrophages from RBM25-deficient mice revealed that the transcriptional enhancement of proinflammatory genes (including Il1b, Il6, and Cxcl10) was coupled with histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac modifications as well as hypoxia inducible factor-1α (HIF-1α) activity. Furthermore, RBM25 directly bound to and mediated the 14th exon skipping of ATP citrate lyase (Acly) pre-mRNA, resulting in two distinct Acly isoforms, Acly Long (Acly L) and Acly Short (Acly S). In proinflammatory macrophages, Acly L was subjected to protein lactylation on lysine 918/995, whereas Acly S did not, which influenced its affinity for metabolic substrates and subsequent metabolic activity. RBM25 deficiency overwhelmingly increased the expression of the Acly S isoform, enhancing glycolysis and acetyl-CoA production for epigenetic remodeling, macrophage overactivation and tissue inflammatory injury. Finally, macrophage-specific deletion of RBM25 led to inflammaging, including spontaneous arthritis in various joints of mice and inflammation in multiple organs, which could be relieved by pharmacological inhibition of Acly. Overall, targeting the RBM25-Acly splicing axis represents a potential strategy for modulating macrophage responses in autoimmune arthritis and aging-associated inflammation.

Acute pancreatitis (AP) is a non-infectious pancreatic enzyme-induced disorder, a life-threatening inflammatory condition that can cause multi-organ dysfunction, characterized by high morbidity and mortality. Several therapies have been employed to target this disorder; however, few happen to be effectively employable even in the early phase. PFKFB3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) is a critical regulator of glycolysis and is upregulated under inflammatory, mitogenic, and hypoxia conditions. Essential information on the targeting of the inflammatory pathway will present the termination of the disorder and recovery. Herein we investigated the protective function of KAN0438757, a potent inhibitor of PFKFB3, and its mechanism of impeding AP induced in mice. KAN0438757 was confirmed to activate the Nrf2/HO-1 inflammatory signaling pathways in response to caerulein induced acute pancreatitis (CAE-AP) and fatty acid ethyl ester induced severe acute pancreatitis (FAEE-SAP). Additionally, KAN0438757 alleviated the inflammatory process in infiltrated macrophage via the Nrf2/HO-1 inflammatory signaling pathway and demonstrated a significant effect on the growth of mice with induced AP. And more importantly, KAN0438757 displayed negligible toxicity in vivo. Taken together our data suggest KAN0438757 directly suppresses the inflammatory role of PFKFB3 and induces a protective role via the Nrf2/HO-1 pathway, which could prove as an excellent therapeutic platform for SAP amelioration.

Type I interferons (IFN-I) are pleiotropic factors endowed with multiple activities that play important roles in innate and adaptive immunity. Although many studies indicate that IFN-I inducers exert favorable effects on broad-spectrum antivirus, immunomodulation, and anti-tumor activities by inducing endogenous IFN-I and IFN-stimulated genes, their function in bone homeostasis still needs further exploration. Here, our study demonstrates 2 distinct IFN-I inducers, diABZI and poly(I:C), as potential therapeutics to alleviate osteolysis and osteoporosis. First, IFN-I inducers suppress the genes that control osteoclast (OC) differentiation and activity in vitro. Moreover, diABZI alleviates bone loss in Ti particle-induced osteolysis and ovariectomized -induced osteoporosis in vivo by inhibiting OC differentiation and function. In addition, the inhibitory effects of IFN-I inducers on OC differentiation are not observed in macrophages derived from Ifnar1-/-mice, which indicate that the suppressive effect of IFN-I inducers on OC is IFNAR-dependent. Mechanistically, RNAi-mediated silencing of IRF7 and IFIT3 in OC precursors impairs the suppressive effect of the IFN-I inducers on OC differentiation. Taken together, these results demonstrate that IFN-I inducers play a protective role in bone turnover by limiting osteoclastogenesis and bone resorption through the induction of OC-specific mediators via the IFN-I signaling pathway.

Acute pancreatitis (AP) is among the most common hospital gastrointestinal diagnoses; understanding the mechanisms underlying the severity of AP is critical for development of new treatment options for this disease. Here, we evaluate the biological function of phosphofructo-2kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in AP pathogenesis in 2 independent genetically engineered mouse models of AP. PFKFB3 was elevated in AP and severe AP (SAP), and KO of Pfkfb3 abrogated the severity of alcoholic SAP (FAEE-SAP). Using a combination of genetic, pharmacological, and molecular studies, we defined the interaction of PFKFB3 with inositol 1,4,5-trisphosphate receptor (IP3R) as a key event mediating this phenomenon. Further analysis demonstrated that the interaction between PFKFB3 and IP3R promotes FAEE-SAP severity by altering intracellular calcium homeostasis in acinar cells. Together, our results support a PFKFB3driven mechanism controlling AP pathobiology and define this enzyme as a therapeutic target to ameliorate the severity of this condition.

Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell-mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor -interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell-recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell-produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

ACE2 is a major receptor for cellular entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2 binding, strategies to flexibly and sufficiently reduce ACE2 levels for the prevention of SARS-CoV-2 infection have not been explored. Here, we reveal vitamin C (VitC) administration as a potent strategy to prevent SARS-CoV-2 infection. VitC reduces ACE2 protein levels in a dose-dependent manner, while even a partial reduction in ACE2 levels can greatly inhibit SARS-CoV-2 infection. Further studies reveal that USP50 is a crucial regulator of ACE2 levels. VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination of ACE2 at Lys788 and subsequent degradation of ACE2 without affecting its transcriptional expression. Importantly, VitC administration reduces host ACE2 levels and greatly blocks SARS-CoV-2 infection in mice. This study reveals that ACE2 protein levels are down-regulated by an essential nutrient, VitC, thereby enhancing protection against infection of SARS-CoV-2 and its variants.

Enterovirus 71 (EV71) and Coxsackie A16 (CVA16) are two major causative agents of hand, foot, and mouth disease (HFMD) in young children. However, the mechanisms regulating the replication and pathogenesis of EV71/CVA16 remain incompletely understood. We performed a genome-wide CRISPR-Cas9 knockout screen and identified Ragulator as a mediator of EV71-induced apoptosis and pyroptosis. The Ragulator-Rag complex is required for EV71 and CVA16 replication. Upon infection, the Ragulator-Rag complex recruits viral 3D protein to the lysosomal surface through the interaction between 3D and RagB. Disruption of the lysosome-tethered Ragulator-Rag-3D complex significantly impairs the replication of EV71/CVA16. We discovered a novel EV71 inhibitor, ZHSI-1, which interacts with 3D and significantly reduces the lysosomal tethering of 3D. ZHSI-1 treatment significantly represses replication of EV71/CVA16 as well as virus-induced pyroptosis associated with viral pathogenesis. Importantly, ZHSI-1 treatment effectively protects against EV71 infection in neonatal and young mice. Thus, our study indicates that targeting lysosome-tethered Ragulator-Rag-3D may be an effective therapeutic strategy for HFMD.

Abstract Over-activated osteoclast (OC) is a major cause of diseases related to bone loss. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. However, the mechanisms that determine osteoclast function and lifespan are limited. Here, our findings were presented indicating that the newly characterized gene X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (Xaf1) was an important interferon-stimulated gene for termination of osteoclastogenesis via apoptosis induction. We showed that Xaf1 ablation enhanced osteoclast generation in vitro. Xaf1 knockout increased osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (XIAP inhibitor) suppressed osteoclast formation. Mechanistically, Xaf1 deletion decreased osteoclast apoptosis via increasing interaction between XIAP and caspase-3/7. Collectively, our data illustrated an essential role of Xaf1 in the regulation of osteoclastogenesis in both osteoporosis and osteolysis models and highlighted its underlying mechanism.

Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD 1/2) are important cytosolic pattern recognition receptors that initiate host immune response. The dysregulation of NOD signaling is highly associated with inflammatory bowel disease (IBD) that needs novel treatment options. Receptor-interacting protein kinase 2 (RIPK2) is a critical mediator of NOD signaling and considered a promising therapeutic target for IBD treatment. However, there are currently no RIPK2 inhibitors available for clinical use. Here, we report the discovery and characterization of Zharp2-1 as a novel and potent RIPK2 inhibitor that effectively blocks RIPK2 kinase function and NOD-mediated NF-κB/MAPK activation in both human and mouse cell lines. Zharp2-1 exhibits significantly superior solubility compared to the non-prodrug form of the advanced RIPK2 inhibitor prodrug GSK2983559. The improved solubility combined with favorable in vitro metabolic stability translated to excellent in vivo pharmacokinetic profiles for Zharp2-1. In addition, Zharp2-1 demonstrates better effects than GSK2983559 in inhibiting the muramyl dipeptide (MDP)-induced production of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) and MDP-induced peritonitis in mice. Furthermore, Zharp2-1 markedly reduces Listeria monocytogenes infection-induced cytokines release in both human and mouse cells. Importantly, Zharp2-1 significantly ameliorates DNBS-induced colitis in rats and suppressed pro-inflammatory cytokine release in intestinal specimens from IBD patients. Collectively, our findings indicate that Zharp2-1 is a promising RIPK2 inhibitor with the potential to be further developed for IBD therapy.

Almost all cases of cervical cancer (CC) can be attributed to high-risk human papillomavirus (HPVs) infections in keratinocytes. However, it is unknown whether HPV invades immune cells such as macrophages and T cells. We analyzed the single-cell transcriptome of the CC and its adjacent tissues and found that HPV16 genes, including E1, E6, and E7, expressed in the macrophages and CD8+ T cells in addition to the malignant cells. HPV16+ macrophages highly expressed the genes that promote cell adhesion and the favorable genes such as WAS, IQCB1, MYO1F, and PDZD11 in CC prognosis. The transcription factor KLF5 potentially accounted for the induction of these protective genes and thus facilitated the infiltration of the immune cells in tumor tissues. Our single-cell transcriptome analysis suggests the potential value of the HPV16+ macrophage in CC prognosis. However, extensive experimental studies investigating the characteristics and functions of the HPV+ immune cells are still required.

SUMMARY ACE2 is a major receptor for cell entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2’s binding, how to efficiently and flexibly control ACE2 levels for prevention of SARS-CoV-2 infection has not been explored. Here, we revealed Vitamin C (VitC) administration as an effective strategy to prevent SARS-CoV-2 infection. VitC reduced ACE2 protein levels in a dose-dependent manner, while partial reduction of ACE2 can greatly restrict SARS-CoV-2 infection. Further studies uncovered that USP50 is a crucial regulator of ACE2 protein levels, and VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination at Lys788 and degradation of ACE2, without disrupting ACE2 transcriptional expression. Importantly, VitC administration reduced host ACE2 and largely blocked SARS-CoV-2 infection in mice. This study identified an in vivo ACE2 balance controlled by both USP50 and an essential nutrient VitC, and revealed a critical role and application of VitC in daily protection from SARS-CoV-2 infection. Highlights VitC reduces ACE2 protein levels in a dose-dependent manner VitC and USP50 regulate K48-linked ubiquitination at Lys788 of ACE2 VitC blocks the interaction between USP50 and ACE2 VitC administration lowers host ACE2 and prevents SARS-CoV-2 infection in vivo Abstract Figure The deubiquitinase USP50 controls ACE2 protein stability and levels, while Vitamin C blocks the USP50-ACE2 interaction and therefore results in ACE2 degradation, offering a flexible and efficient approach to protection of the host from SARS-CoV-2 infection.

Mixed lineage kinase domain-like protein (MLKL) is the terminal effector of necroptosis, a form of regulated necrosis. Optimal activation of necroptosis, which eliminates infected cells, is critical for antiviral host defense. MicroRNAs (miRNAs) regulate the expression of genes involved in various biological and pathological processes. However, the roles of miRNAs in necroptosis-associated host defense remain largely unknown. We screened a library of miRNAs and identified miR-324-5p as the most effective suppressor of necroptosis. MiR-324-5p downregulates human MLKL expression by specifically targeting the 3 ' UTR in a seed region-independent manner. In response to interferons (IFNs), miR-324-5p is downregulated via the JAK/STAT signaling pathway, which removes the posttranscriptional suppression of MLKL mRNA and facilitates the activation of necroptosis. In influenza A virus (IAV)-infected human primary macrophages, IFNs are induced, leading to the downregulation of miR-324-5p. MiR-324-5p overexpression attenuates IAV-associated necroptosis and enhances viral replication, whereas deletion of miR-324-5p potentiates necroptosis and suppresses viral replication. Hence, miR-324-5p negatively regulates necroptosis by manipulating MLKL expression, and its downregulation by IFNs orchestrates optimal activation of necroptosis in host antiviral defense.

XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.