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The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs), whose role in antiviral immunity is elusive. Here, we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state, GLUT4 is trapped at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9, TUG). Following RNA virus infection, GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs, preventing them from activating IFN-β responses. UBXN9 deletion prompts constitutive GLUT4 translocation, sequestration of RLRs and attenuation of antiviral immunity, whereas GLUT4 deletion heightens RLR signaling. Notably, reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall, our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Junctophilin-2 (JPH2) is traditionally recognized as a cardiomyocyte-enriched structural protein that anchors the junction between the plasma membrane and the endo/sarcoplasmic reticulum, facilitating excitation-induced cardiac contraction. In this study, we uncover a novel function of JPH2 as a double-stranded RNA (dsRNA)-binding protein, which forms complexes with dsRNA both in vitro and in cells. Stimulation by cytosolic dsRNA enhances the interaction of JPH2 with the dsRNA sensor MDA5. Notably, JPH2 inhibits MDA5's binding to its dsRNA ligand, likely by sequestering the dsRNA. Silencing JPH2 in cardiomyocytes increased the interaction between MDA5 and its dsRNA ligands, activated the MAVS/TBK1 signaling, and triggered spontaneous interferon-beta (IFNb1) production in the absence of foreign pathogen. Mouse hearts deficient in JPH2 exhibited upregulation of innate immune signaling cascade. Collectively, these findings identify JPH2 as a regulator of dsRNA sensing and highlight its role in suppressing the automatic activation of innate immune responses in cardiomyocytes, suggesting the cytosolic surface of the endo/sarcoplasmic reticulum as a hub for dsRNA sequestration., Competing Interests: Declaration of competing interest We have nothing to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important RNA virus that has caused huge economic losses to swine industry in the whole world. Ubiquitin specific protease 8 (USP8), a pivotal regulator of protein degradation, intricately contributes to orchestrating the delicate balance of various biological processes through its deubiquitinating activity. However, the role of USP8 in antiviral immune response to PRRSV remains elusive. In the study, by means of overexpressing USP8, we identified that USP8 suppressed the replication of PRRSV, while reducing USP8 expression using siRNA significantly led to the promotion of PRRSV replication. And USP8 facilitates the production of IFN-β and some IFN-stimulated genes (ISGs) during PRRSV infection. Mechanistically, USP8 promoted mitochondrial antiviral signaling protein (MAVS)-mediated IFN-β signaling. Moreover, USP8 interacted with MAVS and exerted anti-PRRSV effects in a MAVS-dependent manner. This study highlights the importance of USP8 in regulating PRRSV replication, which may enhance our comprehension of its role in innate immunity and its impact on viral replication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Porcine hemagglutinating encephalomyelitis (PHE), caused by a betacoronavirus named porcine hemagglutinating encephalomyelitis virus (PHEV), is a highly fatal disease of pigs characterized by nonsuppurative encephalitis. Activation of astrocytes is a hallmark of viral encephalomyelitis; however, the mechanism of PHEV-induced astrocyte activation is currently unknown. Based on mouse model, we show that PHEV infection led to astrogliosis in mouse brain and brain slice cultures (BSCs), as indicated by increased expression of glial fibrillary acidic protein (GFAP). PHEV can neither infect nor activate primary astrocytes in vitro, indicating that activation of astrocytes maybe mediated by factors secreted from viral infected neurons but not by direct viral infection of astrocytes. PHEV infection results in increased interferon (IFN) response in later stage, we thereafter focused on whether IFN-β can activate astrocytes after PHEV infection similar to other neurotropic viruses. IFN-β treatment resulted in both the upregulation of GFAP and activation-associated cytokines/chemokines in mouse primary astrocytes. Furthermore, the addition of IFN-β neutralization antibody prevented PHEV-infected mouse brain tissue homogenate from activating astrocytes. Taken together, IFN-β triggers the activation of astrocytes in the central nervous system (CNS) following PHEV infection. Further understanding of the role of activated astrocytes during PHEV infection may provide new insights for treatment this disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Non-POU domain-containing octamer-binding protein (NONO) is a multi-functional nuclear protein which belongs to the Drosophila behavior/human splicing (DBHS) protein family. NONO is known to regulate multiple important biological processes including host antiviral immune response. However, whether NONO can inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication is less well understood. In this study, we demonstrated that swine NONO (sNONO) inhibited PRRSV replication, via increasing expression of IFN-β, whereas NONO knockdown or knockout in PAM-KNU cells was more susceptible to PRRSV infection. As an IRF3 positive regulation factor, NONO promoted IFN-β expression by enhancing activation of IRF3. During PRRSV infection, NONO further up-regulated IRF3-mediated IFN-β expression by interacting with PRRSV N protein. Mechanistically, NONO functioned as a scaffold protein to detect PRRSV N protein and formed N-NONO-IRF3 complex in the nucleus. Interestingly, it was found that the NONO protein reversed the inhibitory effect of PRRSV N protein on type I IFN signaling pathway. Taken together, our study provides a novel mechanism for NONO to increase the IRF3-mediated IFN-β activation by interacting with the viral N protein to inhibit PRRSV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila , Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFNβ, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFNβ has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNβ signaling but not IFNγ signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFNβ-mediated signaling. Cells downregulated their surface levels of IFNα/β receptor 1 (IFNAR1) in response to LPS, which may be mediating our observed inhibition. Lastly, we examined this inhibition in the context of TLR4-deficient BMDMs as well as TLR4 RNA interference and we observed a loss of inhibition with LPS stimulation as well as STm infection. In summary, we show that macrophages exposed to STm have reduced IFNβ signaling via crosstalk with TLR4 signaling, which may be mediated by reduced host cell surface IFNAR1, and that IFNβ signaling does not affect cell-autonomous host defense against STm., Competing Interests: The authors declare no conflict of interest.

Anti-drug antibodies (ADA) reduce the efficacy of immunotherapies in multiple sclerosis (MS) and are associated with increased disease progression risk. Blood biomarkers predicting immunogenicity to biopharmaceuticals represent an unmet clinical need. Patients with relapsing remitting (RR)MS were recruited before (baseline), three, and 12 (M12) months after commencing interferon-beta treatment. Neutralising ADA-status was determined at M12, and patients were stratified at baseline according to their M12 ADA-status (ADA-positive/ADA-negative). Patients stratified as ADA-positive were characterised by an early dampened response to interferon-beta (prior to serum ADA detection) and distinct proinflammatory transcriptomic/proteomic peripheral blood signatures enriched for 'immune response activation' including phosphoinositide 3-kinase-γ and NFκB-signalling pathways both at baseline and throughout the treatment course, compared to ADA-negative patients. These immunogenicity-associated proinflammatory signatures significantly overlapped with signatures of MS disease severity. Thus, whole blood molecular profiling is a promising tool for prediction of ADA-development in RRMS and could provide insight into mechanisms of immunogenicity., Competing Interests: Declaration of competing interest MA is now employed by Nxera Pharma UK, Translational Sciences, Granta Park Steinmetz Building, Cambridge, United Kingdom. KEW is now employed by Nucleome Therapeutics, Oxford, United Kingdom. EKH has received honoraria/research support from Biogen, Merck Serono, Novartis, Roche, and Teva; has served as a member of advisory boards for Actelion, Biogen, Celgene, Merck Serono, Novartis, and Sanofi Genzyme; has been supported by the Czech Ministry of Education – project Cooperatio LF1, research area Neuroscience, and the project National Institute for Neurological Research (Programme EXCELES, ID project No LX22NPO5107) – funded by the European Union-Next Generation EU. RF has received speaker honoraria and hospitality from Merck, Biogen, TEVA, Novartis, Genzyme, Abbvie, Merz and Ipsen. PN has received speaker honoraria and consultant fees from Biogen, Novartis, Merck, and Roche. The remaining authors (LC, PD, CC, IPT, ECJ) declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-β expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the research reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-β) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Type I IFNs play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-β Abs from PBMCs of individuals treated with IFN-α or IFN-β, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-β1a-specific signaling and able to block lipopolysaccharide or S100 calcium-binding protein A14-induced IFN-β signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-β suggests potential for diverse research and clinical applications., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Type I interferon (IFN) induces a large number of interferon-stimulated genes (ISGs) expression to inhibit PRRSV infection. To survive in the host, PRRSV has evolved multiple strategies to antagonize host innate immune response. Previous studies have reported that PRRSV N protein decreases the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress IFN-β production. However, whether other PRRSV proteins inhibit the antiviral function of TRIM25 is less well understood. In this study, we first found that PRRSV NSP1α decreased ISGylation of TRIM25. Meanwhile, NSP1α significantly suppressed TRIM25-mediated IFN-β production to promote PRRSV replication. Further studies demonstrated that PRRSV NSP1α reduced the protein level of TRIM25 in proteasome system but did not regulate the transcription level of TRIM25. In addition, the function of NSP1α in TRIM25 degradation did not rely on its papain-like cysteine protease activity. Taken together, PRRSV NSP1α antagonizes the antiviral response of TRIM25 by mediating TRIM25 degradation to promote PRRSV replication. Our data identify TRIM25 as a natural target of PRRSV NSP1α and reveal a novel mechanism that PRRSV induces TRIM25 degradation and inhibits host antiviral immune response., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-β production and signaling. In particular NS4A was very effective at suppressing IFN-β production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-β production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a polyarticular synovitis. In recent years, elderly onset rheumatoid arthritis (EORA) has been increasing. Treg cells in RA have been reported to be dysfunctional, but the relationship between aging and their functional changes is unclear. Here, we found that Treg cells from EORA patients had increased percentages, but decreased activity compared to those from younger onset RA (YORA) patients. In experiments using arthritis model mice, decreased suppressive function and oxygen consumption rate (OCR) were observed in Treg cells only from old arthritic mice. Furthermore, type I interferon (IFN) signaling was upregulated in Treg cells from old GIA mice, and IFN-β decreased the suppressive function of Treg cells. Our findings demonstrate that increased type I IFN signaling in old Treg cells is induced only in the arthritic environment and relates to decreased suppressive function of Treg cells, gets involved in EORA., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Allergic asthma is characterized by increased type 2 inflammation, including eosinophils. Subjects with allergic asthma have recurrent symptoms due to their constant exposure to environmental allergens, such as house dust mite (HDM), which can be further exacerbated by respiratory infections like rhinovirus. The immunoproteasome (IP) is a proteolytic machinery that is induced by inflammatory mediators during virus infection, but the role of the IP in airway allergic inflammation during rhinovirus infection remains unknown. Wild-type (WT) and IP knockout (KO) mice were challenged with HDM. At 48 h after the last HDM challenge, mice were infected with rhinovirus 1B (RV-A1B) for 24 h. After HDM and RV-A1B treatment, IP KO (vs. WT) mice had significantly more lung eosinophils and neutrophils, as well as a significantly higher viral load, but less IFN-beta expression, compared to WT mice. A TLR3 agonist polyinosinic-polycytidylic acid (Poly I:C) treatment after RV-A1B infection in HDM-challenged IP KO mice significantly increased IFN-beta expression and reduced viral load, with a minimal effect on the number of inflammatory cells. Our data suggest that immunoproteasome is an important mechanism functioning to prevent excessive inflammation and viral infection in allergen-exposed mice, and that Poly I:C could be therapeutically effective in enhancing the antiviral response and lessening the viral burden in lungs with IP deficiency., Competing Interests: The authors declare no conflicts of interest.

mRNA vaccines against SARS-CoV-2 have revolutionized vaccine development, but their immunological mechanisms are not fully understood. Here, we investigate injection site responses of mRNA vaccines by generating a comprehensive single-cell transcriptome profile upon lipid nanoparticle (LNP) or LNP-mRNA challenge in female BALB/c mice. We show that LNP-induced stromal pro-inflammatory responses and mRNA-elicited type I interferon responses dominate the initial injection site responses. By tracking the fate of delivered mRNA, we discover that injection site fibroblasts are highly enriched with the delivered mRNA and that they express IFN-β specifically in response to the mRNA component, not to the LNP component of mRNA vaccines. Moreover, the mRNA-LNP, but not LNP alone, induces migratory dendritic cells highly expressing IFN-stimulated genes (mDC_ISGs) at the injection site and draining lymph nodes. When co-injected with LNP-subunit vaccine, IFN-β induces mDC_ISGs at the injection site, and importantly, it substantially enhances antigen-specific cellular immune responses. Furthermore, blocking IFN-β signaling at the injection site significantly decreases mRNA vaccine-induced cellular immune responses. Collectively, these data highlight the importance of injection site fibroblasts and IFN-β signaling during early immune responses against the mRNA vaccine and provide detailed information on the initial chain of immune reactions elicited by mRNA vaccine injection., (© 2024. The Author(s).)

We recently revealed that activated STING is secreted into RAB22A-induced extracellular vesicles (R-EVs) and promotes antitumor immunity in cancer cells. Whether mesenchymal stem cell (MSC)-derived R-EVs containing activated STING can be used as a novel antitumor immunotherapy remains unclear, as MSC-derived EVs are promising cell-free therapeutics due to their superior biocompatibility and safety, as well as low immunogenicity. Here, we report that induced pluripotent stem cell (iPSC)-derived MSCs can generate R-EVs with a size and mechanism of formation that are similar to those of R-EVs produced from cancer cells. Furthermore, these MSC-derived R-EVs containing activated STING induced IFNβ expression in recipient THP-1 monocytes and antitumor immunity in mice. Our findings reveal that the use of MSC-derived R-EVs containing activated STING is a promising cell-free strategy for antitumor immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-β, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-β levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-β and NF-κB in DEFs stimulated by 5'ppp dsRNA.

Fundamental to mammalian intrinsic and innate immune defenses against pathogens is the production of Type I and Type II interferons, such as IFN-β and IFN-γ, respectively. The comparative effects of IFN classes on the cellular proteome, protein interactions, and virus restriction within cell types that differentially contribute to immune defenses are needed for understanding immune signaling. Here, a multilayered proteomic analysis, paired with biochemical and molecular virology assays, allows distinguishing host responses to IFN-β and IFN-γ and associated antiviral impacts during infection with several ubiquitous human viruses. In differentiated macrophage-like monocytic cells, we classified proteins upregulated by IFN-β, IFN-γ, or pro-inflammatory LPS. Using parallel reaction monitoring, we developed a proteotypic peptide library for shared and unique ISG signatures of each IFN class, enabling orthogonal confirmation of protein alterations. Thermal proximity coaggregation analysis identified the assembly and maintenance of IFN-induced protein interactions. Comparative proteomics and cytokine responses in macrophage-like monocytic cells and primary keratinocytes provided contextualization of their relative capacities to restrict virus production during infection with herpes simplex virus type-1, adenovirus, and human cytomegalovirus. Our findings demonstrate how IFN classes induce distinct ISG abundance and interaction profiles that drive antiviral defenses within cell types that differentially coordinate mammalian immune responses.

The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-β response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-β response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-β response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-β and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-β promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-β promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNβ induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

India saw a spike in COVID-19 cases in early 2023, and this wave of infection was attributed to XBB sublineages of SARS-CoV-2 Omicron variant. The impact of XBB wave was significantly shorter with low burden of severe cases or hospitalization as compared with previous SARS-CoV-2 variants of concern. Although a combination of old and new mutations in the spike region of XBB.1.16 variant led to a drastic reduction in the ability of antibodies from prior immunity to neutralize this virus, additional nonspike mutations suggested a possible change in its ability to suppress innate immune responses. In this study, we tested the sensitivity of Delta, BA.2.75, and XBB.1.16 variants to interferon-β (IFN-β) treatment and found that XBB.1.16 variant was most sensitive to IFN-β. We next tested the ability of serum antibodies from healthy individuals to neutralize XBB.1.16. We showed that most of the individuals with hybrid immunity maintained a low but significant level of neutralizing antibodies to XBB.1.16 variant. Therefore, our observations indicated that both hybrid immunity because of natural infection and enhanced sensitivity to IFNs may have contributed to the low impact of XBB.1.16 infections in India.

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work., (© 2024. The Author(s).)

Background: Signaling by toll-like receptors (TLRs) initiates important immune responses against viral infection. The role of TLRs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well elucidated. Thus, we investigated the interaction of TLRs agonists and SARS-COV-2 antigens with immune cells in vitro., Material & Methods: 30 coronavirus disease 2019 (COVID-19) patients (15 severe and 15 moderate) and 10 age and sex-matched healthy control (HC) were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and activated with TLR3, 7, 8, and 9 agonists, the spike protein (SP) of SARS-CoV-2, and the receptor binding domain (RBD) of SP. Frequencies of CD3 + IFN-β + T cells, and CD3 + IFN-γ + T cells were evaluated by flow cytometry. Interferon (IFN)-β gene expression was assessed by qRT-PCR., Results: The frequency of CD3 + IFN-β + T cells was higher in PBMCs from moderate (p < 0.0001) and severe (p = 0.009) patients at baseline in comparison with HCs. The highest increase in the frequency of CD3 + IFN-β + T cells in cell from moderate patients was induced by TLR8 agonist and SP (p < 0.0001 for both) when compared to HC, while, the highest increase of the frequency of CD3 + IFN-β + T cells in sample of severe patients was seen with TLR8 and TLR7 agonists (both p = 0.002). The frequency of CD3 + IFN-γ + T cells was significantly increased upon stimulation with TLR agonists in cell from patients with moderate and severe COVID-19, compared with HC (all p < 0.01), except with TLR7 and TLR8 agonists. The TLR8 agonist did not significantly increase the frequency of CD3 + IFN-γ + T cells in PBMCs of severe patients, but did so in cells from patients with moderate disease (p = 0.01). Moreover, IFN-β gene expression was significantly upregulated in CD3 + T cells from moderate (p < 0.0001) and severe (p = 0.002) COVID-19 patients, compared to HC after stimulation with the TLR8 agonist, while, stimulation of T cells with SP, significantly up-regulated IFN-β mRNA expression in cells from patients with moderate (p = 0.0003), but not severe disease., Conclusion: Stimulation of PBMCs from COVID-19 patients, especially patients with moderate disease, with TLR8 agonist and SP increased the frequency of IFN-β-producing T cells and IFN-β gene expression., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shahid Beheshti medical University. Published by Elsevier Inc. All rights reserved.)

Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-β (IFNβ) by MSCs. IFNβ reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The infection caused by porcine epidemic diarrhea virus (PEDV) is associated with high mortality in piglets worldwide. Host factors involved in the efficient replication of PEDV, however, remain largely unknown. Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5 (EF-hand protein calmodulin-like 5) following PEDV infection. A further study unveiled a biphasic increase of CALML5 in 2 and 12 ​h after viral infection. Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge. Moreover, CALML5 depletion reduced PEDV mRNA and protein levels, leading to a one-order-of-magnitude decrease in virus titer. At the early stage of PEDV infection, CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins. CALML5 depletion also suppressed IFN-β and IL-6 production in the PEDV-infected cells, thereby indicating its involvement in negatively regulating the innate immune response. Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction., Competing Interests: Conflict of interest All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recurrent respiratory papillomatosis (RRP) is a rare benign tumor caused mainly by the infection of the respiratory tract epithelial cells by the human papillomavirus (HPV) type 6/11. However, the specific mechanisms underlying the inhibition of the host's innate immune response by HPV remain unclear. For this purpose, we employed single-cell RNA sequencing to analyze the states of various immune cells in RRP samples post-HPV infection and utilized a cellular model of HPV infection to elucidate the mechanisms by which HPV evades the innate immune system in RRP. The results revealed distinct immune cell heterogeneity in RRP and demonstrated that HPV11 E7 can inhibit the phosphorylation of the stimulator of interferon genes protein, thereby circumventing the body's antiviral response. In vitro co-culture experiments demonstrated that stimulation of macrophages to produce interferon-beta induced the death of HPV-infected epithelial cells, also reducing HPV viral levels. In summary, our study preliminarily identifies the potential mechanisms by which HPV evades the host's antiviral immune response, as well as the latent antiviral functions exhibited by activated macrophages. This research serves as an initial exploration of antiviral immune evasion in RRP, laying a solid foundation for investigating immunotherapeutic approaches for the disease.IMPORTANCESurgical tumor reduction is the most common treatment for recurrent respiratory papillomatosis (RRP). One of the characteristics of RRP is its persistent recurrence, and multiple surgeries are usually required to control the symptoms. Recently, some adjuvant therapies have shown effectiveness, but none of them can completely clear human papillomavirus (HPV) infection, and thus, a localized antiviral immune response is significant for disease control; after all, HPV infection is limited to the epithelium. Inhibition of interferon-beta (IFN-β) secretion by HPV11 E7 viral proteins in epithelial cells by affecting stimulator of interferon genes phosphorylation may account for the persistence of low-risk HPV replication in the RRP. Moreover, suppression of the IFN-I pathway in RRP cell types might provide clues regarding the hyporeactive function of local immune cells. However, activation of macrophage groups to produce IFN-β can still destroy HPV-infected cells., Competing Interests: The authors declare no conflict of interest.

African swine fever (ASF) is an acute hemorrhagic and devastating infectious disease affecting domestic pigs and wild boars. It is caused by the African swine fever virus (ASFV), which is characterized by genetic diversity and sophisticated immune evasion strategies. To facilitate infection, ASFV encodes multiple proteins to antagonize host innate immune responses, thereby contributing to viral virulence and pathogenicity. The molecular mechanisms employed by ASFV-encoded proteins to modulate host antiviral responses have not been comprehensively elucidated. In this study, it was observed that the ASFV MGF505-6R protein, a member of the multigene family 505 (MGF505), effectively suppressed the activation of the interferon-beta (IFN-β) promoter, leading to reduced mRNA levels of antiviral genes. Additional evidence has revealed that MGF505-6R antagonizes the cGAS-STING signaling pathway by interacting with the stimulator of interferon genes (STING) for degradation in the autophagy-lysosomal pathway. The domain mapping revealed that the N-terminal region (1-260aa) of MGF505-6R is the primary domain responsible for interacting with STING, while the CTT domain of STING is crucial for its interaction with MGF505-6R. Furthermore, MGF505-6R also inhibits the activation of STING by reducing the K63-linked polyubiquitination of STING, leading to the disruption of STING oligomerization and TANK binding kinase 1 (TBK1) recruitment, thereby impairing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Collectively, our study elucidates a novel strategy developed by ASFV MGF505-6R to counteract host innate immune responses. This discovery may offer valuable insights for further exploration of ASFV immune evasion mechanisms and antiviral strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yao, Cao, Li, Hu, Rong, Yin, Tian, Hu, Li and Qian.)

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-β (IFN-β) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-β production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-β production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-β production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Callon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Importance: Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-β with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance., Competing Interests: DEG is a member of advisory boards for GSK and Takeda Pharmaceuticals.

Herpetic stromal keratitis (HSK) is a painful and vision-impairing disease caused by recurrent HSV-1 infection of the cornea. The virus replication in the corneal epithelium and associated inflammation play a dominant role in HSK progression. Current HSK treatments targeting inflammation or virus replication are partially effective and promote HSV-1 latency, and long-term use can cause side effects. Thus, understanding molecular and cellular events that control HSV-1 replication and inflammation is crucial for developing novel HSK therapies. In this study, we report that ocular HSV-1 infection induces the expression of IL-27, a pleiotropic immunoregulatory cytokine. Our data indicate that HSV-1 infection stimulates IL-27 production by macrophages. Using a primary corneal HSV-1 infection mouse model and IL-27 receptor knockout mice, we show that IL-27 plays a critical role in controlling HSV-1 shedding from the cornea, the optimum induction of effector CD4+ T cell responses, and limiting HSK progression. Using in vitro bone marrow-derived macrophages, we show that IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, IFN-β production, and IFN-stimulated gene expression after HSV-1 infection. Furthermore, we report that IL-27 is critical for macrophage survival, Ag uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Metabolic rewiring underlies the effector functions of macrophages 1-3 , but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, one of the most highly infectious animal viruses throughout the world. The JAK-STAT signaling pathway is a highly conserved pathway for IFN-β-induced antiviral gene expression. Previous studies have shown that FMDV can strongly suppress the innate immune response. Moreover, although STAT1 and STAT2 (STAT1/2) have been well established in JAK-STAT signaling-induced antiviral gene expression, whether FMDV proteins inhibit IFN-β-induced JAK-STAT signaling remains poorly understood. In this study, we described the Lb leader protease (Lbpro) of FMDV as a candidate for inhibiting IFN-β-induced signaling transduction via directly interacting with STAT1/2. We further showed that Lbpro colocalized with STAT1/2 to inhibit their nuclear translocation. Importantly, Lbpro cleaved STAT1/2 to inhibit IFN-β-induced signal transduction, whereas the catalytically inactive mutant of LC51A (Lbpro with cysteine substituted with alanine at amino acid residue 51) had no effect on the stability of STAT1/2 proteins. The cleavage of the STAT1/2 proteins was also determined during FMDV infection in vitro. Lbpro could cleave the residues between 252 and 502 aa for STAT1 and the site spanning residues 140 - 150 aa (QQHEIESRIL) for STAT2. The in vivo results showed that Lbpro can cleave STAT1/2 in pigs. Overall, our findings suggest that FMDV Lbpro-mediated targeting of STAT1/2 may reveal a novel mechanism for viral immune evasion., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Pseudorabies virus (PRV) is a member of the genus Varicellovirus, family Herpesviridae and causes Aujeszky's disease to lead to huge economic losses in the global pig industry. The Non-POU domain-containing octamer-binding protein (NONO), as a Drosophila behavior/human splicing (DBHS) protein, plays a key role in multiple biological functions in cells, including transcriptional regulation, RNA splicing, DNA repair and so on. However, whether swine NONO (sNONO) inhibits PRV infection is less understood. In this study, we showed that sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. After PRV infection, sNONO enhanced the activation of IFN-β promoter and IFN-β expression. Furthermore, knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. Taken together, we have first elucidated the anti-PRV function and mechanism of sNONO, which may provide a new strategy for preventing DNA virus infection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifnβ1 transcription in the brain of mice infected with Plasmodium berghei ANKA ( Pba ). This STING1/IFNβ-mediated response increases brain CXCL10 governing the extent of brain leukocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFNβ-reporter and STING1-deficient Pba -infected mice, which were significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba -infected erythrocytes activated the STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFNβ in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments, and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin, triggering brain endothelial inflammatory responses via the STING1/IFNβ/CXCL10 axis crucial to CM pathogenesis and lethality.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-β (IFN-β) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A 4 (15-epi-LXA 4 ) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli -evoked lung injury, and suppresses production of IFN-β and the proresolving lipid mediators 15-epi-LXA 4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-β delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-β accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA 4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-β-mediated resolution. These findings point to a pivotal role of IFN-β in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-β-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Since the discovery of oncogenes, there has been tremendous interest to understand their mechanistic basis and to develop broadly actionable therapeutics. Some of the most frequently activated oncogenes driving diverse cancers are c-MYC, EGFR, HER2, AKT, KRAS, BRAF, and MEK. Using a reductionist approach, we explored how cellular proteomes are remodeled in isogenic cell lines engineered with or without these driver oncogenes. The most striking discovery for all oncogenic models was the systematic downregulation of scores of antiviral proteins regulated by type 1 interferon. These findings extended to cancer cell lines and patient-derived xenograft models of highly refractory pancreatic cancer and osteosarcoma driven by KRAS and MYC oncogenes. The oncogenes reduced basal expression of and autocrine stimulation by type 1 interferon causing remarkable convergence on common phenotypic and functional profiles. In particular, there was dramatically lower expression of dsRNA sensors including DDX58 (RIG-I) and OAS proteins, which resulted in attenuated functional responses when the oncogenic cells were treated with the dsRNA mimetic, polyI:C, and increased susceptibility to infection with an RNA virus shown using SARS-CoV-2. Our reductionist approach provides molecular and functional insights connected to immune evasion hallmarks in cancers and suggests therapeutic opportunities., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The interleukin-12 (IL-12) family comprises the only heterodimeric cytokines mediating diverse functional effects. We previously reported a striking bimodal IL-12p70 response to lipopolysaccharide (LPS) stimulation in healthy donors. Herein, we demonstrate that interferon β (IFNβ) is a major upstream determinant of IL-12p70 production, which is also associated with numbers and activation of circulating monocytes. Integrative modeling of proteomic, genetic, epigenomic, and cellular data confirms IFNβ as key for LPS-induced IL-12p70 and allowed us to compare the relative effects of each of these parameters on variable cytokine responses. Clinical relevance of our findings is supported by reduced IFNβ-IL-12p70 responses in patients hospitalized with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or chronically infected with hepatitis C (HCV). Importantly, these responses are resolved after viral clearance. Our systems immunology approach defines a better understanding of IL-12p70 and IFNβ in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection., Competing Interests: Declaration of interests M.L.A. is a current employee of Hibio, who had no influence on the study design or reporting. D.D. declares previous grant support from Rules Based Medicine that was not implicated in this study. The other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-β. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-β promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-β (TRIF)., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Virus infection triggers intricate signal cascade reactions to activate the host innate immunity, which leads to the production of type I interferon (IFN-I). Herpes simplex virus 1 (HSV-1), a human-restricted pathogen, is capable of encoding over 80 viral proteins, and several of them are involved in immune evasion to resist the host antiviral response through the IFN-I signaling pathway. Here, we determined that HSV-1 UL31, which is associated with nuclear matrix and is essential for the formation of viral nuclear egress complex, could inhibit retinoic acid-inducible gene I (RIG-I)-like receptor pathway-mediated interferon beta (IFN-β)-luciferase (Luc) and (PRDIII-I)4-Luc (an expression plasmid of IFN-β positive regulatory elements III and I) promoter activation, as well as the mRNA transcription of IFN-β and downstream interferon-stimulated genes (ISGs), such as ISG15, ISG54, ISG56, etc., to promote viral infection. UL31 was shown to restrain IFN-β activation at the interferon regulatory factor 3 (IRF3)/IRF7 level. Mechanically, UL31 was demonstrated to interact with TANK binding kinase 1 (TBK1), inducible IκB kinase (IKKi), and IRF3 to impede the formation of the IKKi-IRF3 complex but not the formation of the IRF7-related complex. UL31 could constrain the dimerization and nuclear translocation of IRF3. Although UL31 was associated with the CREB binding protein (CBP)/p300 coactivators, it could not efficiently hamper the formation of the CBP/p300-IRF3 complex. In addition, UL31 could facilitate the degradation of IKKi and IRF3 by mediating their K48-linked polyubiquitination. Taken together, these results illustrated that UL31 was able to suppress IFN-β activity by inhibiting the activation of IKKi and IRF3, which may contribute to the knowledge of a new immune evasion mechanism during HSV-1 infection. IMPORTANCE The innate immune system is the first line of host defense against the invasion of pathogens. Among its mechanisms, IFN-I is an essential cytokine in the antiviral response, which can help the host eliminate a virus. HSV-1 is a double-stranded DNA virus that can cause herpes and establish a lifelong latent infection, due to its possession of multiple mechanisms to escape host innate immunity. In this study, we illustrate for the first time that the HSV-1-encoded UL31 protein has a negative regulatory effect on IFN-β production by blocking the dimerization and nuclear translocation of IRF3, as well as promoting the K48-linked polyubiquitination and degradation of both IKKi and IRF3. This study may be helpful for fully understanding the pathogenesis of HSV-1.

Type-I interferon (IFN-I) signals exert a critical role in disease progression during viral infections. However, the immunomodulatory mechanisms by which IFN-I dictates disease outcomes remain to be fully defined. Here we report that IFN-I signals mediate thymic atrophy in viral infections, with more severe and prolonged loss of thymic output and unique kinetics and subtypes of IFN-α/β expression in chronic infection compared to acute infection. Loss of thymic output was linked to inhibition of early stages of thymopoiesis (DN1-DN2 transition, and DN3 proliferation) and pronounced apoptosis during the late DP stage. Notably, infection-associated thymic defects were largely abrogated upon ablation of IFNαβR and partially mitigated in the absence of CD8 T cells, thus implicating direct as well as indirect effects of IFN-I on thymocytes. These findings provide mechanistic underpinnings for immunotherapeutic strategies targeting IFN-1 signals to manipulate disease outcomes during chronic infections and cancers., (Copyright © 2021. Published by Elsevier Inc.)

IL-15 exhibits pleiotropic effects on NK and CD8 + T cells and contributes to host protection or immunopathology during infection. Although both type I IFNs and IFN-γ upregulate IL-15 expression, their effects on IL-15 upregulation and underlying mechanisms have not been compared comprehensively. In addition, little is known about trans -presentation of IL-15 by epithelial cells to lymphocytes. In this study, we analyzed the expression of IL-15 and IL-15Rα in the human hepatocyte-derived Huh-7 cell line after stimulation with IFN-α, IFN-β, or IFN-γ using RT-PCR, flow cytometry, and confocal microscopy. We also performed knockdown experiments to investigate the signaling pathway involved in IL-15 upregulation. IFN-γ more potently upregulated IL-15 expression in Huh-7 cells than IFN-α and IFN-β. Knockdown experiments revealed that IFN-γ- and IFN-β-induced IL-15 expression relied on IFN regulatory factor 1 (IRF1), which is upregulated by STAT1 and IFN-stimulated gene factor 3, respectively. Inhibitor of κB kinase α/β was also involved in IFN-γ-induced upregulation of IL-15. Furthermore, human NK cells were activated by coculture with IFN-γ-treated Huh-7 cells, which was abrogated by knocking down IL-15Rα in IFN-γ-treated Huh-7 cells, indicating that IFN-γ-induced IL-15 on Huh-7 cells activates NK cells via trans -presentation. In summary, our data demonstrate that IFN-γ potently elicits IL-15 trans -presentation by epithelial cells via IRF1. These data also suggest that the IFN-γ-IRF1-IL-15 axis may be a regulatory target for the treatment of diseases with IL-15 dysregulation., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Zika virus (ZIKV) infection can be associated with neurological pathologies, such as microcephaly in newborns and Guillain-Barre syndrome in adults. Effective therapeutics are currently not available. As such, a comprehensive understanding of virus-host interactions may guide the development of medications for ZIKV. Here we report a human genome-wide overexpression screen to identify host factors that regulate ZIKV infection and find TMEM120A as a ZIKV restriction factor. TMEM120A overexpression significantly inhibits ZIKV replication, while TMEM120A knockdown increases ZIKV infection in cell lines. Moreover, Tmem120a knockout in mice facilitates ZIKV infection in primary mouse embryonic fibroblasts (MEF) cells. Mechanistically, the antiviral activity of TMEM120A is dependent on STING, as TMEM120A interacts with STING, promotes the translocation of STING from the endoplasmic reticulum (ER) to ER-Golgi intermediate compartment (ERGIC) and enhances the phosphorylation of downstream TBK1 and IRF3, resulting in the expression of multiple antiviral cytokines and interferon-stimulated genes. In summary, our gain-of-function screening identifies TMEM120A as a key activator of the antiviral signaling of STING., (© 2022. The Author(s).)

Type I interferons (IFNs) are the first frontline of the host innate immune response against invading pathogens. Herein, we characterized an unknown protein encoded by phospholipase A2 inhibitor and LY6/PLAUR domain-containing (PINLYP) gene that interacted with TBK1 and induced type I IFN in a TBK1- and IRF3-dependent manner. Loss of PINLYP impaired the activation of IRF3 and production of IFN-β induced by DNA virus, RNA virus, and various Toll-like receptor ligands in multiple cell types. Because PINLYP deficiency in mice engendered an early embryonic lethality in mice, we generated a conditional mouse in which PINLYP was depleted in dendritic cells. Mice lacking PINLYP in dendritic cells were defective in type I IFN induction and more susceptible to lethal virus infection. Thus, PINLYP is a positive regulator of type I IFN innate immunity and important for effective host defense against viral infection., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.

Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-β induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-β induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo . Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS -/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.

The cGAS/STING/TBK1 (cyclic guanine monophosphate-AMP synthase/stimulator of interferon genes/Tank-binding kinase 1) innate immunity pathway is activated during human cytomegalovirus (HCMV) productive (lytic) replication in fully differentiated cells and during latency within incompletely differentiated myeloid cells. While multiple lytic-phase HCMV proteins neutralize steps along this pathway, none of them are expressed during latency. Here, we show that the latency-associated protein UL138 inhibits the cGAS/STING/TBK1 innate immunity pathway during transfections and infections, in fully differentiated cells and incompletely differentiated myeloid cells, and with loss of function and restoration of function approaches. UL138 inhibits the pathway downstream of STING but upstream of interferon regulatory factor 3 (IRF3) phosphorylation and NF-κB function and reduces the accumulation of interferon beta mRNA during both lytic and latent infections. IMPORTANCE While a cellular restriction versus viral countermeasure arms race between innate immunity and viral latency is expected, few examples have been documented. Our identification of the first HCMV latency protein that inactivates the cGAS/STING/TBK1 innate immune pathway opens the door to understanding how innate immunity, or its neutralization, impacts long-term persistence by HCMV and other latent viruses.

Interferons (IFNs) are one of the hallmarks of host antiviral immunity. IFNs exert their antiviral activities through the induction of IFN-stimulated genes (ISGs) and antiviral proteins; however, the mechanism by which ISGs inhibit adenovirus (Ad) replication is not clearly understood. IFNs repress Ad immediate early gene expression and, consequently, all subsequent aspects of the viral life cycle. In this study, we found that IFN-induced protein with tetratricopeptide repeats 3, IFIT3 (ISG60), restricts Ad replication. IFIT3 repressed Ad E1A immediate early gene expression but did not alter Ad genome entry into the nucleus. Expression of IFIT3 led to phosphorylation of TBK1, IRF3, and STAT1; increased expression of IFNβ and ISGs; and required IFIT1 and IFIT2 partner proteins. During RNA virus infections, it is known that IFIT3 stimulates IFN production through mitochondrial antiviral signaling (MAVS)-mediated activation of TBK1 which synergizes activation of IRF3 and NF-κB. MAVS or TBK1 depletion in cells expressing IFIT3 blocked IFN signaling and reversed the Ad replication restriction. In addition, STING depletion phenocopied the effect suggesting that IFIT3 activates the STING pathway with cross talk to the MAVS pathway. This occurs independently of viral pathogen-associated molecular patterns (PAMPs). These results demonstrate that the expression of a single ISG, IFIT3, activates IFN signaling and establishes a cellular antiviral state independent of viral PAMPs. IMPORTANCE IFITs belong to a family of IFN-induced proteins that have broad antiviral functions, primarily studied with RNA viruses leaving a gap of knowledge on the effects of these proteins on DNA viruses. In this study we show that IFIT3, with its partner proteins IFIT1 and IFIT2, specifically restricts replication of human Ad, a DNA virus, by stimulating IFNβ production via the STING and MAVS pathways. This effect enhanced the IFN response and is independent of viral PAMPs. These results reveal a novel mechanism of activation of IFN signaling to enhance cellular antiviral responses.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity., Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients., Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses., Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1β, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-β expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections., Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-β expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects., Competing Interests: MH reports personal fees from Astra Zeneca, personal fees from Novartis, outside the submitted work. CP reports grants and personal fees from AZ, grants and personal fees from Novartis, grants and personal fees from GlaxoSmithKline, grants and personal fees from TEVA, grants and personal fees from GSK, grants and personal fees from Chiesi, grants from Pharmaxis, grants and personal fees from Sanofi, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nieto-Fontarigo, Tillgren, Cerps, Sverrild, Hvidtfeldt, Ramu, Menzel, Sander, Porsbjerg and Uller.)

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-β in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Chen et al.)