RNA sensing via the RIG‐I‐like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1‐deficient cells, unedited self RNAs form base‐paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG‐I‐like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi‐Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG‐I‐like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1‐deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5‐dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications.     

Inhibitor of IκB kinase activity,BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

P21 activated kinase‐1 (PAK1) in macrophages is required for promotion of Th17 cell response during helminth infection

CD4⁺T cells differentiate into distinct functional effector and inhibitory subsets are facilitated by distinct cytokine cues present at the time of antigen recognition. Maintaining a balance between T helper 17 (Th17) and regulatory T (Treg) cells are critical for the control of the immunopathogenesis of liver diseases. Here, by using the mouse model of helminth Schistosoma japonicum (S japonicum) infection, we show that the hepatic mRNA levels of P21‐activated kinase 1 (PAK1), a key regulator of the actin cytoskeleton, adhesion and cell motility, are significantly increased and associated with the development of liver pathology during S japonicum infection. In addition, PAK1‐deficient mice are prone to suppression of Th17 cell responses but increased Treg cells. Furthermore, PAK1 enhances macrophage activation through promoting IRF1 nuclear translocation in an NF‐κB‐dependent pathway, resulting in promoting Th17 cell differentiation through inducing IL‐6 production. These findings highlight the importance of PAK1 in macrophages fate determination and suggest that PAK1/IRF1 axis‐dependent immunomodulation can ameliorate certain T cell–based immune pathologies.     

Activation of the STING‐IRF3 pathway involved in psoriasis with diabetes mellitus

Psoriasis and type 2 diabetes mellitus (T2DM) share similar inflammatory pathways in their pathogenesis. The stimulator of interferon genes (STING)‐interferon regulatory factor 3 (IRF3) pathway has recently been shown to play an important role in immune and metabolic diseases. In this study, we investigated the activation of the STING‐IRF3 pathway in human immortalized keratinocytes (HaCaT) cells treated with palmitic acid (PA) and imiquimod (IMQ). Additionally, we detected the STING‐IRF3 pathway in diabetic mice with imiquimod (IMQ)‐induced psoriasis and assessed the potential of STING inhibitor C‐176. Furthermore, skin samples from patients with psoriasis and diabetes were collected for immunohistochemical analysis. The results indicated that the STING‐IRF3 pathway was activated in HaCaT cells. Moreover, the STING pathway was also found to be induced in the skin tissue of diabetic mice with psoriasis; the inflammatory responses were ameliorated by treatment with C‐176. In the skin tissue samples of patients with psoriasis and diabetes, immunohistochemistry showed that the expression levels of STING and phosphorylated IRF3 were also significantly increased. Thus, we conclude that the STING‐IRF3 pathway is involved in the inflammatory response in the manifestation of psoriasis with T2DM. Inhibition of the activation of the STING pathway can ameliorate the development of psoriasis in diabetes and could be targeted for the development of therapeutic agents for these conditions.     

RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.     

Epstein-Barr virus LF2: an antagonist to type I interferon

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway, which is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Despite its association with significant human health problems, activities of Epstein-Barr virus (EBV), a human tumor-inducing herpesvirus, to evade host IFN-mediated innate immunity have not been well characterized. To search for EBV genes that block IFN signal transduction, we carried out a screening of EBV open reading frames for their abilities to block IFN-α/β-mediated luciferase expression upon Sendai virus infection. This screening demonstrates that EBV LF2 tegument protein specifically interacts with the central inhibitory association domain of IRF7, and this interaction leads to inhibition of the dimerization of IRF7, which suppresses IFN-α production and IFN-mediated immunity. This demonstrates a novel immune evasion mechanism of EBV LF2 in blocking cellular IRF7-mediated innate immunity.     

Soluble Interleukin-6 Receptor-Mediated Innate Immune Response to DNA and RNA Viruses

The interleukin-6 (IL-6) receptor, which exists as membrane-bound and soluble forms, plays critical roles in the immune response. The soluble IL-6 receptor (sIL6R) has been identified as a potential therapeutic target for preventing coronary heart disease. However, little is known about the role of this receptor during viral infection. In this study, we show that sIL6R, but not IL-6, is induced by viral infection via the cyclooxygenase-2 pathway. Interestingly, sIL6R, but not IL-6, exhibited extensive antiviral activity against DNA and RNA viruses, including hepatitis B virus, influenza virus, human enterovirus 71, and vesicular stomatitis virus. No synergistic effects on antiviral action were observed by combining sIL6R and IL-6. Furthermore, sIL6R mediated antiviral action via the p28 pathway and induced alpha interferon (IFN-α) by promoting the nuclear translocation of IFN regulatory factor 3 (IRF3) and NF-κB, which led to the activation of downstream IFN effectors, including 2′,5′-oligoadenylate synthetase (OAS), double-stranded RNA-dependent protein kinase (PKR), and myxovirus resistance protein (Mx). Thus, our results demonstrate that sIL6R, but not IL-6, plays an important role in the host antiviral response.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease caused by both genetic and environmental factors. Genome scans in families with SLE point to multiple potential chromosomal regions that harbor SLE susceptibility genes, and association studies in different populations have suggested several susceptibility alleles for SLE. Increased production of type I interferon (IFN) and expression of IFN-inducible genes is commonly observed in SLE and may be pivotal in the molecular pathogenesis of the disease. We analyzed 44 single-nucleotide polymorphisms (SNPs) in 13 genes from the type I IFN pathway in 679 Swedish, Finnish, and Icelandic patients with SLE, in 798 unaffected family members, and in 438 unrelated control individuals for joint linkage and association with SLE. In two of the genes—the tyrosine kinase 2 (TYK2) and IFN regulatory factor 5 (IRF5) genes—we identified SNPs that displayed strong signals in joint analysis of linkage and association (unadjusted P<10^-7) with SLE. TYK2 binds to the type I IFN receptor complex and IRF5 is a regulator of type I IFN gene expression. Thus, our results support a disease mechanism in SLE that involves key components of the type I IFN system.     

Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1‐1, which contains a single WW domain, and YAP1‐2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF‐induced epithelial‐mesenchymal transition (EMT) in non‐small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1‐1 and YAP1‐2 isoforms—although when compared to YAP1‐1, YAP1‐2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF‐induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1‐1 stable expression cells exhibited a stronger migration ability than YAP1‐2 expressing cells. However, upon EGF treatment, YAP1‐2 stable cells showed more robust migration than YAP1‐1 expressing cells. The protein stability and nuclear localization of YAP1‐2 were preferentially enhanced with EGF treatment. Moreover, EGF‐induced EMT and YAP1‐2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1‐2 is the main isoform that is functionally relevant in promoting EGF‐induced EMT and ultimately NSCLC progression.     

GSK3α, not GSK3β, drives hippocampal NMDAR‐dependent LTD via tau‐mediated spine anchoring

Glycogen synthase kinase‐3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3β, simply because of its prevalent expression in the brain. Consequently, the importance of isoform‐specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor‐dependent long‐term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock‐down in mouse hippocampus and with novel isoform‐selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule‐binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α‐induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3β in this process.     

pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC‐I antigen presentation and stress granule signaling are enhanced in IRGM‐deficient cells, indicating a robust cell‐intrinsic antiviral immune state. Consistently, IRGM‐depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS‐CoV‐2, CHIKV, and Zika virus.