HPV vaccines induce trained immunity and modulate pro-inflammatory cytokine expression in response to secondary Toll-like receptor stimulations.

Cervical cancer is caused mostly by human papillomavirus (HPV), and several HPV vaccines have been developed to prevent its onset. Vaccines include antigens as well as adjuvants, with adjuvants playing an important role in activating the innate immune responses necessary for inducing adaptive immunological responses. Recent research has shown the presence of trained immunity inside the innate immune system. However, trained immunity conferred by HPV vaccinations is not well understood. In this work, we explored the innate immune responses and trained immunity caused by two HPV vaccines, Cervarix and Gardasil. Cervarix includes monophosphoryl lipid A and an aluminum adjuvant, and it significantly increased the expression of IL-6 and IFN-ß mRNAs in RAW264.7 cells. On the contrary, Gardasil, which only includes an aluminum adjuvant, exhibited little cytokine expression but increased the expression of TLRs. Furthermore, Cervarix significantly increased IL-1ß secretion from mouse macrophages, while Gardasil only mildly induced IL-1ß secretion. Interestingly, initial stimulation with Gardasil enhanced the expression of IL-6 and TNF-α mRNAs upon secondary stimulation with TLR ligands, indicating that Gardasil induced trained immunity in macrophages. Moreover, Gardasil injection into mice resulted in enhanced TNF-α production in sera following secondary TLR stimulation. Our findings suggest that HPV vaccinations have the ability to induce trained immunity that modulate TLR ligand responses.

K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response.

Type I interferons (IFNs) exhibit strong antiviral activity and induce the expression of antiviral proteins. Since excessive expression of type I IFNs is harmful to the host, their expression should be turned off at the appropriate time. In this study, we find that post-translational modification of LGP2, a member of the RIG-I-like receptor family, modulates antiviral innate immune responses. The LGP2 protein undergoes K63-linked polyubiquitination in response to cytoplasmic double-stranded RNAs or viral infection. Our mass spectrometry analysis reveals the K residues ubiquitinated by the Riplet ubiquitin ligase. LGP2 ubiquitination occurs with a delay compared to RIG-I ubiquitination. Interestingly, ubiquitination-defective LGP2 mutations increase the expression of type I IFN at a late phase, whereas the mutant proteins attenuate other antiviral proteins, such as SP100, PML, and ANKRD1. Our data indicate that delayed polyubiquitination of LGP2 fine-tunes RIG-I-dependent antiviral innate immune responses at a late phase of viral infection.

Circulating extracellular vesicle microRNAs associated with adverse reactions, proinflammatory cytokine, and antibody production after COVID-19 vaccination.

mRNA-based vaccines have been used globally to eradicate the coronavirus-disease 2019 (COVID-19) pandemic. Vaccine efficacy and adverse reactions depend on immune responses, such as proinflammatory cytokine production and lymphocyte activation. We conducted a prospective cohort study to investigate relationships among specific antibody titers, adverse reactions, proinflammatory cytokine production, and immune-regulatory microRNA (miRNA) levels in serum extracellular vesicles (EVs) after COVID-19 vaccination (BNT162b2). Local adverse reactions after the second dose, such as local pain and swelling, were less correlated with those of systemic symptoms, such as fever and muscle pain, whereas serum TNF-α levels were associated with systemic adverse reactions and with specific antibody titers. Interestingly, EV miR-92a-2-5p levels in sera were negatively correlated with degrees of adverse reactions, and EV miR-148a levels were associated with specific antibody titers. Our data suggest a potential of circulating EV miRNAs as biomarkers for vaccine efficacy and adverse reactions.

TICAM-1/TRIF associates with Act1 and suppresses IL-17 receptor-mediated inflammatory responses.

TICAM-1 (also called TRIF) is the sole adaptor of TLR3 that recognizes double-stranded RNA. Here, we report that TICAM-1 is involved not only in TLR3 signaling but also in the cytokine receptor IL-17RA signaling. We found that TICAM-1 bound to IL-17R adaptor Act1 to inhibit the interaction between IL-17RA and Act1. Interestingly, TICAM-1 knockout promoted IL-17RA/Act1 interaction and increased IL-17A-mediated activation of NF-κB and MAP kinases, leading to enhanced expression of inflammatory cytokines and chemokines upon IL-17A stimulation. Moreover, Ticam-1 knockout augmented IL-17A-mediated CXCL1 and CXCL2 expression in vivo, resulting in accumulation of myeloid cells. Furthermore, Ticam-1 knockout enhanced delayed type hypersensitivity and exacerbated experimental autoimmune encephalomyelitis. Ticam-1 knockout promoted accumulation of myeloid and lymphoid cells in the spinal cord of EAE-induced mice. Collectively, these data indicate that TICAM-1 inhibits the interaction between IL-17RA and Act1 and functions as a negative regulator in IL-17A-mediated inflammatory responses.

Export of RNA-derived modified nucleosides by equilibrative nucleoside transporters defines the magnitude of autophagy response and Zika virus replication.

RNA contains a wide variety of posttranscriptional modifications covalently attached to its base or sugar group. These modified nucleosides are liberated from RNA molecules as the consequence of RNA catabolism and released into extracellular space, but the molecular mechanism of extracellular transport and its pathophysiological implications have been unclear. In the present study, we discovered that RNA-derived modified nucleosides are exported to extracellular space through equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2), with ENT1 showing higher preference for modified nucleosides than ENT2. Pharmacological inhibition or genetic deletion of ENT1 and ENT2 significantly attenuated export of modified nucleosides thereby resulting in their accumulation in cytosol. Using mutagenesis strategy, we identified an amino acid residue in ENT1 that is involved in the discrimination of unmodified and modified nucleosides. In ENTs-deficient cells, the elevated levels of intracellular modified nucleosides were closely associated with an induction of autophagy response as evidenced by increased LC3-II level. Importantly, we performed a screening of modified nucleosides capable of inducing autophagy and found that 1-methylguanosine (m1G) was sufficient to induce LC3-II levels. Pathophysiologically, defective export of modified nucleosides drastically induced Zika virus replication in an autophagy-dependent manner. In addition, we also found that pharmacological inhibition of ENTs by dilazep significantly induced Zika virus replication. Collectively, our findings highlight RNA-derived modified nucleosides as important signaling modulators that activate autophagy response and indicate that defective export of these modified nucleoside can have profound consequences for pathophysiology.

RIG-I-Like Receptor-Mediated Recognition of Viral Genomic RNA of Severe Acute Respiratory Syndrome Coronavirus-2 and Viral Escape From the Host Innate Immune Responses.

RIG-I-like receptors (RLR), RIG-I and MDA5, are cytoplasmic viral RNA sensors that recognize viral double-stranded RNAs and trigger signals to induce antiviral responses, including type I interferon production. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the coronavirus disease 2019 pandemic. However, the RLR role in innate immune response to SARS-CoV-2 has not been fully elucidated. Here, we studied the roles of RLR in cytokine expression responding to SARS-CoV-2 and found that not only MDA5 but also RIG-I are involved in innate immune responses in some types of human cells. Transfection of total RNAs extracted from SARS-CoV-2-infected cells into epithelial cells induced IFN-ß, IP-10, and Ccl5 mRNA expression. The cytokine expression was reduced by knockout of either RIG-I or MDA5, suggesting that both proteins are required for appropriate innate immune response to SARS-CoV-2. Two viral genomic RNA regions strongly induced type I IFN expression, and a 200-base fragment of viral RNA preferentially induced type I IFN in a RIG-I-dependent manner. In contrast, SARS-CoV-2 infectious particles hardly induced cytokine expression, suggesting viral escape from the host response. Viral 9b protein inhibited RIG-I and MAVS interaction, and viral 7a protein destabilized the TBK1 protein, leading to attenuated IRF-3 phosphorylation required for type I IFN expression. Our data elucidated the mechanism underlying RLR-mediated response to SARS-CoV-2 infection and viral escape from the host innate immune response.

miR-451a levels rather than human papillomavirus vaccine administration is associated with the severity of murine experimental autoimmune encephalomyelitis.

Human papilloma virus (HPV) vaccine is currently the most effective prophylaxis to prevent cervical cancer. However, concerns regarding its potential severe adverse reactions have limited the vaccination rate. HPV vaccines have been determined to contain adjuvants which induce inflammation by the innate immune system and are crucial for triggering adaptive immunity. MicroRNA-451a (miR-451a) is located within circulating extracellular vesicles (EVs) and regulates the innate immune response. In this study, we examined the effect of HPV vaccines and EV miR-451a on murine experimental autoimmune encephalomyelitis (EAE), which is an autoimmune disorder that affects the central nervous system. Although HPV vaccine induced pro-inflammatory cytokine expression and macrophage cell death, it failed to exacerbate mouse EAE, whereas circulating EV miR-451a levels were associated with the severity of EAE. Since miR-451a knockout exhibited only marginal effect on the murine EAE clinical score, our data suggest that miR-451a levels reflect an unknown condition associated with EAE severity. Interestingly, excessive uptake of glucose increased EV miR-451a levels both in vitro and in vivo and also exacerbated mouse EAE. Therefore, environmental factors that increase EV miR-451a levels exacerbate the autoimmune disorder more than the HPV vaccine. These observations provide evidence for the safety of HPV vaccines.

Aging-Associated Extracellular Vesicles Contain Immune Regulatory microRNAs Alleviating Hyperinflammatory State and Immune Dysfunction in the Elderly.

Aging-associated changes in the immune system often lead to immune dysfunction; however, the mechanisms that underlie this phenomenon have yet to be fully elucidated. This study found that the microRNA-192 (miR-192) is an aging-associated immune regulatory microRNA whose concentration was significantly increased in aged extracellular vesicles (EVs) due to the hyperinflammatory state of aged mice. Interestingly, EV miR-192 exhibited anti-inflammatory effects on macrophages. In our aged mouse model, aging was associated with prolonged inflammation in the lung upon stimulation with inactivated influenza whole virus particles (WVP), whereas EV miR-192 alleviated the prolonged inflammation associated with aging. The hyperinflammatory state of aged mice resulted in reduced production of specific antibodies and efficacy of vaccination with WVP; however, EV miR-192 attenuated this hyperinflammatory state and improved vaccination efficacy in aged mice. Our data indicate that aged EVs constitute a negative feedback loop that alleviates aging-associated immune dysfunction.

Cytoplasmic dsRNA induces the expression of OCT3/4 and NANOG mRNAs in differentiated human cells.

Cytoplasmic dsRNA is recognized by RNA helicase RIG-I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), triggering induction of the innate immune response via the mitochondrial antiviral signaling protein (MAVS). In contrast, extracellular dsRNA is internalized into endosomes and recognized by Toll-like receptor 3 (TLR3), which triggers signaling via the Toll-like receptor adaptor molecule 1 (TICAM-1). Poly(I:C) is a synthetic dsRNA analog and increases the expression of octamer-binding protein 3/4 (OCT3/4), NANOG, and SRY-box (SOX) mRNAs during pluripotency induction. However, the mechanism underlying this increase is unclear. Here, we focused on the mechanism of poly(I:C)-induced expression of stem cell-specific genes in human somatic cells. Addition of poly(I:C) to human fibroblast culture medium did not increase OCT3/4 mRNA expression, but poly(I:C) transfection markedly increased OCT3/4 expression and induced nuclear localization of the OCT3/4 protein, implying that not TLR3, but RIG-I and MDA5 are required for OCT3/4 expression. Moreover, although cytoplasmic dsRNA increased OCT3/4 mRNA, cytoplasmic dsDNAs, such as salmon sperm DNA and poly(dA:dT), did not. Interestingly, the expression of NANOG, SOX2, Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc was also increased by cytoplasmic dsRNA. Of note, siRNAs that silenced MAVS and interferon regulatory factor 1 (IRF1) expression reduced OCT3/4 levels after stimulation with poly(I:C); however, an NF-κB inhibitor and siRNA-mediated knockdown of proto-oncogene c-Jun did not significantly reduce the mRNA levels. We conclude that cytoplasmic dsRNA increases the expression of stem cell-specific genes in human somatic cells in a MAVS- and IRF1-dependent manner.

Attenuation of the Innate Immune Response against Viral Infection Due to ZNF598-Promoted Binding of FAT10 to RIG-I.

Excessive innate immune response is harmful to the host, and aberrant activation of the cytoplasmic viral RNA sensors RIG-I and MDA5 leads to autoimmune disorders. ZNF598 is an E3 ubiquitin ligase involved in the ribosome quality control pathway. It is also involved in the suppression of interferon (IFN)-stimulated gene (ISG) expression; however, its underlying mechanism is unclear. In this study, we show that ZNF598 is a negative regulator of the RIG-I-mediated signaling pathway, and endogenous ZNF598 protein binds to RIG-I. ZNF598 ubiquitin ligase activity is dispensable for the suppression of RIG-I signaling. Instead, ZNF598 delivers a ubiquitin-like protein FAT10 to the RIG-I protein, resulting in the inhibition of RIG-I polyubiquitination, which is required for triggering downstream signaling to produce type I IFN. Moreover, ZNF598-mediated suppression is abrogated by FAT10 knockout. Our data elucidate the mechanism by which ZNF598 inhibits RIG-I-mediated innate immune response.

Immune-regulatory microRNA expression levels within circulating extracellular vesicles correspond with the appearance of local symptoms after seasonal flu vaccination.

Extracellular vesicles (EVs) contain microRNAs (miRNAs) that regulate the innate immune responses, such as the production of pro-inflammatory cytokines. The excessive production of pro-inflammatory cytokines after vaccination can cause local adverse reactions, such as pain, itching, swelling, and redness. Previous studies have shown that circulating EV miR-451a regulates innate immune responses, and miR-451a levels in serum EVs are negatively correlated with the pro-inflammatory cytokine expression levels in response to the influenza vaccine. Since excessive pro-inflammatory cytokine production is a cause of the local adverse reactions to vaccination, we investigated whether miR-451a levels in serum EVs correlate with local symptoms at the vaccination site, such as pain, itching, swelling, and redness. Interestingly, miR-451a levels in serum EVs were inversely correlated with the number of symptoms after vaccination. We determined the level of several other immune-regulatory miRNAs in serum EVs. Using the immune-regulatory miRNA levels of miR-22, miR-29a, miR-451a, and miR-107, we calculated a normalized miRNA level for each healthy donor and found that the normalized miRNA levels were significantly correlated with the number of local symptoms after vaccination. Our data indicated that immune-regulatory miRNA levels in serum EVs can be used as biomarkers to assess local symptoms after influenza vaccination.

MicroRNA-451a in extracellular, blood-resident vesicles attenuates macrophage and dendritic cell responses to influenza whole-virus vaccine.

The innate immune system is important for the efficacy of vaccines, but excessive innate immune responses can cause adverse reactions after vaccination. Extracellular vesicles (EVs) are enriched in the blood and can deliver functional RNAs, such as microRNAs (miRNAs), to recipient cells, thereby mediating intercellular communication. However, the role of EVs in controlling the innate immune responses to vaccines has not been fully elucidated. Here, we found that miR-451a is abundant in human serum EVs and that its presence in blood-circulating EVs affects the innate immune responses of macrophages and dendritic cells to inactivated whole-virus vaccines (WV) against influenza. miR-451a in human serum EVs was stable for a week in healthy subjects, and its levels gradually fluctuated over several months. miR-451a within serum EVs was internalized into serum-cultured macrophages and dendritic cells and reduced endogenous 14-3-3ζ protein levels and decreased the expression of type I IFN and interleukin 6 in response to WV stimulation. miR-451a levels in blood-circulating EVs were positively correlated with intracellular miR-451a levels in mouse splenic CD11c+ cells and inversely correlated with the innate immune response to inactivated WV in vivo These findings suggest that miR-451a in circulating EVs is internalized into recipient cells in vivo and that this internalization results in an attenuation of the innate immune response to WV. Moreover, a microarray analysis identified several other miRNAs that affect the macrophage response to inactivated WV. Our results reveal that miRNAs in circulating EVs significantly modify the responses of macrophages and dendritic cells to inactivated WV.

Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells.

Pattern-recognition receptors (PRRs) recognizes viral RNAs and trigger the innate immune responses. Toll-like receptor 3 (TLR3), a PRR, recognizes viral double-stranded RNA (dsRNA) in endolysosomes, whereas cytoplasmic dsRNA is sensed by another PRR, MDA5. TLR3 and MDA5 utilize TICAM-1 and MAVS, respectively, to trigger the signal for inducing innate immune responses. Extracellular vesicles (EVs) include the exosomes and microvesicles; an accumulating body of evidence has shown that EVs delivers functional RNA, such as microRNAs (miRNAs), to other cells and thus mediate intercellular communications. Therefore, EVs carrying miRNAs affect innate immune responses in macrophages and dendritic cells. However, the mechanism underlying the regulation of miRNA levels in EVs remains unclear. To elucidate the mechanism, we sought to reveal the pathway that control miRNA expression levels in EVs. Here, we found that TLR3 stimulation increased miR-21 levels in EVs released from various types of human cells. Ectopic expression of the TLR3 adaptor, TICAM-1, increased miR-21 levels in EVs but not intracellular miR-21 levels, suggesting that TICAM-1 augmented sorting of miR-21 to EVs. In contrast, the MDA5 adaptor, MAVS, did not increase miR-21 levels in EVs. The siRNA for TICAM-1 reduced EV miR-21 levels after stimulation of TLR3. Collectively, our data indicate a novel role of the TLR3-TICAM-1 pathway in controlling miR-21 levels in EVs.

Aureobasidium pullulans-cultured fluid induces IL-18 production, leading to Th1-polarization during influenza A virus infection.

Several microbial molecules with pathogen-associated molecular patterns stimulate host innate immune responses. The innate immune system plays a crucial role in activating acquired immune response via cytokine production and antigen presentation. Previous studies have shown that Aureobasidium pullulans-cultured fluid (AP-CF), which contains ß-glucan, exhibits adjuvant activity and renders mice resistance to influenza A virus infection; however, the underlying mechanism remains elusive. In this study, we investigated the innate immune response to AP-CF. We found that intraperitoneal administration of AP-CF increased the serum level of IL-18 and the number of splenic IFN-γ producing CD4+ cells during influenza A virus infection. The adjuvant effect of AP-CF was distinct from that of alum, which is known to have the ability to stimulate a Th2 immune response. In addition, AP-CF injection barely increased the number of peritoneal neutrophils and inflammatory macrophages, whereas alum injection markedly increased the number of neutrophils and inflammatory macrophages, suggesting that AP-CF is a weak inducer of inflammation compared to alum. AP-CF induced IL-18 production by DC2.4 cells, a dendritic cell line, and by peritoneal exudate cells that include peritoneal macrophages. Collectively, our findings indicate that AP-CF is an adjuvant that promotes the Th1 response during influenza A virus infection.

Zyxin stabilizes RIG-I and MAVS interactions and promotes type I interferon response.

RIG-I and MDA5 are cytoplasmic viral RNA sensors that belong to the RIG-I-like receptors (RLRs), which induce antiviral innate immune responses, including the production of type I interferon and other pro-inflammatory cytokines. After recognition of viral RNA, the N-terminal caspase activation and recruitment domains (CARDs) of RIG-I and MDA5 bind to a CARD in the MAVS adaptor molecule, resulting in MAVS oligomerization and downstream signaling. To reveal the molecular mechanism of MAVS-dependent signaling, we performed a yeast two-hybrid screening and identified zyxin as a protein that binds to MAVS. Zyxin co-immunoprecipitated with MAVS in human cells. A proximity ligation assay showed that zyxin and MAVS partly co-localized on mitochondria. Ectopic expression of zyxin augmented MAVS-mediated IFN-ß promoter activation, and knockdown of zyxin (ZYX) attenuated the IFN-ß promoter activation. Moreover, ZYX knockdown reduced the expression of type I IFN and an interferon-inducible gene after stimulation with polyI:C or influenza A virus RNA. Interestingly, physical interactions between RLRs and MAVS were abrogated by ZYX knockdown. These observations indicate that zyxin serves as a scaffold for the interactions between RLRs and MAVS.

Recognition of Viral RNA by Pattern Recognition Receptors in the Induction of Innate Immunity and Excessive Inflammation During Respiratory Viral Infections.

The innate immune system is the first line of defense against virus infection that triggers the expression of type I interferon (IFN) and proinflammatory cytokines. Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns, resulting in the induction of innate immune responses. Viral RNA in endosomes is recognized by Toll-like receptors, and cytoplasmic viral RNA is recognized by RIG-I-like receptors. The host innate immune response is critical for protection against virus infection. However, it has been postulated that an excessive inflammatory response in the lung caused by the innate immune response is harmful to the host and is a cause of lethality during influenza A virus infection. Although the deletion of genes encoding PRRs or proinflammatory cytokines does not improve the mortality of mice infected with influenza A virus, a partial block of the innate immune response is successful in decreasing the mortality rate of mice without a loss of protection against virus infection. In addition, morbidity and mortality rates are influenced by other factors. For example, secondary bacterial infection increases the mortality rate in patients with influenza A virus and in animal models of the disease, and environmental factors, such as cigarette smoke and fine particles, also affect the innate immune response. In this review, we summarize recent findings related to the role of PRRs in innate immune response during respiratory viral infection.

Extracellular Vesicles Deliver Host and Virus RNA and Regulate Innate Immune Response.

The innate immune system plays a crucial role in controlling viral infection. Pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-I-like receptors, sense viral components called pathogen-associated molecular patterns (PAMPs) and trigger signals to induce innate immune responses. Extracellular vesicles (EVs), including exosomes and microvesicles, deliver functional RNA and mediate intercellular communications. Recent studies have revealed that EVs released from virus-infected cells deliver viral RNA to dendritic cells and macrophages, thereby activating PRRs in recipient cells, which results in the expression of type I interferon and pro-inflammatory cytokines. On the other hand, EVs transfer not only viral RNA but also host microRNAs to recipient cells. Recently, infection of hepatocytes with hepatitis B virus (HBV) was shown to affect microRNA levels in EVs released from virus-infected cells, leading to attenuation of host innate immune response. This suggests that the virus utilizes the EVs and host microRNAs to counteract the antiviral innate immune responses. In this review, we summarize recent findings related to the role of EVs in antiviral innate immune responses.

Regulation of RIG-I Activation by K63-Linked Polyubiquitination.

RIG-I is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded RNA (dsRNA). Influenza A virus, hepatitis C virus, and several other pathogenic viruses are mainly recognized by RIG-I, resulting in the activation of the innate immune responses. The protein comprises N-terminal two caspase activation and recruitment domains (2CARDs), an RNA helicase domain, and the C-terminal domain (CTD). The CTD recognizes 5'-triphosphate viral dsRNA. After recognition of viral dsRNA, the protein harbors K63-linked polyubiquitination essential for RIG-I activation. First, it was reported that TRIM25 ubiquitin ligase delivered K63-linked polyubiquitin moiety to the 2CARDs. The polyubiquitin chain stabilizes a structure called the 2CARD tetramer, in which four 2CARDs assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (MAVS) protein on mitochondria. MAVS aggregation then triggers the signal to induce the innate immune responses. However, subsequent studies have reported that Riplet, MEX3C, and TRIM4 ubiquitin ligases are also involved in K63-linked polyubiquitination and the activation of RIG-I. MEX3C and TRIM4 mediate polyubiquitination of the 2CARDs. By contrast, Riplet ubiquitinates the CTD. The physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. In this review, we summarize recent findings related to K63-linked polyubiquitination and propose a model that could reconcile current contradictory theories. We also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection.

Extracellular Vesicles Including Exosomes Regulate Innate Immune Responses to Hepatitis B Virus Infection.

The innate immune system is essential for controlling viral infection. Hepatitis B virus (HBV) persistently infects human hepatocytes and causes hepatocellular carcinoma. However, the innate immune response to HBV infection in vivo remains unclear. Using a tree shrew animal model, we showed that HBV infection induced hepatic interferon (IFN)-γ expression during early infection. Our in vitro study demonstrated that hepatic NK cells produced IFN-γ in response to HBV only in the presence of hepatic F4/80(+) cells. Moreover, extracellular vesicles (EVs) released from HBV-infected hepatocytes contained viral nucleic acids and induced NKG2D ligand expression in macrophages by stimulating MyD88, TICAM-1, and MAVS-dependent pathways. In addition, depletion of exosomes from EVs markedly reduced NKG2D ligand expression, suggesting the importance of exosomes for NK cell activation. In contrast, infection of hepatocytes with HBV increased immunoregulatory microRNA levels in EVs and exosomes, which were transferred to macrophages, thereby suppressing IL-12p35 mRNA expression in macrophages to counteract the host innate immune response. IFN-γ increased the hepatic expression of DDX60 and augmented the DDX60-dependent degradation of cytoplasmic HBV RNA. Our results elucidated the crucial role of exosomes in antiviral innate immune response against HBV. ACCESSION NUMBER: Accession number of RNA-seq data is DRA004164 (DRA in DDBJ).

Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response.

Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.