Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca(2+) level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca(2+) did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca(2+) flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice.

The gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here, we found that disruption of nasal bacteria by intranasal application of antibiotics before influenza virus infection enhanced the virus-specific antibody response in a MyD88-dependent manner. Similarly, disruption of nasal bacteria by lysozyme enhanced antibody responses to intranasally administered influenza virus hemagglutinin (HA) vaccine in a MyD88-dependent manner, suggesting that intranasal application of antibiotics or lysozyme could release bacterial pathogen-associated molecular patterns (PAMPs) from disrupted nasal bacteria that act as mucosal adjuvants by activating the MyD88 signaling pathway. Since commensal bacteria in the nasal mucosal surface were significantly lower than those in the oral cavity, intranasal administration of HA vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to an intranasally administered HA vaccine. Finally, we demonstrated that oral bacteria combined with an intranasal vaccine protect from influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our results reveal the role of nasal bacteria in the induction of the virus-specific adaptive immunity and provide clues for developing better intranasal vaccines. IMPORTANCE Intranasal vaccination induces the nasal IgA antibody which is protective against respiratory viruses, such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, understanding how mucosal immune responses are elicited following viral infection is important for developing better vaccines. Here, we focused on the role of nasal commensal bacteria in the induction of immune responses following influenza virus infection. To deplete nasal bacteria, we intranasally administered antibiotics to mice before influenza virus infection and found that antibiotic-induced disruption of nasal bacteria could release bacterial components which stimulate the virus-specific antibody responses. Since commensal bacteria in nasal mucosa were significantly lower than those in the oral cavity, intranasal administration of split virus vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to the intranasally administered vaccine. Therefore, both integrity and amounts of nasal bacteria may be critical for an effective intranasal vaccine.

In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO2 (4-8 nm) and CuxO (1-2 nm or less). Their extraordinarily small size leads to high dispersity and good optical transparency, besides large active surface area. Those photocatalysts can be applied to white and translucent latex paints. Although Cu2O clusters involved in the paint coating undergo gradual aerobic oxidation in the dark, the oxidized clusters are re-reduced under > 380 nm light. The paint coating inactivated the original and alpha variant of novel coronavirus under irradiation with fluorescent light for 3 h. The photocatalysts greatly suppressed binding ability of the receptor binding domain (RBD) of coronavirus (the original, alpha and delta variants) spike protein to the receptor of human cells. The coating also exhibited antivirus effects on influenza A virus, feline calicivirus, bacteriophage Qβ and bacteriophage M13. The photocatalysts would be applied to practical coatings and lower the risk of coronavirus infection via solid surfaces.

Plasmacytoid dendritic cells (pDC) sense viral RNA through toll-like receptor 7 (TLR7), form self-adhesive pDC-pDC clusters, and produce type I interferons. This cell adhesion enhances type I interferon production, but little is known about the underlying mechanisms. Here we show that MyD88-dependent TLR7 signaling activates CD11a/CD18 integrin to induce microtubule elongation. TLR7+ lysosomes then become linked with these microtubules through the GTPase Arl8b and its effector SKIP/Plekhm2, resulting in perinuclear to peripheral relocalization of TLR7. The type I interferon signaling molecules TRAF3, IKKα, and mTORC1 are constitutively associated in pDCs. TLR7 localizes to mTORC1 and induces association of TRAF3 with the upstream molecule TRAF6. Finally, type I interferons are secreted in the vicinity of cell-cell contacts between clustered pDCs. These results suggest that TLR7 needs to move to the cell periphery to induce robust type I interferon responses in pDCs.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6-2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Influenza virus infection is recognized by the innate immune system through Toll like receptor (TLR) 7 and retinoic acid inducible gene I. These two recognition pathways lead to the activation of type I interferons and resistance to infection. In addition, TLR signals are required for the CD4 T cell and IgG2a, but not cytotoxic T lymphocyte, responses to influenza virus infection. In contrast, the role of NOD-like receptors (NLRs) in viral recognition and induction of adaptive immunity to influenza virus is unknown. We demonstrate that respiratory infection with influenza virus results in the activation of NLR inflammasomes in the lung. Although NLRP3 was required for inflammasome activation in certain cell types, CD4 and CD8 T cell responses, as well as mucosal IgA secretion and systemic IgG responses, required ASC and caspase-1 but not NLRP3. Consequently, ASC, caspase-1, and IL-1R, but not NLRP3, were required for protective immunity against flu challenge. Furthermore, we show that caspase-1 inflammasome activation in the hematopoietic, but not stromal, compartment was required to induce protective antiviral immunity. These results demonstrate that in addition to the TLR pathways, ASC inflammasomes play a central role in adaptive immunity to influenza virus.

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Immune responses to vaccines require direct recognition of pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs) on dendritic cells (DCs). Unlike vaccination, infection by a live pathogen often impairs DC function and inflicts additional damage on the host. Here we found that after infection with live influenza A virus, signaling through the interleukin 1 receptor (IL-1R) was required for productive priming of CD8(+) T cells, but signaling through the PRRs TLR7 and RIG-I was not. DCs activated by IL-1 in trans were both required and sufficient for the generation of virus-specific CD8(+) T cell immunity. Our data demonstrate a critical role for a bystander cytokine in the priming of CD8(+) T cells during infection with a live virus.

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via β-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

The AIM2 inflammasome is activated by DNA, leading to caspase-1 activation and release of pro-inflammatory cytokines interleukin 1β (IL-1β) and IL-18, which are critical mediators in host innate immune responses against various pathogens. Some viruses employ strategies to counteract inflammasome-mediated induction of pro-inflammatory cytokines, but their in vivo relevance is less well understood. Here we show that the herpes simplex virus 1 (HSV-1) tegument protein VP22 inhibits AIM2-dependent inflammasome activation. VP22 interacts with AIM2 and prevents its oligomerization, an initial step in AIM2 inflammasome activation. A mutant virus lacking VP22 (HSV-1ΔVP22) activates AIM2 and induces IL-1β and IL-18 secretion, but these responses are lost in the absence of AIM2. Additionally, HSV-1ΔVP22 infection results in diminished viral yields in vivo, but HSV-1ΔVP22 replication is largely restored in AIM2-deficient mice. Collectively, these findings reveal a mechanism of HSV-1 evasion of the host immune response that enables efficient viral replication in vivo.