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.

Glycoprotein B (gB) is one of the essential components for infection by herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with glycoprotein D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have been identified, specific molecules that mediate HSV-1 infection by associating with gB have not been elucidated. Here, we found that paired immunoglobulin-like type 2 receptor (PILR) alpha associates with gB, and cells transduced with PILRalpha become susceptible to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing both HVEM and PILRalpha was blocked by either anti-PILRalpha or anti-HVEM antibody. Our results demonstrate that cellular receptors for both gB and gD are required for HSV-1 infection and that PILRalpha plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.

Cyclic phosphatidic acid (cPA) has an inhibitory effect on the autotaxin (ATX)/lysophosphatidic acid (LPA) axis, which has been implicated to play an important role in the progression of fibrosis in systemic sclerosis (SSc). The purpose of this study is to assess the antifibrotic activity of cPA for the treatment of SSc using SSc skin fibroblasts and an animal model of bleomycin-induced skin fibrosis.

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

HIV-1 latent reservoirs harbouring silenced but replication-competent proviruses are a major obstacle against viral eradication in infected patients. The "shock and kill" strategy aims to reactivate latent provirus with latency reversing agents (LRAs) in the presence of antiretroviral drugs, necessitating the development of effective and efficient LRAs. We screened a chemical library for potential LRAs and identified two dual Polo-like kinase (PLK)/bromodomain inhibitors, BI-2536 and BI-6727 (volasertib), which are currently undergoing clinical trials against various cancers. BI-2536 and BI-6727 significantly reactivated silenced HIV-1 provirus at both the mRNA and protein level in two latently infected model cell lines (ACH2 and U1). BI-2536 dramatically reactivated transcription of latent HIV-1 provirus in peripheral blood mononuclear cells derived from infected patients. Long terminal repeat activation by the inhibitors was associated with bromodomain rather than PLK inhibition. We also found that BI-2536 synergistically activates the latent provirus in combination with SAHA, a histone deacetylase inhibitor, or the non-tumour-promoting phorbol ester prostratin. Our findings strongly suggest that BI-2536 and BI-6727 are potent LRAs for the "shock and kill" HIV-1 eradication strategy.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Systemic sclerosis (SSc; scleroderma) is an autoimmune connective tissue disease that affects the skin and subcutaneous tissue, in addition to the internal organs of the whole body. Onset in childhood is uncommon; however, both patients with childhood-onset and adult-onset SSc are positive for anti-nuclear antibodies (ANAs).Detection of SSc-related anti-nuclear antibodies is often useful for predicting clinical features, disease course, and outcomes.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress major histocompatibility complex class I (MHC-I) expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 but not influenza virus infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC-I, the Omicron subvariants exhibited a greater ability to suppress surface MHC-I expression. We identified a common mutation in the E protein of Omicron that further suppressed MHC-I expression. Collectively, our data suggest that in addition to escaping from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition.

Phosphodiesterase (PDE) 4 inhibitors have been reported to suppress the progression of dermal fibrosis in patients with systemic sclerosis (SSc); however, the precise mechanisms remain to be elucidated. Therefore, we conducted experiments focusing on the antifibrotic and anti-inflammatory effects of apremilast using dermal fibroblasts derived from patients with SSc and an SSc mouse model. Dermal fibroblasts derived from healthy controls and patients with SSc were incubated with apremilast in the presence or absence of 10 ng/ml transforming growth factor (TGF)-β1 for the measurement of intracellular cAMP levels and evaluation of mRNA and protein expression. A bleomycin-induced dermal fibrosis mouse model was used to evaluate the inhibitory effects of apremilast on the progression of dermal fibrosis. Intracellular cAMP levels were significantly reduced in dermal fibroblasts derived from patients with SSc compared with those derived from healthy controls. Apremilast reduced the mRNA expression of profibrotic markers and the protein expression of type I collagen and Cellular Communication Network Factor 2 (CCN2) in dermal fibroblasts. Additionally, apremilast inhibited the progression of dermal fibrosis in mice, partly by acting on T cells. These results suggest that apremilast may be a potential candidate for treating dermal fibrosis in SSc.

Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.

Accumulating evidence has shown that several non-HLA genes are involved in the susceptibility to polymyositis/dermatomyositis. This study aimed to investigate the involvement of C8orf13-BLK, one of the strongest candidate genes for autoimmune diseases, in susceptibility to polymyositis/dermatomyositis in the Japanese population. A possible gene-gene interaction between C8orf13-BLK and STAT4, which we recently showed to be associated with Japanese polymyositis/dermatomyositis, was also analyzed.

Systemic sclerosis (SSc) is a multi-organ fibrotic disease that affects the skin and various internal organs. Therapeutic strategies for tissue fibrosis have not been established; however, aberrantly activated fibroblasts in affected lesions are key targets for modulating fibrosis. Recently, increased intracellular cyclic GMP (cGMP) levels were demonstrated to improve fibrosis levels in various diseases. The purpose of this study was to assess the anti-fibrotic properties of cGMP in cultured fibroblasts from patients with SSc. The phosphodiesterase (PDE) 5 inhibitor sildenafil increased the intracellular cGMP levels in skin fibroblasts in a dose-dependent manner. Sildenafil treatment also significantly decreased the expression of several pro-fibrotic factors that were upregulated by TGF-β1 treatment in SSc skin fibroblasts. These inhibitory effects occurred via non-canonical TGF-β signaling. Our findings revealed that sildenafil might be a novel strategy to treat tissue fibrosis and vasculopathy in SSc.

The herpes simplex virus 1 (HSV-1) UL7 gene is highly conserved among herpesviridae. Since the construction of recombinant HSV-1 with a mutation in the UL7 gene has not been reported, the involvement of HSV-1 UL7 in viral replication has been unclear. In this study, we succeeded in generating a UL7 null HSV-1 mutant virus, MT102, and characterized it. Our results were as follows. (i) In Vero cells, MT102 was replication-competent, but formed smaller plaques and yielded 10- to 100-fold fewer progeny than the wild-type virus, depending on the multiplicity of infection. (ii) Using mass spectrometry-based proteomics technology, we identified a cellular mitochondrial protein, adenine nucleotide translocator 2 (ANT2), as a UL7-interacting partner. (iii) When ANT2 was transiently expressed in COS-7 cells infected with HSV-1, ANT2 was specifically co-precipitated with UL7. (iv) Cell fractionation experiments with HSV-1-infected cells detected the UL7 protein in both the mitochondrial and cytosolic fractions, whereas ANT2 was detected only in the mitochondrial fraction. These results indicate the importance of HSV-1 UL7's involvement in viral replication and demonstrate that it interacts with ANT2 in infected cells. The potential biological significance of the interaction between UL7 and ANT2 is discussed.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/β, and the viruses efficiently infected the HCs in the IFN-α/β receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

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.

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.

Astrocytes are the most abundant glial cell type in the brain, where they participate in various homeostatic functions. Transcriptomically, diverse astrocyte subpopulations play distinct roles during development and disease progression. However, the biochemical identification of astrocyte subtypes, especially by membrane surface protein glycosylation, remains poorly investigated. Protein tyrosine phosphatase receptor type zeta (PTPRZ) is a highly expressed membrane protein in CNS glia cells that can be modified with diverse glycosylation, including the unique HNK-1 capped O-mannosyl (O-Man) core M2 glycan mediated by brain-specific branching enzyme GnT-IX. Although PTPRZ modified with HNK-1 capped O-Man glycans (HNK-1-O-Man+ PTPRZ) is increased in reactive astrocytes of demyelination model mice, whether such astrocytes emerge in a broad range of disease-associated conditions or are limited to conditions associated with demyelination remains unclear. Here, we show that HNK-1-O-Man+ PTPRZ localizes in hypertrophic astrocytes of damaged brain areas in patients with multiple sclerosis. Furthermore, we show that astrocytes expressing HNK-1-O-Man+ PTPRZ are present in two demyelination mouse models (cuprizone-fed mice and a vanishing white matter disease model), while traumatic brain injury does not induce glycosylation. Administration of cuprizone to Aldh1l1-eGFP and Olig2KICreER/+ ;Rosa26eGFP mice revealed that cells expressing HNK-1-O-Man+ PTPRZ are derived from cells in the astrocyte lineage. Notably, GnT-IX but not PTPRZ mRNA was up-regulated in astrocytes isolated from the corpus callosum of cuprizone model mice. These results suggest that the unique PTPRZ glycosylation plays a key role in the patterning of demyelination-associated astrocytes.