DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation.

The IL-6 amplifier was originally discovered as a mechanism for the enhanced activation of NF-κB in non-immune cells. In the IL-6 amplifier, IL-6-STAT3 and NF-κB stimulation is followed by an excessive production of IL-6, chemokines, and growth factors to develop chronic inflammation preceding the development of inflammatory diseases. Previously, using a shRNA-mediated genome-wide screening, we found that DEAD-Box Helicase 6 (DDX6) is a candidate positive regulator of the amplifier. Here, we investigate whether DDX6 is involved in the pathogenesis of inflammatory diseases via the IL-6 amplifier. We found that DDX6-silencing in non-immune cells suppressed the NF-κB pathway and inhibited activation of the IL-6 amplifier, while the forced expression of DDX6 enhanced NF-κB promoter activity independent of the RNA helicase activity of DDX6. The imiquimod-mediated dermatitis model was suppressed by the siRNA-mediated gene downregulation of DDX6. Furthermore, silencing DDX6 significantly reduced the TNF-α-induced phosphorylation of p65/RelA and IκBα, nuclear localization of p65, and the protein levels of IκBα. Mechanistically, DDX6 is strongly associated with p65 and IκBα, but not TRADD, RIP, or TRAF2, suggesting a novel function of DDX6 as an adaptor protein in the NF-κB pathway. Thus, our findings demonstrate a possible role of DDX6 beyond RNA metabolism and suggest DDX6 is a therapeutic target for inflammatory diseases.

High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore.

A digital platform that can rapidly and accurately diagnose pathogenic viral variants, including SARS-CoV-2, will minimize pandemics, public anxiety, and economic losses. We recently reported an artificial intelligence (AI)-nanopore platform that enables testing for Wuhan SARS-CoV-2 with high sensitivity and specificity within five minutes. However, which parts of the virus are recognized by the platform are unknown. Similarly, whether the platform can detect SARS-CoV-2 variants or the presence of the virus in clinical samples needs further study. Here, we demonstrated the platform can distinguish SARS-CoV-2 variants. Further, it identified mutated Wuhan SARS-CoV-2 expressing spike proteins of the delta and omicron variants, indicating it discriminates spike proteins. Finally, we used the platform to identify omicron variants with a sensitivity and specificity of 100% and 94%, respectively, in saliva specimens from COVID-19 patients. Thus, our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants.

Metal-ion transporter SLC39A8 is required for brain manganese uptake and accumulation.

Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using 54Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased 54Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood-brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity.

Computer model of IL-6-dependent rheumatoid arthritis in F759 mice.

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method.

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients.

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn's disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Dupuytren's contracture-associated SNPs increase SFRP4 expression in non-immune cells including fibroblasts to enhance inflammation development.

Dupuytren's contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and ßTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.

Gateway reflexes, neuronal circuits that regulate the autoreactive T cells in organs having blood barriers.

Gateway reflexes are neural circuits that maintain homeostasis of the immune system. They form gateways for autoreactive T cells to infiltrate the central nervous system in a noradrenaline-dependent manner despite the blood-brain barrier. This mechanism is critical not only for maintaining organ homeostasis but also for inflammatory disease development. Gateway reflexes can be regulated by environmental or artificial stimuli including electrical stimulation, suggesting that the infiltration of immune cells can be controlled by bioelectronic medicine. In this review, we describe the discovery of gateway reflexes and their future directions with special focus on bioelectronic medicine.

Sjögren's syndrome-associated SNPs increase GTF2I expression in salivary gland cells to enhance inflammation development.

Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammation with lymphoid infiltration and destruction of the salivary glands. Although many genome-wide association studies have revealed disease-associated risk alleles, the functions of the majority of these alleles are unclear. Here, we show previously unrecognized roles of GTF2I molecules by using two SS-associated single nucleotide polymorphisms (SNPs), rs73366469 and rs117026326 (GTF2I SNPs). We found that the risk alleles of GTF2I SNPs increased GTF2I expression and enhanced nuclear factor-kappa B (NF-κB) activation in human salivary gland cells via the NF-κB p65 subunit. Indeed, the knockdown of GTF2I suppressed inflammatory responses in mouse endothelial cells and in vivo. Conversely, the over-expression of GTF2I enhanced NF-κB reporter activity depending on its p65-binding N-terminal leucine zipper domain. GTF2I is highly expressed in the human salivary gland cells of SS patients expressing the risk alleles. Consistently, the risk alleles of GTF2I SNPs were strongly associated with activation of the IL-6 amplifier, which is hyperactivation machinery of the NF-κB pathway, and lymphoid infiltration in the salivary glands of SS patients. These results demonstrated that GTF2I expression in salivary glands is increased in the presence of the risk alleles of GTF2I SNPs, resulting in activation of the NF-κB pathway in salivary gland cells. They also suggest that GTF2I could be a new therapeutic target for SS.

Biliary excretion of excess iron in mice requires hepatocyte iron import by Slc39a14.

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.

Characterization of in vitro models of SLC30A10 deficiency.

Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of 54Mn import and export in mutant cells. While impaired 54Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired 54Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated 54Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.

Gateway Reflex and Mechanotransduction.

The gateway reflex explains how autoreactive CD4+ T cells cause inflammation in tissues that have blood-barriers, such as the central nervous system and retina. It depends on neural activations in response to specific external stimuli, such as gravity, pain, stress, and light, which lead to the secretion of noradrenaline at specific vessels in the tissues. Noradrenaline activates NFkB at these vessels, followed by an increase of chemokine expression as well as a reduction of tight junction molecules to accumulate autoreactive CD4+ T cells, which breach blood-barriers. Transient receptor potential vanilloid 1 (TRPV1) molecules on sensory neurons are critical for the gateway reflex, indicating the importance of mechano-sensing. In this review, we overview the gateway reflex with a special interest in mechanosensory transduction (mechanotransduction).

How COVID-19 induces cytokine storm with high mortality.

The newly emerging coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, but has rapidly spread all over the world. Some COVID-19 patients encounter a severe symptom of acute respiratory distress syndrome (ARDS) with high mortality. This high severity is dependent on a cytokine storm, most likely induced by the interleukin-6 (IL-6) amplifier, which is hyper-activation machinery that regulates the nuclear factor kappa B (NF-κB) pathway and stimulated by the simultaneous activation of IL-6-signal transducer and activator of transcription 3 (STAT3) and NF-κB signaling in non-immune cells including alveolar epithelial cells and endothelial cells. We hypothesize that IL-6-STAT3 signaling is a promising therapeutic target for the cytokine storm in COVID-19, because IL-6 is a major STAT3 stimulator, particularly during inflammation. We herein review the pathogenic mechanism and potential therapeutic targets of ARDS in COVID-19 patients.

Multiple developmental pathways lead to the generation of CD4 T-cell memory.

Long-term immunological memory mediated by CD4 T cells provides a rapid protection against previously encountered pathogens or antigens. However, it is still controversial how memory CD4 T cells are generated and maintained. Unclear definitions of T-cell memory may be partially responsible for this controversy. It is becoming clear that diverse pathways are responsible for the differentiation and long-term persistence of memory T cells. We herein discuss the diversity of memory cell generation, describing a novel population of resting memory CD4 T cells and their precursors.

Salmonella SiiE prevents an efficient humoral immune memory by interfering with IgG+ plasma cell persistence in the bone marrow.

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin ß1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin ß1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.

Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memory CD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM) and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that, following a critical number of cell divisions, memory precursors downregulate CCR7 and upregulate IL-2Rß, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM.

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Disruption of the mouse Slc39a14 gene encoding zinc transporter ZIP14 is associated with decreased bone mass, likely caused by enhanced bone resorption.

Osteoclasts are bone-resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast-mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14-knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14-KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14-KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14 mRNA was expressed in normal osteoclasts both in vivo and in vitro, but receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis was not impaired in bone marrow-derived macrophages prepared from Zip14-KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14-KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14-KO mice is due to a cell-autonomous or a non-cell-autonomous osteoclast defect.