Biology subjects

Okuzaki, D.

Publications and source records attributed to Okuzaki, D..

16 recordsLinked to original sources

bioRxiv2025

Hypomorphic Lig4 gene mutation in mice predisposes to Th1-skewing intestinal inflammation

Dysregulation of DNA double-strand break (DSB) repair leads to adaptive immunodeficiency, whereas the remaining lymphocytes are aberrantly activated and provoke inflammations. However, no model mice were available to consistently manifest inflammation under defective DSB repair. We generated mutant mice carrying a missense mutation p.W447C in the gene encoding DNA ligase IV (LIG4), critical for DSB repair. Lig4W447C/W447C mice showed growth retardation and severe intestinal inflammations under adaptive immunodeficiency. The inflammations were featured by marked infiltration of T helper type 1 (Th1) cells and macrophages and was dependent on lymphocytes. When Ifng was deleted, Th2 and Th17 instead of Th1 cells drove the inflammations. Lig4W447C/W447C mice showed expansion of oligoclonal T cells with T cell receptor repertoire skewed towards more proximal 3 V and 5 J gene segments. Thus, our novel hypomorphic Lig4 mutant mice show that defective DSB repair leads to Th1-dependent intestinal inflammations under severe adaptive immunodeficiency.

immunology↗

bioRxiv2025

Immunological characterization of peritoneal exudate cells in liver cirrhosis patients

BACKGROUND AND AIMSLiver cirrhosis (LC) is the end stage of liver fibrosis caused by various chronic liver diseases. Patients with LC often develop ascites containing peritoneal exudate cells (PECs). However, those cells have not been fully immunologically characterized. In this study, we clarify immune cell profiles of PECs from patients with LC. APPROACH AND RESULTSPECs were collected from patients with LC or patients receiving continuous ambulatory peritoneal dialysis (CAPD) as a non-cirrhotic control and subjected to single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq) and flowcytometry analyses. Analysis of scRNA-seq revealed that dendritic cells (DCs) and macrophages were major populations in CAPD patient-derived PECs, while those cells were decreased and T cells were most abundant in LC patient-derived PECs. Notably, FCGR3A-expressing macrophages were dominant over DCs and GATA6-expressing macrophages in LC patient-derived PECs. Bulk RNA-seq analysis further clarified expression of a set of genes was up- or down-regulated along with LC severity. Especially, expression of T cell signature genes was featured by its increase at Child-Pugh class B, but decrease at Child-Pugh class C. Flowcytometry analysis showed increase of T cells, decrease of DCs and macrophages, and increased expression of CD16 and CD163 in CD1cintCD14high cells corresponding to FCGR3A-expressing macrophages in LC patient-derived PECs. CONCLUSIONSIn LC patient-derived PECs, myeloid and T cell populations and their gene expression profiles were fluctuated with severity. Our findings should contribute to further development of diagnosis or therapeutic maneuver for LC.

immunology↗

bioRxiv2024

Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma

Myasthenia gravis (MG) is known to be epidemiologically associated with abnormalities of the thymus, an organ that maintains central tolerance. However, due to the complexity of the thymus, specific characteristics related to the pathogenesis of MG remain elusive. In our study, we attempted to narrow down the features associated with MG using spatial transcriptome analysis of thymoma and thymic hyperplasia samples. We found that the majority of thymomas were constituted by the cortical region, whereas the medullary region was localized in comparatively restricted areas. Moreover, the medullary region contained polygenic enrichment, MG-specific germinal center structures, and a supporting immune microenvironment. Additionally, neuromuscular medullary thymic epithelial cells (nmTECs), previously identified as MG-specific autoantigen-producing cells, were situated at the cortico-medullary junction. The immune microenvironment in the medulla was characterized by a specific chemokine pattern and specific immune cells, such as CCR7+ migratory dendritic cells (migDCs) and effector regulatory T (Treg) cells. Furthermore, similar germinal center structures and immune microenvironments were observed in the medulla during thymic hyperplasia. This study indicates that the medulla and junction areas are related to the pathology of MG, suggesting that these areas should be the focus of future studies on MG pathogenesis and drug targeting.

immunology↗

bioRxiv2023

NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA

Chronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA (rcDNA) levels in hepatocellular carcinoma. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA levels while knockdown or knockout of the gene encoding N4BP1 rescued rcDNA levels. N4BP1 possesses the KH-like and RNase domains and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and degraded both the 3.5 kb and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by promoting the degradation of 3.5 kb and 2.1/2.4 kb HBV RNA. ImportanceThere is still a large number of HBV-infected people in the world today because of no curative treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related to HCC progression. This novel factor can potentially become a key to new HBV treatments.

microbiology↗

bioRxiv2023

SiCR: Web application for single-cell repertoire analysis and immune profiling

BackgroundSingle-cell RNA sequencing (scRNA-seq) allows analysis of complete sequences of antigen receptors in individual cells. However, it is a complex technique that requires multiple analyses to obtain accurate results. Although several user-friendly tools for scRNA-seq are available, none are specifically designed for immune profiling. ResultsWe developed a web application called SiCR that is based on the Shiny framework of the R package and specializes in single-cell immune profiling. SiCR allows clustering and cell typing required for both general single-cell and immune profiling analyses, such as predicting whether the chronotype is expanding in each group and the antigen the expanding chronotype targets. These analyses can be performed using a cursor control. SiCR also allows for detailed figure settings, enabling immediate publication of results. ConclusionsSiCR is a comprehensive workbench that can be used by biologists for single-cell immune profiling. Currently, it is the only web application that allows single-cell repertoire analysis using both raw and preprocessed data. Moreover, SiCR significantly reduces the time and effort required to analyze and interpret information in single-cell immune profiling. Therefore, SiCR is a potential reference application for interactive analysis and investigation of biological data, especially for immune profiling.

bioinformatics↗

bioRxiv2023

A stress sensor IRE1α is required for bacterial exotoxin-induced inflammasome activation in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1{beta} (IL-1{beta}), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarified the roles of IRE1, an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1{beta} production from RPMs. In RPMs, CTB is incorporated into ER and induced ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1-deficient RPMs showed a significant impairment of CT- or CTB-induced IL-1{beta} production, indicating that IRE1 was required for CT- or CTB-induced IL-1{beta} production from RPMs. This study first demonstrates the critical roles of IRE1 in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages. One sentence summaryIRE1 is required for NLRP3 and pyrin-mediated IL-1{beta} production

immunology↗

bioRxiv2023

Classification of patients with COVID-19 by blood RNA endotype: A prospective cohort study

BackgroundAlthough the development of vaccines has considerably reduced the severity of COVID-19, its incidence is still high. Hence, a targeted approach based on RNA endotypes of a population should be developed to help design biomarker-based therapies for COVID-19. ObjectivesWe evaluated the major RNAs transcribed in blood cells during COVID-19 using PCR to further elucidate its pathogenesis and determine predictive phenotypes in COVID-19 patients. Study designIn a discovery cohort of 40 patients with COVID-19, 26,354 RNAs were measured on day 1 and day 7. Five RNAs associated with disease severity and prognosis were derived. In a validation cohort of 153 patients with COVID-19 treated in the intensive care unit, we focused on prolactin (PRL), and toll-like receptor 3 (TLR3) among RNAs, which have a strong association with prognosis, and evaluated the accuracy for predicting survival of PRL-to-TL3 ratios (PRL/TLR3) with the areas under the ROC curves (AUC). The validation cohort was divided into two groups based on the cut-off value in the ROC curve with the maximum AUC. The two groups were defined by high PRL/TLR3 (n=47) and low PRL/TLR3 groups (n=106) and the clinical outcomes were compared. ResultsIn the validation cohort, the AUC for PRL/TLR3 was 0.79, showing superior prognostic ability compared to severity scores such as APACHE II and SOFA. The high PRL/TLR3 group had a significantly higher 28-day mortality than the low PRL/TLR3 group (17.0% vs 0.9%, P<0.01). ConclusionsA new RNA endotype classified using high PRL/TLR3 was associated with mortality in COVID-19 patients.

genetics↗

bioRxiv2023

HDAC1-3 inhibition reduces CCR2 expression and immunosuppressive function of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) play a critical role in cancer progression and resistance, making them significant targets for cancer immunotherapy. Although epigenetic regulation by histone deacetylases (HDACs) regulates cell fate and function, the specific roles of HDACs in modulating MDSCs remain poorly understood. We aimed to examine the effects and underlying mechanisms of HDAC on MDSCs using various HDAC inhibitors. HDAC1-3 inhibitors were found to reduce the expression of CCR2, a chemokine receptor that mediates the migration of monocytic (M-)MDSCs to tumors and attenuated the immunosuppressive activity of MDSCs. In an orthotopic hepatocellular carcinoma (HCC) murine model, HDAC1-3 inhibitors reduced the infiltration of M-MDSCs, increased the number of natural killer cells in tumors, and suppressed tumor growth. Our results also suggest that HDAC1-3 inhibitors potentiate the antitumor effects of anti-programmed cell death protein 1 antibodies. To elucidate the molecular mechanisms underlying the inhibition of MDSCs by HDAC1-3 inhibitors, ATAC-seq and RNA-seq analyses were performed. We identified 115 genes that were epigenetically upregulated by HDAC1-3 inhibitors, related to transcriptional regulation and ubiquitination. HDAC1-3 inhibitors further reduced CCR2 protein expression by enhancing ubiquitination-mediated degradation. Our findings reveal a novel mechanism of action of HDAC1-3 inhibitors in MDSCs and suggest a potential combination strategy with immunotherapy for the clinical translation of HCC.

immunology↗

bioRxiv2023

Single-cell transcriptome landscape of circulating CD4+ T cell populations in human autoimmune diseases

CD4+ T cells are a key mediator of various autoimmune diseases; however, how they contribute to disease development remains obscure primarily because of their cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations by decomposition-based transcriptome characterization together with canonical clustering strategies. This approach identified 12 independent transcriptional gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell data sets of over 1.8M peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloged cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

immunology↗

bioRxiv2022

A novel TGF-β-responsive enhancer regulates SRC expression and epithelial-mesenchymal transition-associated cell migration

The non-receptor tyrosine kinase SRC is overexpressed and/or hyperactivated in various human cancers, and facilitates cancer progression by promoting invasion and metastasis. However, the mechanisms underlying SRC upregulation are poorly understood. In this study, we demonstrated that transforming growth factor-{beta} (TGF-{beta}) induces SRC expression at the transcriptional level by activating a novel intragenic SRC enhancer. In the human breast epithelial cell line MCF10A, TGF-{beta}1 stimulation upregulated the SRC1A promoter, resulting in increased SRC mRNA and protein levels. Chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that SMAD complex is recruited to three enhancer regions ~15 kb upstream and downstream of the SRC promoter, and one of them is capable of activating the SRC promoter in response to TGF-{beta}. In addition, JUN, which is a member of the activator protein (AP)-1 family, also localizes to the enhancer and regulates TGF-{beta}-induced SRC expression. Furthermore, the total amount of active SRC also increased, coinciding with the TGF-{beta}-induced SRC expression. In TGF-{beta}-induced epithelial-mesenchymal transition (EMT), TGF-{beta}-induced SRC upregulation plays a crucial role in EMT-associated cell migration by activating the SRC/focal adhesion kinase (FAK) circuit. Overall, these results suggest that TGF-{beta}-induced SRC upregulation promotes cancer cell invasion and metastasis in a subset of human malignancies.

molecular biology↗

bioRxiv2022

Paradoxical tumor suppressive role of the musculoaponeurotic fibrosarcoma gene in colorectal cancer

Somatic cell reprogramming using the microRNAs miR200c, miR-302s, and miR-369s leads to increased expression of cyclin-dependent kinase inhibitors in human colorectal cancer (CRC) cells and suppressed tumor growth. Here, we investigated whether these microRNAs inhibit colorectal tumorigenesis in CPC;Apc mice, which are prone to colon and rectal polyps. Repeated administration of microRNAs inhibited polyp formation. Microarray analysis indicated that c-MAF, which reportedly shows oncogene-like behavior in multiple myeloma and T-cell lymphoma, decreased in tumor samples but increased in microRNA-treated normal mucosa. Immunohistochemistry identified downregulation of c-MAF as an early tumorigenesis event in CRC, with low c-MAF expression associated with poor prognosis. Of note, c-MAF expression and p53 protein levels were inversely correlated in CRC samples. c-MAF knockout led to enhanced tumor formation in azoxymethane/dextran sodium sulfate-treated mice, with activation of cancer-promoting genes. c-MAF may play a tumor-suppressive role in CRC development.

molecular biology↗

bioRxiv2021

Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG is frequently developed in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-seq, we identified ectopic expression of neuromuscular molecules in MG-associated thymoma (MG-thymoma). These molecules were originated from a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we named neuromuscular mTECs (nmTECs). MG-thymoma also exhibited microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicted the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma was further confirmed by immunohistochemically and by cellular composition estimation from MG-thymoma transcriptome. Altogether, this study suggests that nmTECs play a significant role in MG pathogenesis via ectopic expression of neuromuscular molecules.

immunology↗

bioRxiv2020

Generation of human bronchial organoids for SARS-CoV-2 research

Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved human bronchial epithelial cells and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. Furthermore, the gene expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq analysis. In conclusion, we succeeded in generating hBO that can be used for SARS-CoV-2 research and COVID-19 drug discovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/115600v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@13a6908org.highwire.dtl.DTLVardef@1c59300org.highwire.dtl.DTLVardef@362167org.highwire.dtl.DTLVardef@1cb31ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

CDCP1 controls compensatory renal growth by integrating Src and Met signaling

Hepatocyte growth factor (HGF) controls various biological responses, including morphogenesis, organ regeneration, and cancer invasion, by activating its receptor, Met. However, the mechanisms that precisely control diverse Met signaling remain unclear. Here, we identified CUB domain-containing protein 1 (CDCP1) as a crucial element of HGF signaling. In MDCK cysts, HGF induced Src activation via CDCP1 upregulation, and CDCP1 ablation abrogated HGF responses, i.e., extended invasive cell protrusions and promoted cell growth/proliferation. Mechanistically, CDCP1 upregulation promoted Src recruitment into lipid rafts to activate Met-associated STAT3. In breast cancer cells in which Met and CDCP1 were co-upregulated, CDCP1 knockdown suppressed HGF-induced cell invasion. Furthermore, in vivo analysis showed that CDCP1 ablation suppressed compensatory renal growth by attenuating Met-STAT3 signaling. These findings demonstrate that CDCP1 plays a crucial role in controlling HGF responses by integrating Src and Met-STAT3 signaling, and provide new insights into the regulatory mechanisms underlying the multifaceted functions of HGF.

cell biology↗

bioRxiv2019

Transcriptomic profiling of Streptococcus pyogenes M1T1 strain in a mouse model of necrotizing fasciitis

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interaction between host and bacteria affect bacterial gene-expression profiles, but the S. pyogenes gene-expression pattern in necrotizing fasciitis remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by using infected hindlimbs obtained at 24, 48, and 96 h post-infection. The RNA-seq analysis identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs as compared to their expression under in vitro conditions. The consistently enriched genes during infection included 306 genes encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport and vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the mouse model of necrotizing fasciitis (log2 fold-change values: >6.0, >9.4, and >7.1, respectively). Conversely, the consistently downregulated genes included 177 genes, containing genes associated with oxidative-stress response and cell division. These results suggest that S. pyogenes in necrotizing fasciitis changes its metabolism, decreases cell proliferation, and upregulates the expression of major toxins. Our findings could provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis.\n\nAuthor summaryNecrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection, principally caused by a Streptococcus pyogenes. At infection sites in hosts, bacterial pathogens are exposed to drastically changing environmental conditions and alter global gene expression patterns for survival and pathogenesis. However, there is no previous report about transcriptomic profiling of S. pyogenes in the necrotizing fasciitis. Here, we conducted comprehensive gene-expression analyses of S. pyogenes in the mouse model of necrotizing fasciitis at three distinct time points during infection. Our results indicated that S. pyogenes drastically upregulates the expression of virulence-associated genes and shifts metabolic-pathway usage during infection. The high-level expressions in particular of toxins, such as cytolysins, proteases, and nucleases, were observed at infection sites. In addition, the consistently enriched genes identified here included genes for metabolism of arginine and histidine, and carbohydrate uptake and utilization. Conversely, the genes associated with oxidative-stress response and cell division were consistently downregulated in the mouse model of necrotizing fasciitis. These data will provide useful information necessary for establishing novel treatment strategies (166 words).

microbiology↗

bioRxiv2017

Modest static pressure suppresses columnar epithelial cell proliferation in association with cell shape and cytoskeletal modifications

Intraluminal pressure elevation can cause degenerative disorders, such as ileus and hydronephrosis, and the threshold is fairly low and constant, 20-30 cm H2O. We previously devised a novel two-chamber culture system subjecting cells cultured on a semipermeable membrane to increased culture medium height (water pressure up to 60 cm H2O). Here, we cultured several different cell lines using the low static pressure-loadable two-chamber system, and examined cell growth, cell cycle, and cell morphology. Madin-Darby canine kidney (MDCK) columnar epithelial cells were growth-suppressed in a manner dependent on static water pressure ranging from 2-50 cm H2O, without cell cycle arrest at any specific phase. Two other types of columnar epithelial cells exhibited similar phenotypes. By contrast, spherical epithelial and mesenchymal cells were not growth-suppressed, even at 50 cm H2O. Phalloidin staining revealed that 50 cm H2O pressure load vertically flattened and laterally widened columnar epithelial cells and made actin fiber distribution sparse, without affecting total phalloidin intensity per cell. When the mucosal protectant irsogladine maleate (100 nM) was added to 50-cm-high culture medium, MDCK cells were reduced in volume and their doubling time shortened. Cell proliferation and morphology are known to be regulated by the Hippo signaling pathway, but a pressure load of 50 cm H2O did not alter the expression levels of Hippo signaling molecules in columnar epithelial cells, suggesting that this pathway was not involved in the pressure-induced phenotypes. RNA sequencing of MDCK cells showed that a 50 cm H2O pressure load upregulated keratin 14, an intermediate filament, 12-fold. This upregulation was confirmed at the protein level by immunofluorescence, suggesting a role in cytoskeletal reinforcement. These results provide evidence that cell morphology and the cytoskeleton are closely linked to cell growth. Pathological intraluminal pressure elevation may cause mucosal degeneration by acting directly on this linkage.\n\nSummaryWe provide evidence that columnar epithelial cells are growth-suppressed by pressure loads as low as 30 cm H2O, in association with cell-shape flattening and cytoskeletal alterations.

molecular biology↗