A Splice Variant of ASC Regulates IL-1β Release and Aggregates Differently from Intact ASC

The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation. ASC mRNA is encoded by three exons: exons 1 and 3 encode a pyrin domain (PYD) and caspase recruit domain (CARD), respectively, and exon 2 encodes a proline and glycine-rich (PGR) domain. Here, we identified a variant ASC protein (vASC) lacking the PGR domain that was smaller than full length ASC (fASC) derived from fully transcribed mRNA and searched for differences in biochemical and biological nature. Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1β excretion was significantly higher for vASC. There was also a marked structural difference observed in the fibrous aggregates formed by fASC and vASC. These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC

Involvement of CD11b+ GR-1low cells in autoimmune disorder in MRL-Faslpr mouse

金沢大学医薬保健研究域医学系Objective: Myeloid-derived suppressor cells (MDSCs) have been identified as immunosuppressive cells in tumor-related inflammation. However, the pathogenesis of MDSCs for autoimmune disease has not been investigated as yet. The aim of this study was to address whether MDSCs contribute to autoimmune organ injury in lupus-prone mice. Methods: MDSCs were analyzed by flow cytometric staining of CD11b+ GR-1+ in MRL-Faslpr mice. CD4+ T-cell proliferation assay was performed by coculture with CD11b+ GR-1+ splenocytes. The percentage of immunosuppressive cells was examined during disease progression. Expression of chemokine receptor on immunosuppressive cells was analyzed, and chemotaxis assay was performed. Results: CD11b+ GR-1low cells had a suppressive effect on CD4+ T-cell proliferation, which was restored by an arginase-1 inhibitor. CD11b+ GR-1low cells increased in percentage during disease progression in kidney and blood. The number of migrated CD11b+ GR-1low cells increased in the presence of monocyte chemoattractant protein-1/CCL2. Conclusion: We assessed the involvement of CD11b+ GR-1low cells in autoimmune disorder in MRL-Faslpr mice. These cells regulate immunological responses via CCL2/CCR2 signaling. The regulation of immunosuppressive monocytes may provide novel therapeutic strategy for organ damage in autoimmune diseases. © 2010 Japanese Society of Nephrology.

Collagen adhesion gene is associated with blood stream infections caused by methicillin-resistant Staphylococcus aureus

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. Methods: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. Results: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. Conclusions: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection. (c) 2019 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases

CD105 is a more appropriate marker for evaluating angiogenesis in urothelial cancer of the upper urinary tract than CD31 or CD34

Angiogenesis plays an important role in cancer progression in many types of cancer. Evaluation of angiogenesis is often performed, but the optimal methodology for human cancer has not been agreed upon. As adequate evaluation of angiogenesis in cancer tissues might be important for prediction of prognosis and treatment decisions, we evaluated angiogenesis semiquantitatively by assessing microvessel density (MVD) in urothelial cancer of the upper urinary tract (UC-UUT). We compared the performance of three endothelial cell markers (CD31, CD34, and CD105) on formalin-fixed tissues from 122 patients diagnosed with UC-UUT without metastasis. Vascular endothelial growth factor (VEGF)-A expression was also evaluated immunohistochemically. Correlations between MVD with each marker and pT stage, grade, survival, and VEGF-A expression were investigated. Mean (standard deviation) MVD as estimated by immunohistochemical staining with anti-CD31, anti-CD34, and anti-CD105 were 47.1 (17.9)/high-power field (HPF), 70.9 (19.5)/HPF, and 31.2 (16.7)/HPF, respectively. Although all MVDs were significantly associated with pT stage and grade, CD105-MVD showed the strongest association. Similarly, CD105-MVD showed the strongest correlation with VEGF-A expression (r = 0.530, p < 0.001). Although all MVDs were associated with metastasis-free survival and cause-specific survival on univariate analysis, only CD105-MVD was retained as an independent predictor in multivariate analysis including pT stage and grade. CD105-MVD may be the preferred marker for semiquantitative assessment of angiogenesis in patients with UC-UUT

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

新規骨髄由来線維化誘導細胞を介したANCA関連血管炎における臓器線維化機序の解明

金沢大学医薬保健研究域医学系抗好中球細胞質抗体関連血管炎(AAV)症例における，新規骨髄由来細胞 CD45+C1q+CCR8+ cell[precursors of fibroblastic cell(preFC)] の臨床的，生物学的意義を検討した．初発および再燃のAAV 12例の血液を，各種抗体を用いて多重染色し，フローサイトメトリーで解析することで，preFCを同定した．対照群は，健常者 10例，AAV以外の膠原病　10例とし，CD45陽性単核球中のpreFCの割合をAAV症例と比較した．AAV症例において，血液中のpreFC/CD45陽性単核球比は，健常者およびAAV以外の膠原病症例より高値であった．We explored the clinical and biologic significance of the new myeloid-derived fibrosis-inducing cells, CD45+C1q+CCR8+ cell[precursors of fibroblastic cell(preFC)] in the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) cases. We identified preFC in the blood of 12 AAV cases of the onset or relapse by the flow cytometry. Control groups were 10 cases of the other connective tissue diseases except AAV, and 10 healthy volunteers. Moreover, the ratio of preFC/CD45+ mononuclear cells in the AAV cases was increased, compared with the other connective tissue disease cases, or the healthy volunteers.研究課題/領域番号:18K15991, 研究期間(年度):2018-04-01 – 2020-03-31出典：「新規骨髄由来線維化誘導細胞を介したANCA関連血管炎における臓器線維化機序の解明」研究成果報告書　課題番号18K15991（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-18K15991/18K15991seika/)を加工して作

糖尿病における心・腎線維化機序に関わる新規骨髄由来細胞の同定と解析

金沢大学附属病院全身GFP発現マウスの骨髄を野生型B6マウスに移植し，一側尿管結紮（UUO）を行うことで腎線維化モデルを作成した．心，腎に浸潤したGFP+CD45+Sca1+細胞に着目し，Fibroblastsとの共培養やCOL-GFPマウスを用いた骨髄移植による検討にて，CD45+Sca1+細胞の線維化誘導能とコラーゲン産生能を確認した．また CD45+Sca1+細胞よりRNAを抽出し，Genechipによる遺伝子発現解析を行った．共培養とフローサイトメトリーによる検証を経て，CD45+Sca1+細胞の中に更なる亜集団が存在することを示した．この細胞群はヒト末梢血にも存在し，腎機能の増悪に伴って増加した．To chase and identify fibrosis-related cell types, we used unilateral ureteral obstruction model mice, which showed renal and heart fibrosis, in combination with GFP-based tracing systems such as bone-marrow transplantation (BMT). We found BM-derived mononuclear cell cluster of CD45+Sca1+ cells mobilized and accumulated in the fibrotic lesions of kidney and heart using flow cytometry (FCM). The CD45+Sca1+ cells had an activating potential for collagen production of cultured fibroblasts and also produced type 1 collagen by themselves. Genechip analyses and FCM revealed the subpopulation of CD45+Sca1+ cells, which were related to chemotaxis and innate immunity. These cells were also detected in human peripheral blood and increased in proportion to kidney dysfunction. In this study, we succeeded to identify a new myeloid-derived fibrosis-inducing cells, which could provide a new avenue in fibrosis.研究課題/領域番号:15K19449, 研究期間(年度):2015-04-01 – 2017-03-3