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

Liver Injury and Cell Survival in Non-Alcoholic Steatohepatitis Regulated by Sex-Based Difference through B Cell Lymphoma 6

The liver is a crucial organ for maintaining homeostasis in living organisms and is the center of various metabolic functions. Therefore, abnormal metabolic activity, as in metabolic syndrome, leads to pathological conditions, such as abnormal accumulation of lipids in the liver. Inflammation and cell death are induced by several stresses in the fatty liver, namely steatohepatitis. In recent years, an increase in non-alcoholic steatohepatitis (NASH), which is not dependent on excessive alcohol intake, has become an issue as a major cause of liver cirrhosis and liver cancer. There are several recent findings on functional sex-based differences, NASH, and cell stress and death in the liver. In particular, NASH-induced liver injury and tumorigeneses were suppressed by B cell lymphoma 6, the transcriptional factor regulating sex-based liver functional gene expression. In this review, we discuss cell response to stress and lipotoxicity in NASH and its regulatory mechanisms

An in vitro model of polycystic liver disease using genome-edited human inducible pluripotent stem cells

In the developing liver, bile duct structure is formed through differentiation of hepatic progenitor cells (HPC) into cholangiocytes. A subtype of polycystic liver diseases characterized by uncontrolled expansion of bile ductal cells is caused by genetic abnormalities such as in that of protein kinase C substrate 80 K-H (PRKCSH). In this study, we aimed to mimic the disease process in vitro by genome editing of the PRKCSH locus in human inducible pluripotent stem (iPS) cells. A proportion of cultured human iPS cell-derived CD13+CD133+ HPC differentiated into CD13− cells. During the subsequent gel embedding culture, CD13− cells formed bile ductal marker-positive cystic structures with the polarity of epithelial cells. A deletion of PRKCSH gene increased expression of cholangiocytic transcription factors in CD13− cells and the number of cholangiocytic cyst structure. These results suggest that PRKCSH deficiency promotes the differentiation of HPC-derived cholangiocytes, providing a good in vitro model to analyze the molecular mechanisms underlying polycystic diseases. Keywords: Human iPS cells, Polycystic liver disease, Genome editing, Cholangiocyt

Changes of HPC surface marker expression in human iPS cell-derived hepatic progenitor-like colonies.

<p>(<b>A</b>) After 12 days of culture with cytokines, CD13^highCD133⁺ cells were sorted onto GFP-MEFs. After 10–12 days, cells were trypsinized and stained with antibodies against CD13 and CD133. Expression of CD13 and CD133 was analyzed by flow cytometry. (<b>B–E</b>) Colony forming activity of CD13⁺CD133⁺, CD13⁺CD133⁻, CD13⁻CD133⁺, and CD13⁻CD133⁻ fractions of human iPS cell-derived HPCs. At every replating step, cells stained with antibodies against CD13 and CD133 were sorted onto new GFP-MEFs, and their colony-forming activity was analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (duplicate samples). N.D. shows “not determined”.</p

<i>In vitro</i> long-term expansion of CD13^highCD133⁺ cells.

<p>(<b>A</b>) Colonies derived from CD13^highCD133⁺ cells were trypsinized and replated onto MEFs. The number of cells was counted at each replating step. The cells continued to proliferate for more than 1 month. A representative growth curve is shown. Similar results were obtained in two independent experiments. (<b>B</b>) Expression of the proliferation marker Ki67 in human iPS cell-derived hepatocytic colonies. After the 4th passage, the colonies were stained with antibodies against Ki67, HNF4α, and Oct3/4. (<b>C</b>) Human iPS cells were stained with an antibody against Oct3/4.</p

Differentiation from human iPS cells toward hepatic lineage cells.

<p>(<b>A</b>) Schematic of the experimental procedure. Human iPS cells were sequentially stimulated with various cytokines: (1) activin A, (2) basic FGF and BMP-4, and (3) HGF. The cells were cultured in 10% O₂ for days 0–4 and 5% O₂ for days 5–12. (<b>B</b>) After 12 days of culture with or without cytokines, cells were stained with antibodies against AFP and HNF4α. Nuclei were counterstained with DAPI. (<b>C</b>) Expression of cell surface markers in human iPS cell-derived hepatic lineage cells.</p

An <i>In Vitro</i> Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

<div><p>Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult <i>ex vivo</i>. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments <i>in vitro</i>. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an <i>in vitro</i> expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.</p></div

Isolation of HPCs from human iPS cell-derived hepatic lineage cells.

<p>(<b>A</b>) Expression of hepatic progenitor markers in undifferentiated human iPS cells and differentiated cells. After 12 days of culture with or without cytokines, cells were stained with antibodies against CD13 and CD133, and then analyzed by flow cytometry. Ratios of CD13^highCD133⁺ cells are shown. (<b>B</b>) Representative images of a colony derived from a single CD13^highCD133⁺ cell. Colonies were stained with antibodies against AFP and HNF4α. Nuclei were counterstained with DAPI. (<b>C</b>) Culture condition of the human iPS cell-derived hepatic progenitor colony assay. CD13^highCD133⁺ cells were sorted and cultured on MEFs in the presence or absence of A-83-01 (ALK inhibitor) and Y-27632 (ROCK inhibitor). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (triplicate samples). (<b>D</b>) CD13⁻CD133⁻, CD13 weakly single positive, CD13^mid single positive and CD13^highCD133⁺ cells were sorted onto MEFs. The cells were cultured in standard culture media in the presence of A-83-01 and Y-27632. Large colonies (containing more than 100 cells) derived from individual sorted cells were counted. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (triplicate samples).</p

Expressions of hepatic functional genes in differentiated HPCs.

<p>The levels of mRNAs encoding phase 1 and 2 enzymes in human iPS cell-derived HPCs from the 3rd culture, and spheroids derived from human iPS cell-derived HPCs from the 3rd culture are shown as the fold values relative to the levels in uncultured human hepatocytes. Spheroid formation was induced by hanging drop culture in the presence or absence of OSM. The results are represented as the mean colony counts ± SD (spheroid culture, n = 6; HPCs, n = 3; uncultured human hepatocytes, n = 2).</p