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

A Case Report of Autosomal Dominant Polycystic Kidney Disease Under Peritoneal Dialysis With Cyst Infection and Culture-Positive Peritoneal Fluid

Background: Cyst infection is a complication sometimes seen in patients with autosomal dominant polycystic kidney disease (ADPKD) and often shows through a positive blood culture. However, there have been no reports of ADPKD patients whose cyst infection propagate to peritoneal fluid leading to positive peritoneal fluid culture. Case presentation: A 74-year-old Japanese man with ADPKD under peritoneal dialysis (PD) was presented with left flank pain, fever, and chills at our hospital. He did not show any symptoms or signs suggestive of peritonitis. There were no elevated cell counts or polymorphonuclear leucocytes in his PD fluid. There were some complicated cysts found in computed tomography and magnetic resonance imaging examinations. We clinically diagnosed him as having a renal cyst infection rather than PD-related peritonitis. We initiated treatment by administering ceftriaxone with an immediate favorable response. As the possibility of accompanying prostatitis still remained, we switched to intravenous levofloxacin on the second day. On the 10th day, Helicobacter cinaedi was detected in 2 sets of blood culture as well as in PD fluid. We switched back to ceftriaxone and this treatment was entirely successful. Conclusions: This is the first report of H cinaedi cyst infection which propagates to peritoneal fluid in a patient with ADPKD

Ghrelin Protects against Renal Damages Induced by Angiotensin-II via an Antioxidative Stress Mechanism in Mice

<div><p>We explored the renal protective effects by a gut peptide, Ghrelin. Daily peritoneal injection with Ghrelin ameliorated renal damages in continuously angiotensin II (AngII)-infused C57BL/6 mice as assessed by urinary excretion of protein and renal tubular markers. AngII-induced increase in reactive oxygen species (ROS) levels and senescent changes were attenuated by Ghrelin. Ghrelin also inhibited AngII-induced upregulations of transforming growth factor-β (TGF-β) and plasminogen activator inhibitor-1 (PAI-1), ameliorating renal fibrotic changes. These effects were accompanied by concomitant increase in mitochondria uncoupling protein, UCP2 as well as in a key regulator of mitochondria biosynthesis, PGC1α. In renal proximal cell line, HK-2 cells, Ghrelin reduced mitochondria membrane potential and mitochondria-derived ROS. The transfection of UCP2 siRNA abolished the decrease in mitochondria-derived ROS by Ghrelin. Ghrelin ameliorated AngII-induced renal tubular cell senescent changes and AngII-induced TGF-β and PAI-1 expressions. Finally, Ghrelin receptor, growth hormone secretagogue receptor (GHSR)-null mice exhibited an increase in tubular damages, renal ROS levels, renal senescent changes and fibrosis complicated with renal dysfunction. GHSR-null mice harbored elongated mitochondria in the proximal tubules. In conclusion, Ghrelin suppressed AngII-induced renal damages through its UCP2 dependent anti-oxidative stress effect and mitochondria maintenance. Ghrelin/GHSR pathway played an important role in the maintenance of ROS levels in the kidney.</p></div

Amelioration of renal tubular damages and increased renal oxidative stress by Ghrelin in AngII-infused mice.

<p>The effects of Ghrelin treatment on the phenotypes of AngII-infused mice. Blood pressures (A), daily chow intake (B) and body weight (C) were compared among saline-infused mice (NS), AngII-infused mice (AngII), and AngII-infused mice treated with Ghrelin (AngII+Ghrelin) or Hydralazine (AngII+Hydralazine). Serum levels of blood urea nitrogen (BUN, D) and creatinine (E), urinary excretion of protein (F), neutrophil gelatinase-associated lipocalin (NGAL, G), n-acetyl-galactasaminase (NAG, H) were compared among the experimental groups. (I) Representative immunostaining for the Ghrelin receptor (Growth hormone secretagogue receptor, GHSR) is shown in the middle panel. Negative control without using anti-GHSR antibody is shown in the left panel. The staining of GHSR in the kidney of GHSR null mice is also shown in the right panel. Scale bar, 50 µm. G represents glomerulus. (J) Representative immunostaining for 4-Hydroxynonenal-2-nonenal (4HNE) of four experimental groups. Bar graphs represent the quantification of immunostained areas. Scale bar; 50 µm. **p<0.01 vs. NS, *p<0.05 vs. NS, ##p<0.01 vs. AngII, #p<0.05 vs. AngII, N.S. represents no significant difference. n = 8.</p

The amelioration of cellular senescent changes in AngII-treated HK-2 by Ghrelin.

<p>(A) Representative staining of senescence-associated β-Galactosidase (SA β-Gal) in untreated HK-2 cells (control), AngII-treated HK-2 cells (AngII, 1 µM), and AngII-treated with the pretreatment of 10 nM Ghrelin (AngII+Ghrelin), 10 nM Des-acyl-Ghrelin (AngII+Des-acy-Ghrelin), or 1 µM AngII type 1 receptor antagonist, irbesartan (left panel). Bar graphs represent the quantification of stained cells (right panel). (B) The protein expressions of p53 (left) and p21 (right) in HK-2 cells. The representative immunoblotting (upper panel) and the results of densitometry analysis (lower panel) were shown. (C) The expression of TGF-β mRNA in HK-2 cells (upper panel) and the concentration of TGF-β in the medium of HK-2 cells (lower panel). (D) The expression of PAI-1 mRNA in HK-2 cells (upper panel) and the concentration of PAI-1 in the medium of HK-2 cells (lower panel). C; control cells, AngII; HK-2 cells treated with 1 µM of AngII, G1, G10, G100; HK-2 cells treated with 1 nM, 10 nM, and 100 nM of Ghrelin, respectively, Des-G; HK-2 cells treated with 10 nM of Des-acyl-Ghrelin, Irb; HK-2 cells treated with 1 µM of irbesartan **p<0.01 vs. control HK-2 cells, *p<0.05 vs. control HK-2 cells, ##p<0.01 vs. AngII-treated HK-2 cells, #p<0.05 vs. AngII-treated HK-2 cells, n = 8.</p

Mitochondria-derived ROS was reduced by Ghrelin through the induction of UCP2.

<p>(A) The effects of Ghrelin on mitochondria-derived UCP2 mRNA levels. (B) Mitochondrial membrane potential was measured by the specific dye as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094373#s4" target="_blank">Materials and Methods</a>. **p<0.01 vs. control cells, n = 8. (C, D) The effects of Ghrelin on mitochondria-derived ROS levels (C) and mitochondria number (D) in HK-2 cells. **p<0.01 vs. control HK-2 cells, *p<0.05 vs. control, n = 8. (E) The effects of AMP-kinase inhibitor on Ghrelin-induced UCP2 upregulation. Compound C, AMP-kinase inhibitor at the concentrations of 2 and 20 µM was pretreated 30 minutes before the Ghrelin administration to HK-2 cells. **p<0.01 vs. control HK-2 cells, ##p<0.01 vs. HK-2 cells treated with 100 nM of Ghrelin, ¶p<0.05 vs. Ghrelin-treated cell with 2 µM of Compound C administration, n = 8. (F) Knock-down of UCP2 protein and mRNA were shown in the representative immunoblotting (left panel) and real-time PCR (right panel), respectively. **p<0.01 vs. control siRNA-transfected cells, n = 6. (G–I) Mitochondria-derived ROS (G), total cellular ROS (H), and total cellular superoxide (I) were measured after the transfection of UCP2 siRNA or control siRNA. HK-2 cells were transfected with siRNA and treated with or without 100 nM of Ghrelin **p<0.01 vs. control siRNA-transfected cells without Ghrelin. N.S. represents no significant difference. n = 8. (J) The effects of Ghrelin on AngII-induced Mitochondrial ROS production. HK-2 cells were treated with 1 nM, 10 nM, and 100 nM of Ghrelin 30 minutes before the treatment with 1 mM of AngII. respectively. **p<0.01 vs. control cells, *p<0.05 vs. control cells, ##p<0.01 vs. AngII-treated HK-2 cells, #p<0.05 vs. AngII-treated HK-2 cells, n = 8.</p

Phenotype of GHSR-null mice.

<p>(A) The primers used for the genotyping in the PCR (left) and representative results of genotyping (right). The primers used are indicated as arrows. TBC, transcription blocking cassette. (B–J) The phenotype differences among the four experimental groups: WT or GHSR^−/− mice infused with normal saline (NS) or AngII. Blood pressure (B), daily chow intake (C), body weight (D) were compared among the four groups. Serum levels of blood urea nitrogen (BUN, E) and creatinine (F) and urinary excretion of protein (G), neutrophil gelatinase-associated lipocalin (NGAL, H), and n-acetyl-galactasaminase (NAG, I) were compared among the four experimental groups. Urinary excretion of each marker was normalized by that of creatinine. (J) Representative immunostaining for 4-Hydroxynonenal-2-nonenal (4HNE) of four experimental groups. Bar graph represents the quantification of immunostained areas. Scale bar; 50 µm. **p<0.01 vs. WT+NS, *p<0.05 vs. WT+NS, ##p<0.01 vs. GHSR^−/−+NS, #p<0.05 vs. GHSR^−/−+NS, n = 8.</p