Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1–5. 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.
D-galactosamine (D-GalN) is a well-known hepatotoxic agent that causes liver injury. Conversely, hepatic macrophages play a crucial role in maintaining liver tissue integrity. Macrophage functions were investigated in hepatic lesions induced by a single intraperitoneal injection of D-GalN (800 mg/kg body weight [BW]) in 6-week-old F344 rats. Blood and liver samples were examined at 8 hr and on 1, 2, 3, and 5 days postsingle injection (PSI). Hepatic lesions consisting of degeneration/sporadic foci of coagulation necrosis, inflammatory cell reaction, and reparative fibrosis were seen on PSI days 1 and 2, reflected by significantly increased serum levels of aspartate transaminase and alanine transaminase and upregulation of CD68 M1 (tumor necrosis factor-a, interleukin [IL]-6, and interferon-g) and CD163 M2 (transforming growth factor-b1, IL-10, monocyte chemoattractant protein-1, and IL-4) macrophage-related factors. Double immunofluorescence staining on PSI day 2 demonstrated that 82% of hepatic macrophages expressed of CD163/CD68 simultaneously; 65-75% of MHC class II macrophages showed co-expression of CD163 or CD68 and 95% CD204-expressing macrophages reacted to CD163 or CD68. These findings showed that both M1-and M2-macrophages contributed to the development of hepatic lesions induced by D-GalN and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization.
Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.
Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68+ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-g and TNF-α, whereas CD163+ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204+ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B+ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.
Background We aimed to elucidate differences in the characteristics of patients with coronavirus disease 2019 (COVID-19) requiring hospitalization in Japan, by COVID-19 waves, from conventional strains to the Delta variant. Methods We used secondary data from a database and performed a retrospective cohort study that included 3261 patients aged ≥ 18 years enrolled from 78 hospitals that participated in the Japan COVID-19 Task Force between February 2020 and September 2021. Results Patients hospitalized during the second (mean age, 53.2 years [standard deviation {SD}, ± 18.9]) and fifth (mean age, 50.7 years [SD ± 13.9]) COVID-19 waves had a lower mean age than those hospitalized during the other COVID-19 waves. Patients hospitalized during the first COVID-19 wave had a longer hospital stay (mean, 30.3 days [SD ± 21.5], p < 0.0001), and post-hospitalization complications, such as bacterial infections (21.3%, p < 0.0001), were also noticeable. In addition, there was an increase in the use of drugs such as remdesivir/baricitinib/tocilizumab/steroids during the latter COVID-19 waves. In the fifth COVID-19 wave, patients exhibited a greater number of presenting symptoms, and a higher percentage of patients required oxygen therapy at the time of admission. However, the percentage of patients requiring invasive mechanical ventilation was the highest in the first COVID-19 wave and the mortality rate was the highest in the third COVID-19 wave. Conclusions We identified differences in clinical characteristics of hospitalized patients with COVID-19 in each COVID-19 wave up to the fifth COVID-19 wave in Japan. The fifth COVID-19 wave was associated with greater disease severity on admission, the third COVID-19 wave had the highest mortality rate, and the first COVID-19 wave had the highest percentage of patients requiring mechanical ventilation.
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