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

Changing route: aerosol vaccine against tuberculosis

n/

RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

<div><p>RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of <i>RAGE</i> or dominant-negative-<i>RAGE</i> (DN-<i>RAGE</i>) demonstrated that <i>RAGE</i> inhibited cartilaginous matrix production, while DN-<i>RAGE</i> promoted production. Additionally, <i>RAGE</i> regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for <i>RAGE</i>-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor <i>I-κB-SR</i> inhibited <i>RAGE</i>-induced NF-<i>κ</i>B activation, but did not influence inhibition of cartilaginous matrix production by <i>RAGE</i>. The inhibitory action of <i>RAGE</i> was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, <i>L63RhoA</i>, <i>L61Rac</i>, and <i>L61Cdc42</i>, but not by <i>I-κB-SR</i>. <i>Cdx1</i> induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of <i>RAGE</i>. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-<i>κ</i>B-independent and Rho family GTPases-dependent mechanisms.</p></div

From molecules to Young Stellar Clusters: the star formation cycle across the M33 disk

To shed light on the time evolution of local star formation episodes in M33, we study the association between 566 Giant Molecular Clouds (GMCs), identified through the CO (J=2-1) IRAM-all-disk survey, and 630 Young Stellar Cluster Candidates (YSCCs), selected via Spitzer-24~$\mu$m emission. The spatial correlation between YSCCs and GMCs is extremely strong, with a typical separation of 17~pc, less than half the CO(2--1) beamsize, illustrating the remarkable physical link between the two populations. GMCs and YSCCs follow the HI filaments, except in the outermost regions where the survey finds fewer GMCs than YSCCs, likely due to undetected, low CO-luminosity clouds. The GMCs have masses between 2$\times 10^4$ and 2$\times 10^6$ M$_\odot$ and are classified according to different cloud evolutionary stages: inactive clouds are 32$\%$ of the total, classified clouds with embedded and exposed star formation are 16$\%$ and 52$\%$ of the total respectively. Across the regular southern spiral arm, inactive clouds are preferentially located in the inner part of the arm, possibly suggesting a triggering of star formation as the cloud crosses the arm. Some YSCCs are embedded star-forming sites while the majority have GALEX-UV and H$\alpha$ counterparts with estimated cluster masses and ages. The distribution of the non-embedded YSCC ages peaks around 5~Myrs with only a few being as old as 8--10~Myrs. These age estimates together with the number of GMCs in the various evolutionary stages lead us to conclude that 14~Myrs is a typical lifetime of a GMC in M33, prior to cloud dispersal. The inactive and embedded phases are short, lasting about 4 and 2~Myrs respectively. This underlines that embedded YSCCs rapidly break out from the clouds and become partially visible in H$\alpha$ or UV long before cloud dispersal

Cdx1 regulated chondrocytes differentiation.

<p>(A, B) Cdx localization in fetal skeleton. Immunohistochemical analysis was performed using serial section as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone-0108819-g001" target="_blank">Fig.1D, E</a>, Cdx1 expressed from prehypertrophic to hypertrophic zone of femur (double-headed arrow) and B showed magnified image indicated as square in A. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone.0108819.s006" target="_blank">figure S6</a>. C, The interaction between Cdx1 and Ihh promoter by ChIP analysis. Fragmented chromatins of MOCK or <i>Cdx1</i> cells in ATDC5 were immunoprecipitated by FLAG M2 beads. Amplification input DNA was constant. Immunoprecipitated DNA-derived amplification by Flag-M2 beads was detected only in <i>Cdx1</i> cells. (D, E) Ihh and Col10a1 promoter regulation by <i>Cdx1</i>. ATDC5 cells were transiently transfected with indicated promoter constructs with pRL-CMV. DLA was performed 24-h after transfection. Elevated promoter activities of Ihh (D) and Col10a1 (E) in <i>Cdx1</i> cells were observed compared to MOCK control. **P<0.001 vs MOCK. Values are expressed as the mean ±SEM of 4 wells. (F-H) Ihh and Col10a1 regulated by <i>Cdx1</i>. mRNA expression levels were monitored by real-time PCR analysis. **P<0.001 vs MOCK. Values are expressed as the mean ±SEM of 4 wells. I, Cdx1 restored cartilaginous matrix production inhibited by RAGE. Respective adenoviruses were infected at approximately 25 MOI at same time. Similar results were obtained from additional three experiments.</p

NF-κB-independent and Rho family GTPases-dependent mechanisms.

<p>A, RAGE independency of AGEs-induced cartilaginous matrix production. Cells were infected with indicated adenoviruses at approximately 50MOI, then treated with BSA and AGE. 2 days after, cells were stained by alucian blue. B, The inhibitory action to NF-κB activities by <i>I-κB-SR</i>-Ad. Respective adenoviruses were infected at approximately 50 MOI to established stable transfected cells of NF-κB-luc in ATDC5 cells. 24-h after infection, cells were lyzed and analyzed NF-κB activities. Relative luciferase units (RLU) were shown. *P<0.05 vs GFP-Ad, †P<0.05, ††P<0.001 vs <i>RAGE</i>-Ad. n = 8. C, <i>I-κB-SR</i>-Ad failed to rescue <i>RAGE</i>-dependent cartilaginous matrix production in micromass culture. 2 days after infection at approximately 25 MOI, cells were stained by alucian blue. (D,E) Toxin B restored Ihh and Col10a1 promoter activities downregulated by RAGE. ATDC5 cells were transfected with Ihh p1312-luc (D) or Col10a1-luc (E) with pRL-CMV, and respective adenoviruses were infected at approximately 50 MOI at same time. 3-h after gene transfer, cells were treated with 100 pg/ml Toxin B. Values are expressed as the mean ±SEM of 4–8 wells. *P<0.05, **P<0.001 vs GFP-Ad without Toxin B, †P<0.001 vs <i>RAGE</i>-Ad without Toxin B. F, Toxin B restored the inhibition of <i>RAGE</i>-dependent cartilaginous matrix production in micromass culture. Respective adenoviruses were infected at approximately 50 MOI. 3-h after infection, cells were treated with 100 pg/ml Toxin B. (G, H) <i>I-κB-SR</i>-Ad did not influence Ihh (G) and Col10a1 (H) expressions in ATDC5 cells. 2 days after infection at approximately 25 MOI, total RNAs were prepared. *P<0.05 vs GFP-Ad, †P<0.05 vs <i>RAGE</i>-Ad. n = 4. (I, J) Rho GTP ases activation causes the reduction of Ihh and Col10a1 in ATDC5 cells. Expression levels of Ihh (J) and Col10a1 (K) transcripts were monitored by real-time PCR analysis. After confluency, total RNAs were prepared from respective stable transfected cells. Values are expressed as the mean ±SEM of 4 wells. **P<0.001 vs GFP. Similar results were obtained from additional three experiments.</p

RAGE-dependent inhibition of chondrocytes differentiation rescued by RA.

<p>A, RAGE-induced inhibition of cartilaginous matrix production was rescued by RA. Indicated concentrations of RA were added 3 hours after infection of GFP-Ad, or <i>RAGE</i>-Ad at approximately 50 MOI. 2-days later, alucian blue staining was performed and quantitative data were obtained and summarized in B. *P<0.001 vs GFP, †P<0.001 vs <i>RAGE</i>-Ad without RA. Values are expressed as the mean ±SEM of 4 wells. C, Transient induction of Cdx1 by RA. Immunoblot analysis showed that 0.1 µM RA maximally stimulated Cdx1 at 3 h after treatment. D, Comparison of Cdx1 induction in GFP and <i>RAGE</i> cells. Using established stable transfected cells, Cdx1 induction by RA treatment was examined by immunoblot analysis. After confluency, GFP or <i>RAGE</i> cells were treated for 3 h in the absence or the presence of 0.1 µM RA. E, Downregulated Ihh promoter activity by RAGE was partially recovered by RA. 0.1 µM RA was added at 3-h after infection of indicated adenoviruses. *P<0.05 vs GFP-Ad,†P<0.05 vs <i>RAGE</i>-Ad. n = 4.</p

Relationship between Cdx and Ihh, and downregulation by RAGE.

<p>A, Ihh promoter activities regulated by RAGE. Cells were transfected with indicated Ihh-luc constructs (left panel). 3-h after transfection, cells were infected with GFP-Ad, <i>RAGE</i>-Ad, or DN-<i>RAGE</i>-Ad at approximately 50MOI. Ihh promoter activities reduced by <i>RAGE</i>-Ad and increased by DN-<i>RAGE</i>-Ad were monitored in p1312-luc and p994-luc, while in p597-luc, <i>RAGE</i>-Ad and DN-<i>RAGE</i>-Ad did not influence to promoter activity (right panel). Values are expressed as the mean ±SEM of 4–8 wells. *P<0.05 vs GFP-Ad. (B–D) Cdxs regulation by RAGE. cDNA pool was used same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone-0108819-g002" target="_blank">Fig.2</a>. *P<0.05 vs GFP-Ad, †P<0.05 vs <i>RAGE</i>-Ad. Values are expressed as the mean ±SEM of 4 wells. (E–G) NF-κB-independent downregulation of Cdxs by RAGE. cDNA pool was used same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone-0108819-g003" target="_blank">Fig.3G and H</a>. *P<0.05 vs GFP-Ad,†P<0.05 vs <i>RAGE</i>-Ad. Values are expressed as the mean ±SEM of 4 wells. (H–J) Rho GTP ases activation cause the reduction of Cdxs. cDNA pool was used same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone-0108819-g003" target="_blank">Fig.3I and J</a>. **P<0.001 vs GFP. Values are expressed as the mean ±SEM of 4 wells. Similar results were obtained from additional three experiments.</p

Cartilaginous matrix production and gene regulation by RAGE.

<p>(A, B) RAGE did not modulate ALP activities. A, Established stable transfected cells were stained by ALP in ATDC5 cells. ALP activity levels were not influenced by <i>RAGE</i> or DN-<i>RAGE</i>. Transgene levels are shown in B. (C, D) RAGE inhibited cartilaginous matrix production. Staining procedures were mentioned in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#s2" target="_blank">Materials and Methods</a>”. In micromass culture, cells were infected with adenoviruses expressing GFP (GFP-Ad), <i>RAGE</i> (<i>RAGE</i>-Ad), or DN-<i>RAGE</i> (DN-<i>RAGE</i>-Ad) at approximately 50MOI. Transgene levels are shown in D. The value are normalized against Gapdh. (E–P) ATDC5 cells were infected by GFP-Ad, <i>RAGE</i>-Ad, or DN-<i>RAGE</i>-Ad. At day 2 post infection, mRNA expression levels were monitored by real time-PCR analysis using respective specific primer pairs described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#s2" target="_blank">Materials and Methods</a>”. Values are expressed as the mean ±SEM of 4 wells. *P<0.05 vs GFP-Ad, †P<0.001 vs <i>RAGE</i>-Ad. Similar results were obtained from additional three experiments.</p

RAGE expressed in chondrocytes both in <i>in vitro</i> and <i>in vivo</i>.

<p>RAGE expression in primary chondrocytes (A), and in chondrogenic ATDC5 (B). Staining procedures were described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#s2" target="_blank">Materials and Methods</a>”. RAGE antibody positive reactions (Green) were observed in membranous regions. Cells were counter stained by DAPI (Blue). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone.0108819.s005" target="_blank">figure S5</a>. C, RAGE expressions in cultured cells. Primary chondrocytes (PC) and ATDC5 cells were cultured up to confluent and then cells were lyzed. Immunoblot analysis was performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#s2" target="_blank">Materials and Methods</a>”. (D, E) RAGE expression in cartilage. Immunostaining of hindlimb isolated from mouse embryos at 15.5 days post-gestation at low (D) and high (E) magnification (indicated as square in D). Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108819#pone.0108819.s005" target="_blank">figure S5</a>. RAGE expressed from prehypertrophic to hypertrophic zone of femur (double-headed arrow). The arrow shows area of non-specific reaction. F, AGEs (AGE) stimulated chondrogenic ATDC5 cells proliferation. ATDC5 cells were treated with indicated concentrations of BSA or AGE-BSA (AGE) for 48-h, and cell proliferation was evaluated by WST-8 incorporation calorimetrically. Values are expressed as the mean ±SEM of 8–16 wells. *P<0.05 vs BSA alone. (G, H) RAGE dependency of AGEs-induced ATDC5 cells proliferation. To examine RAGE-dependency, we established stable transfected cells by retrovirus infection. G, Respective gene-transferred cells were treated with indicated concentrations of BSA or AGE and cell proliferation were examined. Increased cell proliferation by AGE compared to BSA control in GFP cells was reproduced in <i>RAGE</i>, or DN-<i>RAGE</i> cells at similar levels. Values are expressed as the mean ±SEM of 8–16 wells. *P<0.05 vs BSA alone. The expression levels of RAGE in each infectants are shown in H. (I–J) RAGE promoter activity and RAGE expression regulated by RAGE in ATDC5 cells. I, Respective cells were treated with indicated concentrations of BSA or AGE 3-h after transfection. Dual luciferase activities (DLA) were evaluated at 24-h after transfection. AGEs did not promote RAGE promoter activity in GFP cells, and stimulated in <i>RAGE</i> cells significantly. Suppressed RAGE promoter activity was observed in DN-<i>RAGE</i> cells. Values are expressed as the mean ±SEM of 8 wells. J, Endogenous RAGE expression levels in response to AGE. Cells were treated with indicated concentrations of BSA or AGE. RAGE expression levels were examined by real-time-PCR using primer pair designed to detect endogenous RAGE expression. The value corrected by Gapdh, was expressed as 1 GFP control. Values are expressed as the mean ±SEM of 4 wells. *P<0.05, **P<0.005 vs BSA in GFP cells, †P<0.05 vs AGE in GFP cells. Similar results were obtained from additional three experiments.</p