Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Characterization of vasorelaxant responses to anandamide in the rat mesenteric arterial bed

The endogenous cannabinoid anandamide has recently been identified as a vasorelaxant but the underlying mechanisms are controversial. The vasorelaxant responses to anandamide have now been examined in the rat mesenteric arterial bed. Anandamide caused potent vasorelaxations (pD2 = 6.24 ± 0.06; Rmax = 89.4 ± 2.2 %) which were unaffected by inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester (l-NAME; 300 μm). The responses were also predominantly endothelium independent and were unaffected by the cannabinoid CB1 receptor antagonist SR141716A (1 μm), although at higher concentrations (3 and 10 μm) SR141716A was inhibitory. Both 1 mm ouabain (pD2 = 5.90 ± 0.07; Rmax = 50.4 ± 6.5 %) and 100 μm 18α-glycyrrhetinic acid (pD2 = 6.04 ± 0.14; Rmax = 40.9 ± 5.8 %) opposed anandamide-induced vasorelaxation. However, the gap junction inhibitors carbenoxolone (100 μm) and palmitoleic acid (50 μm) did not affect vasorelaxation to anandamide. Relaxation to anandamide was significantly attenuated by both capsaicin pretreatment to deplete the sensory nerves of neurotransmitters (pD2 = 5.86 ± 0.18; Rmax = 56.3 ± 5.2 %) and the vanilloid antagonist ruthenium red (10 μm; pD2 = 5.64 ± 0.09; Rmax = 33.7 ± 3.9 %). However, these inhibitory effects were prevented by the additional presence of l-NAME, when the relaxation to anandamide was unaffected (pD2 = 6.19 ± 0.07; Rmax = 81.9 ± 2.8 %). The inhibitor of neuronal nitric oxide synthase, 7-nitroindazole, also prevented capsaicin from inhibiting the responses to anandamide. The results of this study point to anandamide acting via several mechanisms, which include the involvement of sensory nerves, but only in the presence of nitric oxide

Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1. We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol. 2. Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED(50(M)) of 0.43±0.15 nmol and a maximum relaxation (R(max(M)) of 89.6±1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED(50(F))=0.72±0.19 nmol and R(max(F))=90.7±0.9%; n=22). 3. In the males, the addition of 100 μM N(G)-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED(50(M))=6.45±3.53 nmol) and significantly (P<0.01) reduced R(max(M)) (79.7±2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED(50(F))=0.89±0.19 nmol, R(max(F))=86.9±2.3%, n=9). 4. Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (R(max(M))=40.3±9.2%, n=4; P<0.001) and female rats by 1.5 fold (R(max(F))=55.3±3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED(50(M))=0.87±0.26 nmol; P<0.01) but not the females (ED(50(F))=4.04±1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups. 5. The cannabinoid receptor antagonist SR141716A (1 μM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED(50(M))=1.41±0.74 nmol, R(max(M))=89.4±2.5%, n=7; ED(50(F))=2.17±0.95 nmol, R(max(F))=89.9±1.8%, n=9). In mesenteries from male rats, in the presence of 100 μM L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED(50(M))=53.8±36.8 nmol) without affecting R(max(M)) (72.4±4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR141716A, significantly (P<0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED(50(F))=6.66±2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased R(max(F)) (70.1±5.5%, n=8). 6. Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds. 7. The continuous presence of sodium nitroprusside (SNP; 20–60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED(50(M))=40.9±19.6 nmol, R(max(M))=79.4±2.5%, n=11). Similarly in mesenteries from female rats, the ED(50(F)) was also significantly (P<0.01) increased 7 fold (6.24±2.02 nmol), while the R(max(F)) was unaffected (81.9±11.0%; n=4). 8. The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males

Characterization and modulation of EDHF-mediated relaxations in the rat isolated superior mesenteric arterial bed

1. We have used the isolated, buffer-perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mM K(+)) to characterize nitric oxide (NO)-independent vasorelaxation which is thought to be mediated by the endothelium-derived hyperpolarizing factor (EDHF). 2. The muscarinic agonists carbachol, acetylcholine (ACh) and methacholine caused dose-related relaxations in preconstricted preparations with ED(50) values of 0.18±0.04 nmol (n=8), 0.05±0.02 nmol (n=6) and 0.26±0.16 nmol (n=5), respectively. In the same preparations N(G)-nitro-L-arginine methyl ester (L-NAME, 100 μM) significantly (P<0.05) decreased the potency of all the agents (ED(50) values in the presence of L-NAME: carbachol, 0.66±0.11 nmol; ACh, 0.28±0.10 nmol; methacholine, 1.97±1.01 nmol). The maximal relaxation to ACh was also significantly (P<0.05) reduced (from 85.3±0.9 to 73.2±3.7%) in the presence of L-NAME. The vasorelaxant effects of carbachol were not significantly altered by the cyclo-oxygenase inhibitor indomethacin (10 μM; n=4). 3. The K(+) channel blocker, tetraethylammonium (TEA, 10 mM) also significantly (P<0.001) reduced both the potency of carbachol (ED(50)=1.97±0.14 nmol in presence of TEA) and the maximum relaxation (R(max)=74.6±3.2% in presence of TEA, P<0.05, n=3). When TEA was added in the presence of L-NAME (n=4), there was a further significant (P<0.001) decrease in the potency of carbachol (ED(50)=22.4±13.5 nmol) relative to that in the presence of L-NAME alone, and R(max) was also significantly (P<0.05) reduced (74.6±4.2%). The ATP-sensitive K(+) channel inhibitor, glibenclamide (10 μM), had no effect on carbachol-induced relaxation (n=9). 4. High extracellular K(+) (60 mM) significantly (P<0.01) reduced the potency of carbachol (n=5) by 5 fold (ED(50): control, 0.16±0.04 nmol; high K(+), 0.88±0.25 nmol) and the R(max) was also significantly (P<0.01) reduced (control, 83.4±2.7%; high K(+), 40.3±9.2%). The residual vasorelaxation to carbachol in the presence of high K(+) was abolished by L-NAME (100 μM; n=5). In preparations preconstricted with high K(+), the potency of sodium nitroprusside was not significantly different from that in preparations precontracted with methoxamine, though the maximal response was reduced (62.4±3.4% high K(+), n=7; 83.1±3.1% control, n=7). 5. In the presence of the cytochrome P450 inhibitor, clotrimazole (1 μM, n=5 and 10 μM, n=4), the dose-response curve to carbachol was significantly shifted to the right 2 fold (P<0.05) and 4 fold (P<0.001) respectively, an effect which was further enhanced in the presence of L-NAME. R(max) was significantly (P<0.01) reduced by the presence of 10 μM clotrimazole alone, being 86.9±2.5% in its absence and 61.8±7.8% in its presence (n=6). 6. In the presence of the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (6 μM), the inhibitory effects of L-NAME on carbachol-induced relaxation were substantially enhanced (ED(50): L-NAME alone, 0.52±0.11 nmol, n=5; L-NAME+8-bromo cyclic GMP, 1.42±0.28 nmol, n=7. R(max): L-NAME alone, 82.2±2.4%; L-NAME+8-bromo cyclic GMP, 59.1±1.8%. P<0.001). These results suggest that the magnitude of the NO-independent component of vasorelaxation is reduced when functional cyclic GMP levels are maintained, suggesting that basal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium-dependent relaxations becomes functionally greater. 7. The present investigation demonstrates that muscaranic receptor-induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO-dependent and independent mechanisms. The L-NAME-insensitive mechanism, most probably occurs via activation of a K(+) conductance and shows the characteristics of EDHF-mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and this may compensate for the loss of NO

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease