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

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 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

The heterogeneity of mast cell tryptase from human lung and skin

There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9–12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis–Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues

Matrix metalloproteinase-2 from bronchial epithelial cells induces the proliferation of subepithelial fibroblasts

BackgroundIn bronchial asthma, subepithelial fibrosis in the conducting airways is associated with increased numbers of subepithelial fibroblasts.ObjectiveThis study examined the hypothesis that MMP-2 from airway epithelial cells induces the proliferation of subepithelial fibroblasts.MethodsUsing primary bronchial epithelial cells MMP-2, MT1-MMP and TIMP-2 mRNA expression were assessed by Northern blotting and RT-PCR. Primary bronchial epithelial cells transfected with constructs encoding pro-MMP-2 and MT1-MMP (MMP-14).ResultsTransfected cells showed enhanced expression of the appropriate mRNA species by RT-PCR and enhanced MMP-2 or MT1-MMP activity by zymography. Active MMP-2 levels in epithelial supernatants were increased most by cotransfection with pro-MMP-2 and MT1-MMP encoding constructs. By measuring tritiated thymidine incorporation, supernatants from transfected cells were found to enhance DNA synthesis of primary airway fibroblast cultures compared with controls. There was a strong correlation (r = 0.9, P < 0.01) between MMP-2 levels in epithelial cell conditioned media and fibroblast proliferation as indicated by DNA synthesis. The MMP inhibitor 1,10-phenanthroline attenuated the increased proliferation, while the addition of exogenous purified MMP-2 alone also increased fibroblast proliferation.ConclusionsOur results support a role for MMP-2 in mediating cross-talk between epithelial cells and myofibroblasts

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the potent collagen-use inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP- 1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasing concentrations with duration of culture on plastic. Expression of TIMP-1, interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CCl4 administration) by ribonuclease protection assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CCl4 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrast, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and Kupffer cells freshly isolated from livers after acute CCl4 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.Articl

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors

Background: Activated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue inhibitors, the TIMPs.Aims: To test the hypothesis that HSC apoptosis is critical to recovery from biliary fibrosis and that soluble growth factors may regulate HSC survival and apoptosis.Methods: Rats (n=15) were subjected to bile duct ligation for 21 days, after which biliodigestive anastomosis was undertaken (n=13). Livers were harvested at fixed time points of recovery for periods of up to 42 days. Numbers of activated HSCs were quantified after alpha smooth muscle actin staining and HSC apoptosis was detected by terminal UDP-nick end labelling (TUNEL) staining and quantified at each time point. HSC apoptosis was quantified in vitro in the presence or absence of insulin-like growth factor (IGF)-1, IGF-2, platelet derived growth factor (PDGF), and transforming growth factor beta 1 (TGF-beta 1).Results: Following biliodigestive anastomosis after 21 days of bile duct ligation, rat liver demonstrated a progressive resolution of biliary fibrosis over 42 days, associated with a fivefold decrease in activated HSC determined by alpha smooth muscle actin staining. TUNEL staining indicated that loss of activated HSC resulted from an increase in the rate of apoptosis during the first two days post biliodigestive anastomosis. Serum deprivation and culture in the presence of 50 µM cycloheximide was associated with an increase in HSC apoptosis which was significantly inhibited by addition of 10 ng/ml and 100 ng/ml IGF-1, respectively (0.05>p, n=5). In contrast, 1 and 10 ng/ml of TGF-beta 1 caused a significant increase in HSC apoptosis compared with serum free controls (p<0.05, n=4). PDGF and IGF-2 were neutral with respect to their effect on HSC apoptosis.Conclusion: HSC apoptosis plays a critical role in the spontaneous recovery from biliary fibrosis. Both survival and apoptosis of HSC are regulated by growth factors expressed during fibrotic liver injury

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

Background: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis.Aims: To determine the effects of relaxin on activated HSC.Methods: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta 1 (TGF-beta 1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively.Results: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta 1 mRNA expression.Conclusion: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation