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1.
iScience ; 25(5): 104223, 2022 May 20.
Article in English | MEDLINE | ID: covidwho-1783436

ABSTRACT

The effect of SARS-CoV-2 infection on placental function is not well understood. Analysis of placentas from women who tested positive at delivery showed SARS-CoV-2 genomic and subgenomic RNA in 22 out of 52 placentas. Placentas from two mothers with symptomatic COVID-19 whose pregnancies resulted in adverse outcomes for the fetuses contained high levels of viral Alpha variant RNA. The RNA was localized to the trophoblasts that cover the fetal chorionic villi in direct contact with maternal blood. The intervillous spaces and villi were infiltrated with maternal macrophages and T cells. Transcriptome analysis showed an increased expression of chemokines and pathways associated with viral infection and inflammation. Infection of placental cultures with live SARS-CoV-2 and spike protein-pseudotyped lentivirus showed infection of syncytiotrophoblast and, in rare cases, endothelial cells mediated by ACE2 and Neuropilin-1. Viruses with Alpha, Beta, and Delta variant spikes infected the placental cultures at significantly greater levels.

2.
Elife ; 112022 03 23.
Article in English | MEDLINE | ID: covidwho-1786253

ABSTRACT

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.


Subject(s)
COVID-19 , SARS-CoV-2 , Blood Coagulation Factors , Humans , Spike Glycoprotein, Coronavirus , Virus Internalization
3.
JCI Insight ; 7(5)2022 03 08.
Article in English | MEDLINE | ID: covidwho-1759583

ABSTRACT

Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram - an FDA-approved drug for alcohol use disorder - dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2-infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and it downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for patients with COVID-19. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in 2 rodent models of lung injury for which treatment options are limited.


Subject(s)
Acute Lung Injury/drug therapy , COVID-19/complications , Disulfiram/pharmacology , Extracellular Traps/drug effects , Lung/immunology , SARS-CoV-2 , Acetaldehyde Dehydrogenase Inhibitors/pharmacology , Acute Lung Injury/etiology , Animals , COVID-19/virology , Disease Models, Animal , Extracellular Traps/immunology , Rodentia
5.
Circ Res ; 130(7): 963-977, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1731376

ABSTRACT

BACKGROUND: Increasing evidence suggests that cardiac arrhythmias are frequent clinical features of coronavirus disease 2019 (COVID-19). Sinus node damage may lead to bradycardia. However, it is challenging to explore human sinoatrial node (SAN) pathophysiology due to difficulty in isolating and culturing human SAN cells. Embryonic stem cells (ESCs) can be a source to derive human SAN-like pacemaker cells for disease modeling. METHODS: We used both a hamster model and human ESC (hESC)-derived SAN-like pacemaker cells to explore the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pacemaker cells of the heart. In the hamster model, quantitative real-time polymerase chain reaction and immunostaining were used to detect viral RNA and protein, respectively. We then created a dual knock-in SHOX2:GFP;MYH6:mCherry hESC reporter line to establish a highly efficient strategy to derive functional human SAN-like pacemaker cells, which was further characterized by single-cell RNA sequencing. Following exposure to SARS-CoV-2, quantitative real-time polymerase chain reaction, immunostaining, and RNA sequencing were used to confirm infection and determine the host response of hESC-SAN-like pacemaker cells. Finally, a high content chemical screen was performed to identify drugs that can inhibit SARS-CoV-2 infection, and block SARS-CoV-2-induced ferroptosis. RESULTS: Viral RNA and spike protein were detected in SAN cells in the hearts of infected hamsters. We established an efficient strategy to derive from hESCs functional human SAN-like pacemaker cells, which express pacemaker markers and display SAN-like action potentials. Furthermore, SARS-CoV-2 infection causes dysfunction of human SAN-like pacemaker cells and induces ferroptosis. Two drug candidates, deferoxamine and imatinib, were identified from the high content screen, able to block SARS-CoV-2 infection and infection-associated ferroptosis. CONCLUSIONS: Using a hamster model, we showed that primary pacemaker cells in the heart can be infected by SARS-CoV-2. Infection of hESC-derived functional SAN-like pacemaker cells demonstrates ferroptosis as a potential mechanism for causing cardiac arrhythmias in patients with COVID-19. Finally, we identified candidate drugs that can protect the SAN cells from SARS-CoV-2 infection.


Subject(s)
COVID-19 , Ferroptosis , Humans , Myocytes, Cardiac/metabolism , SARS-CoV-2 , Sinoatrial Node/metabolism
6.
Cell Rep Med ; 3(2): 100522, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1650891

ABSTRACT

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.


Subject(s)
COVID-19/genetics , COVID-19/pathology , Lung/pathology , SARS-CoV-2 , Transcriptome/genetics , Adult , Aged , Aged, 80 and over , COVID-19/metabolism , COVID-19/virology , Case-Control Studies , Cohort Studies , Female , Gene Expression Regulation , Humans , Influenza, Human/genetics , Influenza, Human/pathology , Influenza, Human/virology , Lung/metabolism , Male , Middle Aged , Orthomyxoviridae , RNA-Seq/methods , Respiratory Distress Syndrome/genetics , Respiratory Distress Syndrome/microbiology , Respiratory Distress Syndrome/pathology , Viral Load
10.
J Leukoc Biol ; 110(1): 21-26, 2021 07.
Article in English | MEDLINE | ID: covidwho-1574077

ABSTRACT

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic RNA virus causing coronavirus disease 2019 (COVID-19) in humans. Although most patients with COVID-19 have mild illness and may be asymptomatic, some will develop severe pneumonia, acute respiratory distress syndrome, multi-organ failure, and death. RNA viruses such as SARS-CoV-2 are capable of hijacking the epigenetic landscape of host immune cells to evade antiviral defense. Yet, there remain considerable gaps in our understanding of immune cell epigenetic changes associated with severe SARS-CoV-2 infection pathology. Here, we examined genome-wide DNA methylation (DNAm) profiles of peripheral blood mononuclear cells from 9 terminally-ill, critical COVID-19 patients with confirmed SARS-CoV-2 plasma viremia compared with uninfected, hospitalized influenza, untreated primary HIV infection, and mild/moderate COVID-19 HIV coinfected individuals. Cell-type deconvolution analyses confirmed lymphopenia in severe COVID-19 and revealed a high percentage of estimated neutrophils suggesting perturbations to DNAm associated with granulopoiesis. We observed a distinct DNAm signature of severe COVID-19 characterized by hypermethylation of IFN-related genes and hypomethylation of inflammatory genes, reinforcing observations in infection models and single-cell transcriptional studies of severe COVID-19. Epigenetic clock analyses revealed severe COVID-19 was associated with an increased DNAm age and elevated mortality risk according to GrimAge, further validating the epigenetic clock as a predictor of disease and mortality risk. Our epigenetic results reveal a discovery DNAm signature of severe COVID-19 in blood potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against SARS-CoV-2.


Subject(s)
COVID-19/genetics , DNA Methylation/genetics , Epigenesis, Genetic , Genome, Human , COVID-19/virology , HIV Infections/genetics , Humans , Influenza, Human/genetics , SARS-CoV-2/physiology
12.
Cell Rep ; 37(6): 109920, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1530684

ABSTRACT

It is urgent to develop disease models to dissect mechanisms regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we derive airway organoids from human pluripotent stem cells (hPSC-AOs). The hPSC-AOs, particularly ciliated-like cells, are permissive to SARS-CoV-2 infection. Using this platform, we perform a high content screen and identify GW6471, which blocks SARS-CoV-2 infection. GW6471 can also block infection of the B.1.351 SARS-CoV-2 variant. RNA sequencing (RNA-seq) analysis suggests that GW6471 blocks SARS-CoV-2 infection at least in part by inhibiting hypoxia inducible factor 1 subunit alpha (HIF1α), which is further validated by chemical inhibitor and genetic perturbation targeting HIF1α. Metabolic profiling identifies decreased rates of glycolysis upon GW6471 treatment, consistent with transcriptome profiling. Finally, xanthohumol, 5-(tetradecyloxy)-2-furoic acid, and ND-646, three compounds that suppress fatty acid biosynthesis, also block SARS-CoV-2 infection. Together, a high content screen coupled with transcriptome and metabolic profiling reveals a key role of the HIF1α-glycolysis axis in mediating SARS-CoV-2 infection of human airway epithelium.


Subject(s)
COVID-19/metabolism , Glycolysis/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung/metabolism , Organoids/metabolism , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/metabolism , HEK293 Cells , Humans , Pluripotent Stem Cells/metabolism , SARS-CoV-2/pathogenicity , Transcriptome/physiology , Vero Cells
14.
Cell Metab ; 33(11): 2174-2188.e5, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1446535

ABSTRACT

Individuals infected with SARS-CoV-2 who also display hyperglycemia suffer from longer hospital stays, higher risk of developing acute respiratory distress syndrome (ARDS), and increased mortality. Nevertheless, the pathophysiological mechanism of hyperglycemia in COVID-19 remains poorly characterized. Here, we show that hyperglycemia is similarly prevalent among patients with ARDS independent of COVID-19 status. Yet among patients with ARDS and COVID-19, insulin resistance is the prevalent cause of hyperglycemia, independent of glucocorticoid treatment, which is unlike patients with ARDS but without COVID-19, where pancreatic beta cell failure predominates. A screen of glucoregulatory hormones revealed lower levels of adiponectin in patients with COVID-19. Hamsters infected with SARS-CoV-2 demonstrated a strong antiviral gene expression program in the adipose tissue and diminished expression of adiponectin. Moreover, we show that SARS-CoV-2 can infect adipocytes. Together these data suggest that SARS-CoV-2 may trigger adipose tissue dysfunction to drive insulin resistance and adverse outcomes in acute COVID-19.

15.
J Virol ; 95(23): e0125721, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1410202

ABSTRACT

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.


Subject(s)
COVID-19/metabolism , Cytokines/metabolism , Interferon Type I/metabolism , SARS-CoV-2 , Transcription Factor RelA/metabolism , Transcriptome , Virus Replication , A549 Cells , Animals , COVID-19/virology , Chlorocebus aethiops , Epigenomics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Host Microbial Interactions , Humans , Signal Transduction , Single-Cell Analysis , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factors/metabolism , Vero Cells
16.
Mol Med ; 27(1): 105, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1403209

ABSTRACT

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.


Subject(s)
Antiviral Agents/pharmacology , Atorvastatin/pharmacology , COVID-19/drug therapy , Lung/drug effects , Organoids/drug effects , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Atorvastatin/chemistry , COVID-19/prevention & control , Cell Line , Coronavirus 3C Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Doxycycline/pharmacology , Drug Approval , Drug Repositioning , Gene Expression Regulation/drug effects , Humans , Lung/virology , Models, Biological , Molecular Docking Simulation , Organoids/virology , Raloxifene Hydrochloride/chemistry , Raloxifene Hydrochloride/pharmacology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Trifluoperazine/chemistry , Trifluoperazine/pharmacology , United States , United States Food and Drug Administration , Vesiculovirus/genetics , Virus Internalization/drug effects
17.
Stem Cell Reports ; 16(9): 2274-2288, 2021 09 14.
Article in English | MEDLINE | ID: covidwho-1360129

ABSTRACT

Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology remains unknown. Here, using an established in vivo hamster model, we demonstrate that SARS-CoV-2 can be detected in cardiomyocytes of infected animals. Furthermore, we found damaged cardiomyocytes in hamsters and COVID-19 autopsy samples. To explore the mechanism, we show that both human pluripotent stem cell-derived cardiomyocytes (hPSC-derived CMs) and adult cardiomyocytes (CMs) can be productively infected by SARS-CoV-2, leading to secretion of the monocyte chemoattractant cytokine CCL2 and subsequent monocyte recruitment. Increased CCL2 expression and monocyte infiltration was also observed in the hearts of infected hamsters. Although infected CMs suffer damage, we find that the presence of macrophages significantly reduces SARS-CoV-2-infected CMs. Overall, our study provides direct evidence that SARS-CoV-2 infects CMs in vivo and suggests a mechanism of immune cell infiltration and histopathology in heart tissues of COVID-19 patients.


Subject(s)
COVID-19/pathology , Chemokine CCL2/metabolism , Heart Injuries/virology , Monocytes/immunology , Myocytes, Cardiac/metabolism , Animals , Cell Communication/physiology , Cell Line , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Humans , Macrophages/immunology , Male , Myocytes, Cardiac/virology , Pluripotent Stem Cells/cytology , Vero Cells
18.
Cell Metab ; 33(8): 1577-1591.e7, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1240259

ABSTRACT

Recent clinical data have suggested a correlation between coronavirus disease 2019 (COVID-19) and diabetes. Here, we describe the detection of SARS-CoV-2 viral antigen in pancreatic beta cells in autopsy samples from individuals with COVID-19. Single-cell RNA sequencing and immunostaining from ex vivo infections confirmed that multiple types of pancreatic islet cells were susceptible to SARS-CoV-2, eliciting a cellular stress response and the induction of chemokines. Upon SARS-CoV-2 infection, beta cells showed a lower expression of insulin and a higher expression of alpha and acinar cell markers, including glucagon and trypsin1, respectively, suggesting cellular transdifferentiation. Trajectory analysis indicated that SARS-CoV-2 induced eIF2-pathway-mediated beta cell transdifferentiation, a phenotype that could be reversed with trans-integrated stress response inhibitor (trans-ISRIB). Altogether, this study demonstrates an example of SARS-CoV-2 infection causing cell fate change, which provides further insight into the pathomechanisms of COVID-19.


Subject(s)
COVID-19/virology , Cell Transdifferentiation , Insulin-Secreting Cells/virology , SARS-CoV-2/pathogenicity , Acetamides/pharmacology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , COVID-19/mortality , Cell Transdifferentiation/drug effects , Chlorocebus aethiops , Cyclohexylamines/pharmacology , Cytokines/metabolism , Eukaryotic Initiation Factor-2/metabolism , Female , Glucagon , Host-Pathogen Interactions , Humans , Insulin/metabolism , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Male , Middle Aged , Phenotype , Signal Transduction , Tissue Culture Techniques , Trypsin/metabolism , Vero Cells , Young Adult
19.
Nature ; 595(7865): 114-119, 2021 07.
Article in English | MEDLINE | ID: covidwho-1207147

ABSTRACT

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.


Subject(s)
COVID-19/pathology , COVID-19/virology , Lung/pathology , SARS-CoV-2/pathogenicity , Single-Cell Analysis , Aged , Aged, 80 and over , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Atlases as Topic , Autopsy , COVID-19/immunology , Case-Control Studies , Female , Fibroblasts/pathology , Fibrosis/pathology , Fibrosis/virology , Humans , Inflammation/pathology , Inflammation/virology , Macrophages/pathology , Macrophages/virology , Macrophages, Alveolar/pathology , Macrophages, Alveolar/virology , Male , Middle Aged , Plasma Cells/immunology , T-Lymphocytes/immunology
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