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2.
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
3.
Nat Genet ; 53(11): 1606-1615, 2021 11.
Article in English | MEDLINE | ID: covidwho-1503871

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target.


Subject(s)
COVID-19/complications , Chromosomes, Human, Pair 3/genetics , Epithelial-Mesenchymal Transition , Lung/virology , Polymorphism, Single Nucleotide , SARS-CoV-2/isolation & purification , Transcription Factors/genetics , COVID-19/transmission , COVID-19/virology , Case-Control Studies , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Genome-Wide Association Study , Humans , Lung/metabolism , Lung/pathology , Male , Transcription Factors/metabolism
4.
Molecules ; 26(21)2021 Nov 06.
Article in English | MEDLINE | ID: covidwho-1502470

ABSTRACT

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.


Subject(s)
Respiratory Mucosa/metabolism , Tissue Engineering/methods , Trachea/transplantation , Cell Differentiation , Cell Proliferation , Epithelial Cells/metabolism , Epithelium/metabolism , Feasibility Studies , Humans , Ki-67 Antigen/analysis , Ki-67 Antigen/genetics , Mucin 5AC/analysis , Mucin 5AC/genetics , Mucous Membrane/metabolism , Primary Cell Culture/methods , Respiratory Mucosa/physiology , Trachea/metabolism , Trachea/physiology
5.
J Virol ; 95(13): e0019221, 2021 06 10.
Article in English | MEDLINE | ID: covidwho-1486499

ABSTRACT

Understanding factors that affect the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is central to combatting coronavirus disease 2019 (COVID-19). The virus surface spike protein of SARS-CoV-2 mediates viral entry into cells by binding to the ACE2 receptor on epithelial cells and promoting fusion. We found that Epstein-Barr virus (EBV) induces ACE2 expression when it enters the lytic replicative cycle in epithelial cells. By using vesicular stomatitis virus (VSV) particles pseudotyped with the SARS-CoV-2 spike protein, we showed that lytic EBV replication enhances ACE2-dependent SARS-CoV-2 pseudovirus entry. We found that the ACE2 promoter contains response elements for Zta, an EBV transcriptional activator that is essential for EBV entry into the lytic cycle of replication. Zta preferentially acts on methylated promoters, allowing it to reactivate epigenetically silenced EBV promoters from latency. By using promoter assays, we showed that Zta directly activates methylated ACE2 promoters. Infection of normal oral keratinocytes with EBV leads to lytic replication in some of the infected cells, induces ACE2 expression, and enhances SARS-CoV-2 pseudovirus entry. These data suggest that subclinical EBV replication and lytic gene expression in epithelial cells, which is ubiquitous in the human population, may enhance the efficiency and extent of SARS-CoV-2 infection of epithelial cells by transcriptionally activating ACE2 and increasing its cell surface expression. IMPORTANCE SARS-CoV-2, the coronavirus responsible for COVID-19, has caused a pandemic leading to millions of infections and deaths worldwide. Identifying the factors governing susceptibility to SARS-CoV-2 is important in order to develop strategies to prevent SARS-CoV-2 infection. We show that Epstein-Barr virus, which infects and persists in >90% of adult humans, increases susceptibility of epithelial cells to infection by SARS-CoV-2. EBV, when it reactivates from latency or infects epithelial cells, increases expression of ACE2, the cellular receptor for SARS-CoV-2, enhancing infection by SARS-CoV-2. Inhibiting EBV replication with antivirals may therefore decrease susceptibility to SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Epithelial Cells/virology , Herpesvirus 4, Human/physiology , SARS-CoV-2/physiology , Virus Internalization , Virus Replication , Angiotensin-Converting Enzyme 2/metabolism , Cell Line , DNA Methylation , Epithelial Cells/metabolism , Gene Expression Regulation , Humans , Promoter Regions, Genetic , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Trans-Activators/metabolism , Virus Activation
6.
Cells ; 10(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470800

ABSTRACT

Pulmonary epithelial cells are widely considered to be the first line of defence in the lung and are responsible for coordinating the innate immune response to injury and subsequent repair. Consequently, epithelial cells communicate with multiple cell types including immune cells and fibroblasts to promote acute inflammation and normal wound healing in response to damage. However, aberrant epithelial cell death and damage are hallmarks of pulmonary disease, with necrotic cell death and cellular senescence contributing to disease pathogenesis in numerous respiratory diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and coronavirus disease (COVID)-19. In this review, we summarise the literature that demonstrates that epithelial damage plays a pivotal role in the dysregulation of the immune response leading to tissue destruction and abnormal remodelling in several chronic diseases. Specifically, we highlight the role of epithelial-derived damage-associated molecular patterns (DAMPs) and senescence in shaping the immune response and assess their contribution to inflammatory and fibrotic signalling pathways in the lung.


Subject(s)
COVID-19/immunology , Epithelium/immunology , Idiopathic Pulmonary Fibrosis/immunology , Lung/immunology , Alarmins , Animals , Cellular Senescence , Coculture Techniques , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Fibrosis/metabolism , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Immunity , Inflammation/metabolism , Ligands , Necroptosis , Necrosis/pathology , Pulmonary Disease, Chronic Obstructive , SARS-CoV-2 , Signal Transduction
7.
Curr Issues Mol Biol ; 43(3): 1212-1225, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438531

ABSTRACT

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air-liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.


Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/metabolism , COVID-19/epidemiology , COVID-19/metabolism , Pandemics , Receptors, Immunologic/metabolism , SARS-CoV-2/metabolism , Severity of Illness Index , Signal Transduction/immunology , Blood Donors , Blotting, Western/methods , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Caco-2 Cells , Epithelial Cells/metabolism , Epithelial Cells/virology , Healthy Volunteers , Humans , Monocytes/metabolism , Monocytes/virology , Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
8.
Mol Biol Rep ; 48(9): 6655-6661, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1432594

ABSTRACT

Severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) is the causative agent of current coronavirus disease 2019 (COVID-19) pandemic. Electrolyte disorders particularly potassium abnormalities have been repeatedly reported as common clinical manifestations of COVID-19. Here, we discuss how SARS-CoV-2 may affect potassium balance by impairing the activity of epithelial sodium channels (ENaC). The first hypothesis could justify the incidence of hypokalemia. SARS-CoV-2 cell entry through angiotensin-converting enzyme 2 (ACE2) may enhance the activity of renin-angiotensin-aldosterone system (RAAS) classical axis and further leading to over production of aldosterone. Aldosterone is capable of enhancing the activity of ENaC and resulting in potassium loss from epithelial cells. However, type II transmembrane serine protease (TMPRSS2) is able to inhibit the ENaC, but it is utilized in the case of SARS-CoV-2 cell entry, therefore the ENaC remains activated. The second hypothesis describe the incidence of hyperkalemia based on the key role of furin. Furin is necessary for cleaving both SARS-CoV-2 spike protein and ENaC subunits. While the furin is hijacked by the virus, the decreased activity of ENaC would be expected, which causes retention of potassium ions and hyperkalemia. Given that the occurrence of hypokalemia is higher than hyperkalemia in COVID-19 patients, the first hypothesis may have greater impact on potassium levels. Further investigations are warranted to determine the exact role of ENaC in SARS-CoV-2 pathogenesis.


Subject(s)
COVID-19/metabolism , Epithelial Cells/metabolism , Epithelial Sodium Channels/metabolism , Potassium/metabolism , SARS-CoV-2/metabolism , COVID-19/virology , Epithelial Cells/virology , Furin/metabolism , Humans , Pandemics/prevention & control , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
9.
EMBO J ; 40(20): e106765, 2021 10 18.
Article in English | MEDLINE | ID: covidwho-1436404

ABSTRACT

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans and transmitted the virus to ACE2-positive cells. Notably, human primary nasal cells were infected by SARS-CoV-2, and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.


Subject(s)
COVID-19/transmission , Heparan Sulfate Proteoglycans/metabolism , Heparin, Low-Molecular-Weight/pharmacology , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , Chlorocebus aethiops , Dendritic Cells/metabolism , Dendritic Cells/virology , Epithelial Cells/metabolism , Epithelial Cells/virology , Host-Pathogen Interactions , Humans , Mucous Membrane/cytology , Mucous Membrane/virology , SARS-CoV-2/metabolism , Syndecan-1/metabolism , Syndecan-4/metabolism , Vero Cells
11.
Elife ; 102021 04 09.
Article in English | MEDLINE | ID: covidwho-1389777

ABSTRACT

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.


Subject(s)
Epithelial Cells , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Virus Cultivation/methods , Virus Internalization , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Proteolysis , Respiratory System/cytology , Respiratory System/virology , Serine Proteases/metabolism
12.
Cytokine ; 140: 155430, 2021 04.
Article in English | MEDLINE | ID: covidwho-1385381

ABSTRACT

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/prevention & control , Epithelial Cells/drug effects , Gene Expression/drug effects , Interferons/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Adult , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/virology , Cell Line, Tumor , Child , Cytokines/genetics , Epithelial Cells/metabolism , Humans , Interferons/metabolism , Lung/cytology , Middle Aged , SARS-CoV-2/physiology , Ubiquitins/genetics
13.
Proteomics ; 21(2): e2000246, 2021 01.
Article in English | MEDLINE | ID: covidwho-1384281

ABSTRACT

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PLpro ) protease and the so-called main protease (Mpro ), a chymotrypsin-like cysteine protease, also named 3CLpro or non-structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 Mpro , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coronavirus NL63, Human/enzymology , Peptide Fragments/analysis , SARS Virus/enzymology , SARS-CoV-2/enzymology , Viral Proteins/metabolism , COVID-19/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/virology , Epithelial Cells/metabolism , Epithelial Cells/virology , Eukaryotic Initiation Factor-4G/metabolism , Host-Pathogen Interactions , Humans , Lung/metabolism , Lung/virology , Substrate Specificity
14.
Sci Rep ; 11(1): 6621, 2021 03 23.
Article in English | MEDLINE | ID: covidwho-1387468

ABSTRACT

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.


Subject(s)
Epithelial Cells/metabolism , Proteome/analysis , Proteomics/methods , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Cell Culture Techniques , Cell Line , Culture Media/chemistry , Epithelial Cells/cytology , Humans , Mass Spectrometry , Models, Biological , Principal Component Analysis , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology
15.
Sci Rep ; 11(1): 2459, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1387462

ABSTRACT

A deeper understanding of the molecular biology of SARS-CoV-2 infection, including the host response to the virus, is urgently needed. Commonalities exist between the host immune response to viral infections and cancer. Here, we defined transcriptional signatures of SARS-CoV-2 infection involving hundreds of genes common across lung adenocarcinoma cell lines (A549, Calu-3) and normal human bronchial epithelial cells (NHBE), with additional signatures being specific to one or both adenocarcinoma lines. Cross-examining eight transcriptomic databases, we found that host transcriptional responses of lung adenocarcinoma cells to SARS-CoV-2 infection shared broad similarities with host responses to multiple viruses across different model systems and patient samples. Furthermore, these SARS-CoV-2 transcriptional signatures were manifested within specific subsets of human cancer, involving ~ 20% of cases across a wide range of histopathological types. These cancer subsets show immune cell infiltration and inflammation and involve pathways linked to the SARS-CoV-2 response, such as immune checkpoint, IL-6, type II interferon signaling, and NF-κB. The cell line data represented immune responses activated specifically within the cancer cells of the tumor. Common genes and pathways implicated as part of the viral host response point to therapeutic strategies that may apply to both SARS-CoV-2 and cancer.


Subject(s)
COVID-19/genetics , Host Microbial Interactions/physiology , SARS-CoV-2/physiology , A549 Cells , Bronchi/metabolism , COVID-19/metabolism , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Immunity , Lung Neoplasms/pathology , Lung Neoplasms/virology , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Transcription, Genetic , Transcriptome , Virus Replication/genetics
16.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1387615

ABSTRACT

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Proteolysis , Serine Proteases/metabolism , A549 Cells , Cells, Cultured , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Domains , Protein Transport , Respiratory Mucosa/cytology , Serine Proteases/chemistry , Serine Proteases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Trypsin/metabolism
17.
Nat Commun ; 12(1): 5148, 2021 08 26.
Article in English | MEDLINE | ID: covidwho-1376197

ABSTRACT

Coronavirus infection in humans is usually associated to respiratory tract illnesses, ranging in severity from mild to life-threatening respiratory failure. The aryl hydrocarbon receptor (AHR) was recently identified as a host factor for Zika and dengue viruses; AHR antagonists boost antiviral immunity, decrease viral titers and ameliorate Zika-induced pathology in vivo. Here we report that AHR is activated by infection with different coronaviruses, potentially impacting antiviral immunity and lung epithelial cells. Indeed, the analysis of single-cell RNA-seq from lung tissue detected increased expression of AHR and AHR transcriptional targets, suggesting AHR signaling activation in SARS-CoV-2-infected epithelial cells from COVID-19 patients. Moreover, we detected an association between AHR expression and viral load in SARS-CoV-2 infected patients. Finally, we found that the pharmacological inhibition of AHR suppressed the replication in vitro of one of the causative agents of the common cold, HCoV-229E, and the causative agent of the COVID-19 pandemic, SARS-CoV-2. Taken together, these findings suggest that AHR activation is a common strategy used by coronaviruses to evade antiviral immunity and promote viral replication, which may also contribute to lung pathology. Future studies should further evaluate the potential of AHR as a target for host-directed antiviral therapy.


Subject(s)
Coronavirus Infections/metabolism , Coronavirus/physiology , Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Coronavirus Infections/genetics , Coronavirus Infections/virology , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Humans , Male , Receptors, Aryl Hydrocarbon/genetics , SARS-CoV-2/physiology
18.
Viruses ; 13(8)2021 08 12.
Article in English | MEDLINE | ID: covidwho-1355052

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.


Subject(s)
Bronchi/immunology , Bronchi/virology , Immunity, Innate , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , SARS-CoV-2/immunology , Aged , Aging/immunology , Bronchi/cytology , Bronchi/metabolism , COVID-19/immunology , Cell Death/genetics , Cells, Cultured , Cellular Senescence/genetics , Cytokines/biosynthesis , Cytokines/genetics , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Humans , Inflammation , Interferons/biosynthesis , Interferons/genetics , Male , RNA-Seq , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , SARS-CoV-2/physiology , Signal Transduction/genetics
19.
FASEB J ; 35(9): e21798, 2021 09.
Article in English | MEDLINE | ID: covidwho-1334263

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.


Subject(s)
COVID-19/complications , Calgranulin A/physiology , Cytokine Release Syndrome/etiology , Inflammation/etiology , Pandemics , Respiratory Distress Syndrome/etiology , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/pathology , Calgranulin A/blood , Calgranulin A/genetics , Chemokine CXCL11/blood , Cytokine Release Syndrome/genetics , Cytokine Release Syndrome/pathology , Disaccharides/pharmacology , Disaccharides/therapeutic use , Disease Models, Animal , Drug Discovery , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Inflammation/genetics , Inflammation/pathology , Lung/metabolism , Lung/pathology , Lung/virology , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Lymphocyte Antigen 96/physiology , Macaca mulatta , Mice , Mice, Transgenic , Models, Biological , Mutation , Respiratory Distress Syndrome/genetics , Respiratory Distress Syndrome/metabolism , Species Specificity , Sugar Phosphates/pharmacology , Sugar Phosphates/therapeutic use , Toll-Like Receptor 4/physiology , Up-Regulation , Virus Internalization
20.
Int J Mol Sci ; 22(14)2021 Jul 16.
Article in English | MEDLINE | ID: covidwho-1323266

ABSTRACT

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.


Subject(s)
Airway Remodeling/drug effects , Alveolar Epithelial Cells/drug effects , Cigarette Smoking/adverse effects , Epithelial Cells/metabolism , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/pathology , Cell Differentiation/drug effects , Cells, Cultured , Cigarette Smoking/metabolism , Epithelial Cells/drug effects , Humans , Neoplasms, Basal Cell/metabolism , Primary Cell Culture , Pulmonary Disease, Chronic Obstructive/etiology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Smoke , Smoking/adverse effects , Smoking/metabolism
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