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1.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1702075

ABSTRACT

BACKGROUND: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. METHODS: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n = 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). RESULTS: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. CONCLUSIONS: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.


Subject(s)
Lung/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Cytokines/metabolism , Gene Expression Profiling , Humans , Models, Biological , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism
2.
Expert Rev Proteomics ; 18(11): 925-938, 2021 11.
Article in English | MEDLINE | ID: covidwho-1528086

ABSTRACT

INTRODUCTION: The outbreak of the newly discovered human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has disrupted the normal life of almost every civilization worldwide. Studies have shown that the coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 can affect multiple human organs and physiological systems, but the respiratory system remains the primary location for viral infection. AREAS COVERED: We summarize how omics technologies are used in SARS-CoV-2 research and specifically review the current knowledge of COVID-19 from the aspect of human bronchial-pulmonary proteomics. Also, knowledge gaps in COVID-19 that can be fulfilled by proteomics are discussed. EXPERT OPINION: Overall, human bronchial-pulmonary proteomics plays an important role in revealing the dynamics, functions, tropism, and pathogenicity of SARS-CoV-2, which is crucial for COVID-19 biomarker and therapeutic target discoveries. To more fully understand the impact of COVID-19, research from various angles using multi-omics approaches should also be conducted on the lungs as well as other organs.


Subject(s)
Bronchi/metabolism , COVID-19/metabolism , Lung/metabolism , Pandemics , Proteomics , SARS-CoV-2/pathogenicity , Animals , COVID-19/virology , Humans , Molecular Targeted Therapy
3.
Clin Exp Allergy ; 52(2): 324-333, 2022 02.
Article in English | MEDLINE | ID: covidwho-1437986

ABSTRACT

BACKGROUND: Deaths attributed to Coronavirus Disease 2019 (COVID-19) are mainly due to severe hypoxemic respiratory failure. Although the inflammatory storm has been considered the main pathogenesis of severe COVID-19, hypersensitivity may be another important mechanism involved in severe cases, which have a perfect response to corticosteroids (CS). METHOD: We detected the serum level of anti-SARS-CoV-2-spike S1 protein-specific IgE (SP-IgE) and anti-SARS-CoV-2 nucleocapsid protein-specific IgE (NP-IgE) in COVID-19. Correlation of levels of specific IgE and clinical severity were analysed. Pulmonary function test and bronchial provocation test were conducted in early convalescence of COVID-19. We also obtained histological samples via endoscopy to detect the evidence of mast cell activation. RESULT: The levels of serum SP-IgE and NP-IgE were significantly higher in severe cases, and were correlated with the total lung severity scores (TLSS) and the PaO2 /FiO2 ratio. Nucleocapsid protein could be detected in both airway and intestinal tissues, which was stained positive together with activated mast cells, binded with IgE. Airway hyperresponsiveness (AHR) exists in the early convalescence of COVID-19. After the application of CS in severe COVID-19, SP-IgE and NP-IgE decreased, but maintained at a high level. CONCLUSION: Hypersensitivity may be involved in severe COVID-19.


Subject(s)
Bronchi/immunology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Duodenum/immunology , Hypersensitivity/immunology , Immunoglobulin E/immunology , Mast Cells/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Bronchi/metabolism , Bronchi/pathology , COVID-19/metabolism , COVID-19/pathology , COVID-19/physiopathology , Case-Control Studies , Coronavirus Nucleocapsid Proteins/metabolism , Duodenum/metabolism , Duodenum/pathology , Female , Humans , Hypersensitivity/metabolism , Hypersensitivity/pathology , Hypersensitivity/physiopathology , Lung/physiopathology , Male , Mast Cells/metabolism , Mast Cells/pathology , Middle Aged , Mucous Membrane/immunology , Mucous Membrane/metabolism , Mucous Membrane/pathology , Phosphoproteins/immunology , Phosphoproteins/metabolism , Recovery of Function , Respiratory Hypersensitivity/physiopathology , Retrospective Studies , SARS-CoV-2 , Severity of Illness Index , Spike Glycoprotein, Coronavirus/metabolism , Young Adult
4.
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
5.
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
6.
STAR Protoc ; 2(3): 100663, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1275773

ABSTRACT

Patients with chronic lung disease are vulnerable to getting severe diseases associated with SARS-CoV-2 infection. Here, we describe protocols for subculturing and differentiating primary normal human bronchial epithelial (NHBE) cells of patients with chronic obstructive lung disease. The differentiation of NHBE cells in air-liquid interface mimics an in vivo airway and provides an in vitro model for studying SARS-CoV-2 infection. We also describe a protocol for detecting proteins in the sectioned epithelium for detailing SARS-CoV-2 infection-induced pathobiology with a vertical view.


Subject(s)
Bronchi/metabolism , COVID-19/complications , Coronavirus Nucleocapsid Proteins/analysis , Epithelium/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , SARS-CoV-2/isolation & purification , Bronchi/pathology , Bronchi/virology , COVID-19/metabolism , COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , Epithelium/pathology , Epithelium/virology , Humans , Immunohistochemistry , Paraffin Embedding , Phosphoproteins/analysis , Phosphoproteins/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/virology , Virus Replication
7.
Int J Mol Sci ; 22(10)2021 May 20.
Article in English | MEDLINE | ID: covidwho-1244036

ABSTRACT

Genome-wide association studies (GWAS) found locus 3p21.31 associated with severe COVID-19. CCR5 resides at the same locus and, given its known biological role in other infection diseases, we investigated if common noncoding and rare coding variants, affecting CCR5, can predispose to severe COVID-19. We combined single nucleotide polymorphisms (SNPs) that met the suggestive significance level (P ≤ 1 × 10-5) at the 3p21.31 locus in public GWAS datasets (6406 COVID-19 hospitalized patients and 902,088 controls) with gene expression data from 208 lung tissues, Hi-C, and Chip-seq data. Through whole exome sequencing (WES), we explored rare coding variants in 147 severe COVID-19 patients. We identified three SNPs (rs9845542, rs12639314, and rs35951367) associated with severe COVID-19 whose risk alleles correlated with low CCR5 expression in lung tissues. The rs35951367 resided in a CTFC binding site that interacts with CCR5 gene in lung tissues and was confirmed to be associated with severe COVID-19 in two independent datasets. We also identified a rare coding variant (rs34418657) associated with the risk of developing severe COVID-19. Our results suggest a biological role of CCR5 in the progression of COVID-19 as common and rare genetic variants can increase the risk of developing severe COVID-19 by affecting the functions of CCR5.


Subject(s)
COVID-19/genetics , COVID-19/metabolism , Genetic Predisposition to Disease , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Alleles , Bronchi/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19/physiopathology , Chromosomes, Human/genetics , Cohort Studies , Computational Biology , Databases, Genetic , Genome-Wide Association Study , Genotype , Humans , Lung/metabolism , Lung/pathology , Lung/virology , Polymorphism, Single Nucleotide , Whole Exome Sequencing
8.
Am J Pathol ; 191(8): 1374-1384, 2021 08.
Article in English | MEDLINE | ID: covidwho-1240148

ABSTRACT

Patients with coronavirus disease 2019 (COVID-19) who are critically ill develop vascular complications characterized by thrombosis of small, medium, and large vessels. Dysfunction of the vascular endothelium due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated in the pathogenesis of the COVID-19 vasculopathy. Although initial reports suggested that endothelial injury was caused directly by the virus, recent studies indicate that endothelial cells do not express angiotensin-converting enzyme 2, the receptor that SARS-CoV-2 uses to gain entry into cells, or express it at low levels and are resistant to the infection. These new findings, together with the observation that COVID-19 triggers a cytokine storm capable of injuring the endothelium and disrupting its antithrombogenic properties, favor an indirect mechanism of endothelial injury mediated locally by an augmented inflammatory reaction to infected nonendothelial cells, such as the bronchial and alveolar epithelium, and systemically by the excessive immune response to infection. Herein we review the vascular pathology of COVID-19 and critically discuss the potential mechanisms of endothelial injury in this disease.


Subject(s)
COVID-19/metabolism , Cytokine Release Syndrome/metabolism , Endothelium, Vascular/injuries , Endothelium, Vascular/metabolism , SARS-CoV-2/metabolism , Thrombosis/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Bronchi/pathology , COVID-19/complications , COVID-19/pathology , COVID-19/therapy , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/therapy , Endothelium, Vascular/pathology , Humans , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Thrombosis/etiology , Thrombosis/pathology , Thrombosis/therapy
9.
J Med Virol ; 93(3): 1443-1448, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196454

ABSTRACT

Our study intended to longitudinally explore the prediction effect of immunoglobulin A (IgA) on pulmonary exudation progression in COVID-19 patients. The serum IgA was tested with chemiluminescence method. Autoregressive moving average model was used to extrapolate the IgA levels before hospital admission. The positive rate of IgA and IgG in our cohort was 97% and 79.0%, respectively. In this study, the IgA levels peaks within 10-15 days after admission, while the IgG levels peaks at admission. We found that the time difference between their peaks was about 10 days. Viral RNA detection results showed that the positive rate in sputum and feces were the highest. Blood gas analysis showed that deterioration of hypoxia with the enlargement of pulmonary exudation area. And alveolar-arterial oxygen difference and oxygenation index were correlated with IgA and IgG. The results of biopsy showed that the epithelium of lung was exfoliated and the mucosa was edematous. In severe COVID-19 patients, the combination of IgA and IgG can predict the progress of pulmonary lesions and is closely related to hypoxemia and both also play an important defense role in invasion and destruction of bronchial and alveolar epithelium by SARS-CoV-2.


Subject(s)
COVID-19/pathology , COVID-19/virology , Immunoglobulin A/blood , Immunoglobulin G/blood , Sputum/virology , Aged , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Antibodies, Viral/blood , Bronchi/metabolism , Bronchi/virology , COVID-19/blood , COVID-19/metabolism , Female , Humans , Hypoxia/blood , Hypoxia/metabolism , Male , Middle Aged , Mucous Membrane/metabolism , Mucous Membrane/virology , Oxygen/metabolism , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/virology , RNA, Viral/genetics , SARS-CoV-2/genetics
10.
PLoS Comput Biol ; 17(4): e1008860, 2021 04.
Article in English | MEDLINE | ID: covidwho-1175370

ABSTRACT

The COVID-19 pandemic is posing an unprecedented threat to the whole world. In this regard, it is absolutely imperative to understand the mechanism of metabolic reprogramming of host human cells by SARS-CoV-2. A better understanding of the metabolic alterations would aid in design of better therapeutics to deal with COVID-19 pandemic. We developed an integrated genome-scale metabolic model of normal human bronchial epithelial cells (NHBE) infected with SARS-CoV-2 using gene-expression and macromolecular make-up of the virus. The reconstructed model predicts growth rates of the virus in high agreement with the experimental measured values. Furthermore, we report a method for conducting genome-scale differential flux analysis (GS-DFA) in context-specific metabolic models. We apply the method to the context-specific model and identify severely affected metabolic modules predominantly comprising of lipid metabolism. We conduct an integrated analysis of the flux-altered reactions, host-virus protein-protein interaction network and phospho-proteomics data to understand the mechanism of flux alteration in host cells. We show that several enzymes driving the altered reactions inferred by our method to be directly interacting with viral proteins and also undergoing differential phosphorylation under diseased state. In case of SARS-CoV-2 infection, lipid metabolism particularly fatty acid oxidation, cholesterol biosynthesis and beta-oxidation cycle along with arachidonic acid metabolism are predicted to be most affected which confirms with clinical metabolomics studies. GS-DFA can be applied to existing repertoire of high-throughput proteomic or transcriptomic data in diseased condition to understand metabolic deregulation at the level of flux.


Subject(s)
COVID-19/metabolism , Lung/metabolism , Models, Biological , SARS-CoV-2 , Algorithms , Biomass , Bronchi/metabolism , Bronchi/virology , COVID-19/genetics , COVID-19/virology , Cells, Cultured , Computational Biology , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Profiling , Humans , Lung/pathology , Lung/virology , Metabolic Flux Analysis/statistics & numerical data , Metabolic Networks and Pathways/genetics , Metabolomics , Pandemics , Phosphorylation , Protein Interaction Maps , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , Transcriptome
11.
Sci Rep ; 11(1): 7052, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1157913

ABSTRACT

SARS-CoV2 is a previously uncharacterized coronavirus and causative agent of the COVID-19 pandemic. The host response to SARS-CoV2 has not yet been fully delineated, hampering a precise approach to therapy. To address this, we carried out a comprehensive analysis of gene expression data from the blood, lung, and airway of COVID-19 patients. Our results indicate that COVID-19 pathogenesis is driven by populations of myeloid-lineage cells with highly inflammatory but distinct transcriptional signatures in each compartment. The relative absence of cytotoxic cells in the lung suggests a model in which delayed clearance of the virus may permit exaggerated myeloid cell activation that contributes to disease pathogenesis by the production of inflammatory mediators. The gene expression profiles also identify potential therapeutic targets that could be modified with available drugs. The data suggest that transcriptomic profiling can provide an understanding of the pathogenesis of COVID-19 in individual patients.


Subject(s)
Bronchi/metabolism , COVID-19/metabolism , Gene Expression Profiling , Lung/metabolism , Bronchoalveolar Lavage Fluid , COVID-19/blood , COVID-19/virology , Humans , Inflammation Mediators/metabolism , Myeloid Cells/metabolism , Protein Binding , SARS-CoV-2/isolation & purification
12.
J Cell Physiol ; 236(9): 6597-6606, 2021 09.
Article in English | MEDLINE | ID: covidwho-1098899

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/drug therapy , HMGB1 Protein/genetics , Proto-Oncogene Proteins c-akt/genetics , A549 Cells , Bronchi/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19/genetics , COVID-19/pathology , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation/drug effects , Humans , Lung/drug effects , Lung/pathology , RNA, Viral/genetics , Ribonucleosides/administration & dosage , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
13.
Am J Physiol Lung Cell Mol Physiol ; 320(2): L246-L253, 2021 02 01.
Article in English | MEDLINE | ID: covidwho-1088311

ABSTRACT

The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host factor proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, angiotensin-converting enzyme 2 (ACE2), and the proteases, transmembrane serine protease 2 (TMPRSS2) and furin. A number of studies have reported cell-type-specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in an air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-ß on the expression of these proteins in well-differentiated HBE cells. Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells, as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in ALI are a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that the profibrotic mediators, TGF-ß1 and TGF-ß2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.


Subject(s)
Bronchi/metabolism , COVID-19/etiology , Furin/metabolism , SARS-CoV-2 , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/cytology , Bronchi/drug effects , Cell Differentiation , Cells, Cultured , Disease Susceptibility , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Furin/genetics , Gene Expression/drug effects , Host Microbial Interactions/drug effects , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , Models, Biological , Pandemics , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transforming Growth Factor beta1/pharmacology , Transforming Growth Factor beta2/pharmacology , Virus Internalization
14.
Gastroenterology ; 160(5): 1647-1661, 2021 04.
Article in English | MEDLINE | ID: covidwho-1065985

ABSTRACT

BACKGROUND & AIMS: Gastrointestinal (GI) manifestations have been increasingly reported in patients with coronavirus disease 2019 (COVID-19). However, the roles of the GI tract in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We investigated how the GI tract is involved in SARS-CoV-2 infection to elucidate the pathogenesis of COVID-19. METHODS: Our previously established nonhuman primate (NHP) model of COVID-19 was modified in this study to test our hypothesis. Rhesus monkeys were infected with an intragastric or intranasal challenge with SARS-CoV-2. Clinical signs were recorded after infection. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction. Host responses to SARS-CoV-2 infection were evaluated by examining inflammatory cytokines, macrophages, histopathology, and mucin barrier integrity. RESULTS: Intranasal inoculation with SARS-CoV-2 led to infections and pathologic changes not only in respiratory tissues but also in digestive tissues. Expectedly, intragastric inoculation with SARS-CoV-2 resulted in the productive infection of digestive tissues and inflammation in both the lung and digestive tissues. Inflammatory cytokines were induced by both types of inoculation with SARS-CoV-2, consistent with the increased expression of CD68. Immunohistochemistry and Alcian blue/periodic acid-Schiff staining showed decreased Ki67, increased cleaved caspase 3, and decreased numbers of mucin-containing goblet cells, suggesting that the inflammation induced by these 2 types of inoculation with SARS-CoV-2 impaired the GI barrier and caused severe infections. CONCLUSIONS: Both intranasal and intragastric inoculation with SARS-CoV-2 caused pneumonia and GI dysfunction in our rhesus monkey model. Inflammatory cytokines are possible connections for the pathogenesis of SARS-CoV-2 between the respiratory and digestive systems.


Subject(s)
COVID-19/transmission , Gastroenteritis/pathology , Gastrointestinal Tract/pathology , Lung/pathology , Animals , Bronchi/metabolism , Bronchi/pathology , COVID-19/immunology , COVID-19/metabolism , COVID-19/pathology , COVID-19 Nucleic Acid Testing , Caspase 3/metabolism , Cytokines/immunology , Disease Models, Animal , Gastric Mucosa , Gastroenteritis/metabolism , Gastroenteritis/virology , Gastrointestinal Tract/immunology , Gastrointestinal Tract/metabolism , Goblet Cells/pathology , Intestine, Small/metabolism , Intestine, Small/pathology , Ki-67 Antigen/metabolism , Lung/diagnostic imaging , Lung/immunology , Lung/metabolism , Macaca mulatta , Nasal Mucosa , RNA, Viral/isolation & purification , Random Allocation , Rectum/metabolism , Rectum/pathology , SARS-CoV-2 , Trachea/metabolism , Trachea/pathology
15.
Cell Res ; 31(2): 126-140, 2021 02.
Article in English | MEDLINE | ID: covidwho-1015005

ABSTRACT

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.


Subject(s)
COVID-19/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Respiratory Mucosa/cytology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Bronchi/cytology , Bronchi/metabolism , Cell Line , Humans , Lung/cytology , Lung/metabolism , Models, Molecular , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins/analysis , Receptor Protein-Tyrosine Kinases/analysis , Respiratory Mucosa/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Virus Internalization
16.
PLoS One ; 15(12): e0242536, 2020.
Article in English | MEDLINE | ID: covidwho-965821

ABSTRACT

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.


Subject(s)
Bronchi/drug effects , Respiratory Mucosa/drug effects , Tretinoin/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Anti-Inflammatory Agents/pharmacology , Bronchi/cytology , Bronchi/metabolism , Cell Line , Humans , Inflammation/drug therapy , Inflammation/metabolism , Permeability/drug effects , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Tight Junctions/drug effects , Tight Junctions/metabolism
17.
Viruses ; 12(11)2020 10 23.
Article in English | MEDLINE | ID: covidwho-895404

ABSTRACT

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.


Subject(s)
CD13 Antigens/metabolism , Epithelial Cells/metabolism , Porcine Respiratory Coronavirus/physiology , Receptors, Virus/metabolism , Respiratory Mucosa/virology , Viral Tropism , Animals , Bronchi/metabolism , Bronchi/virology , Cell Differentiation , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/virology , Swine , Trachea/metabolism , Trachea/virology , Virus Internalization , Virus Release , Virus Replication
19.
Exp Cell Res ; 395(2): 112204, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-680000

ABSTRACT

BACKGROUND: SARS-CoV2, the agent responsible for the current pandemic, is also causing respiratory distress syndrome (RDS), hyperinflammation and high mortality. It is critical to dissect the pathogenetic mechanisms in order to reach a targeted therapeutic approach. METHODS: In the present investigation, we evaluated the effects of SARS-CoV2 on human bronchial epithelial cells (HBEC). We used RNA-seq datasets available online for identifying SARS-CoV2 potential genes target on human bronchial epithelial cells. RNA expression levels and potential cellular gene pathways have been analyzed. In order to identify possible common strategies among the main pandemic viruses, such as SARS-CoV2, SARS-CoV1, MERS-CoV, and H1N1, we carried out a hypergeometric test of the main genes transcribed in the cells of the respiratory tract exposed to these viruses. RESULTS: The analysis showed that two mechanisms are highly regulated in HBEC: the innate immunity recruitment and the disassembly of cilia and cytoskeletal structure. The granulocyte colony-stimulating factor (CSF3) and dynein heavy chain 7, axonemal (DNAH7) represented respectively the most upregulated and downregulated genes belonging to the two mechanisms highlighted above. Furthermore, the carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7) that codifies for a surface protein is highly specific of SARS-CoV2 and not for SARS-CoV1, MERS-CoV, and H1N1, suggesting a potential role in viral entry. In order to identify potential new drugs, using a machine learning approach, we highlighted Flunisolide, Thalidomide, Lenalidomide, Desoximetasone, xylazine, and salmeterol as potential drugs against SARS-CoV2 infection. CONCLUSIONS: Overall, lung involvement and RDS could be generated by the activation and down regulation of diverse gene pathway involving respiratory cilia and muscle contraction, apoptotic phenomena, matrix destructuration, collagen deposition, neutrophil and macrophages recruitment.


Subject(s)
Bronchi/metabolism , Coronavirus Infections/genetics , Gene Regulatory Networks , Pneumonia, Viral/genetics , Respiratory Mucosa/metabolism , Transcriptome , Bronchi/pathology , COVID-19 , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/metabolism , Coronavirus Infections/metabolism , Drug Discovery/methods , Dyneins/genetics , Dyneins/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Immunity, Innate , Machine Learning , Pandemics , Pneumonia, Viral/metabolism , Up-Regulation
20.
Biochim Biophys Acta Gen Subj ; 1864(10): 129672, 2020 10.
Article in English | MEDLINE | ID: covidwho-601229

ABSTRACT

BACKGROUND: Exposure to PM2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B. METHODS: BEAS-2B cells were exposed to 0, 20, 50, 100 and 150 µg/ml of PM2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1ß was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting. RESULTS: PM2.5 exposure increased the secretion of IL-6, IL-8, and IL-1ß in a concentration-dependent fashion. Moreover, exposure to PM2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM2.5-induced IL-6, IL-8 and IL-1ß secretion, implying that both oxidative stress and EGFR activation were involved in PM2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM2.5-induced EGFR activation, suggesting that oxidative stress was required for PM2.5-induced EGFR activation. CONCLUSION: PM2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.


Subject(s)
Air Pollutants/adverse effects , Epithelial Cells/metabolism , Inflammation Mediators/metabolism , Oxidative Stress , Particulate Matter/adverse effects , Bronchi/cytology , Bronchi/metabolism , Cell Line , Epithelial Cells/cytology , ErbB Receptors/metabolism , Humans , Inflammation/etiology , Inflammation/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism
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