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1.
Respir Res ; 24(1): 130, 2023 May 11.
Article in English | MEDLINE | ID: covidwho-2318274

ABSTRACT

After more than two years the COVID-19 pandemic, that is caused by infection with the respiratory SARS-CoV-2 virus, is still ongoing. The risk to develop severe COVID-19 upon SARS-CoV-2 infection is increased in individuals with a high age, high body mass index, and who are smoking. The SARS-CoV-2 virus infects cells of the upper respiratory tract by entering these cells upon binding to the Angiotensin-converting enzyme 2 (ACE2) receptor. ACE2 is expressed in various cell types in the lung but the expression is especially high in goblet and ciliated cells. Recently, it was shown that next to its full-length isoform, ACE2 also has a short isoform. The short isoform is unable to bind SARS-CoV-2 and does not facilitate viral entry. In the current study we investigated whether active cigarette smoking increases the expression of the long or the short ACE2 isoform. We showed that in active smokers the expression of the long, active isoform, but not the short isoform of ACE2 is higher compared to never smokers. Additionally, it was shown that the expression of especially the long, active isoform of ACE2 was associated with secretory, club and goblet epithelial cells. This study increases our understanding of why current smokers are more susceptible to SARS-CoV-2 infection, in addition to the already established increased risk to develop severe COVID-19.


Subject(s)
COVID-19 , Respiratory Mucosa , Smoking , Humans , Angiotensin-Converting Enzyme 2 , COVID-19/genetics , COVID-19/immunology , Epithelium/metabolism , Pandemics , Peptidyl-Dipeptidase A , Respiratory Mucosa/metabolism , SARS-CoV-2 , Smoking/adverse effects , Spike Glycoprotein, Coronavirus/metabolism
2.
J Biol Chem ; 299(6): 104820, 2023 06.
Article in English | MEDLINE | ID: covidwho-2316300

ABSTRACT

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Cystic Fibrosis , Leukocyte Elastase , Humans , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , Cystic Fibrosis/metabolism , Leukocyte Elastase/metabolism , Protein Binding , Proteomics , Respiratory Mucosa/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
3.
Eur J Pharm Biopharm ; 184: 62-82, 2023 Mar.
Article in English | MEDLINE | ID: covidwho-2235648

ABSTRACT

The intranasal route has been receiving greater attention from the scientific community not only for systemic drug delivery but also for the treatment of pulmonary and neurological diseases. Along with it, drug transport and permeability studies across the nasal mucosa have exponentially increased. Nevertheless, the translation of data from in vitro cell lines to in vivo studies is not always reliable, due to the difficulty in generating an in vitro model that resembles respiratory human physiology. Among all currently available methodologies, the air-liquid interface (ALI) method is advantageous to promote cell differentiation and optimize the morphological and histological characteristics of airway epithelium cells. Cells grown under ALI conditions, in alternative to submerged conditions, appear to provide relevant input for inhalation and pulmonary toxicology and complement in vivo experiments. Different methodologies and a variety of materials have been used to induce ALI conditions in primary cells and numerous cell lines. Until this day, with only exploratory results, no consensus has been reached regarding the validation of the ALI method, hampering data comparison. The present review describes the most adequate cell models of airway epithelium and how these models are differently affected by ALI conditions. It includes the evaluation of cellular features before and after ALI, and the application of the method in primary cell cultures, commercial 3D primary cells, cell lines and stem-cell derived models. A variety of these models have been recently applied for pharmacological studies against severe acute respiratory syndrome-coronavirus(-2) SARS-CoV(-2), namely primary cultures with alveolar type II epithelium cells and organotypic 3D models. The herein compiled data suggest that ALI conditions must be optimized bearing in mind the type of cells (nasal, bronchial, alveolar), their origin and the objective of the study.


Subject(s)
Cell Culture Techniques , Respiratory Mucosa , Humans , Respiratory Mucosa/metabolism , Cell Line , Lung , Nasal Mucosa , Epithelial Cells/metabolism
4.
Exp Biol Med (Maywood) ; 248(3): 271-279, 2023 02.
Article in English | MEDLINE | ID: covidwho-2195301

ABSTRACT

Epidemiological evidence links lower air quality with increased incidence and severity of COVID-19; however, mechanistic data have yet to be published. We hypothesized air pollution-induced oxidative stress in the nasal epithelium increased viral replication and inflammation. Nasal epithelial cells (NECs), collected from healthy adults, were grown into a fully differentiated epithelium. NECs were infected with the ancestral strain of SARS-CoV-2. An oxidant combustion by-product found in air pollution, the environmentally persistent free radical (EPFR) DCB230, was used to mimic pollution exposure four hours prior to infection. Some wells were pretreated with antioxidant, astaxanthin, for 24 hours prior to EPFR-DCB230 exposure and/or SARS-CoV-2 infection. Outcomes included viral replication, epithelial integrity, surface receptor expression (ACE2, TMPRSS2), cytokine mRNA expression (TNF-α, IFN-ß), intracellular signaling pathways, and oxidative defense enzymes. SARS-CoV-2 infection induced a mild phenotype in NECs, with some cell death, upregulation of the antiviral cytokine IFN-ß, but had little effect on intracellular pathways or oxidative defense enzymes. Prior exposure to EPFR-DCB230 increased SARS-CoV-2 replication, upregulated TMPRSS2 expression, increased secretion of the proinflammatory cytokine TNF-α, inhibited expression of the mucus producing MUC5AC gene, upregulated expression of p21 (apoptosis pathway), PINK1 (mitophagy pathway), and reduced levels of antioxidant enzymes. Pretreatment with astaxanthin reduced SARS-CoV-2 replication, downregulated ACE2 expression, and prevented most, but not all EPFR-DCB230 effects. Our data suggest that oxidant damage to the respiratory epithelium may underly the link between poor air quality and increased COVID-19. The apparent protection by antioxidants warrants further research.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , COVID-19/metabolism , Antioxidants/metabolism , Tumor Necrosis Factor-alpha/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Free Radicals/metabolism , Cytokines/metabolism , Respiratory Mucosa/metabolism , Oxidants/metabolism
5.
Am J Physiol Lung Cell Mol Physiol ; 323(3): L240-L250, 2022 09 01.
Article in English | MEDLINE | ID: covidwho-2138198

ABSTRACT

The balance of gas exchange and lung ventilation is essential for the maintenance of body homeostasis. There are many ion channels and transporters in respiratory epithelial cells, including epithelial sodium channel, Na,K-ATPase, cystic fibrosis transmembrane conductance regulator, and some transporters. These ion channels/transporters maintain the capacity of liquid layer on the surface of respiratory epithelial cells and provide an immune barrier for the respiratory system to clear off foreign pathogens. However, in some harmful external environments and/or pathological conditions, the respiratory epithelium is prone to hypoxia, which would destroy the ion transport function of the epithelium and unbalance the homeostasis of internal environment, triggering a series of pathological reactions. Many respiratory diseases associated with hypoxia manifest an increased expression of hypoxia-inducible factor-1, which mediates the integrity of the epithelial barrier and affects epithelial ion transport function. It is important to study the relationship between hypoxia and ion transport function, whereas the mechanism of hypoxia-induced ion transport dysfunction in respiratory diseases is not clear. This review focuses on the relationship between hypoxia and respiratory diseases, as well as dysfunction of ion transport and tight junctions in respiratory epithelial cells under hypoxia.


Subject(s)
Respiration Disorders , Sodium-Potassium-Exchanging ATPase , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Sodium Channels/metabolism , Humans , Hypoxia/metabolism , Ion Transport , Respiration Disorders/metabolism , Respiratory Mucosa/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism
6.
Signal Transduct Target Ther ; 7(1): 255, 2022 07 27.
Article in English | MEDLINE | ID: covidwho-1960331

ABSTRACT

SARS-CoV-2, the culprit pathogen of COVID-19, elicits prominent immune responses and cytokine storms. Intracellular Cl- is a crucial regulator of host defense, whereas the role of Cl- signaling pathway in modulating pulmonary inflammation associated with SARS-CoV-2 infection remains unclear. By using human respiratory epithelial cell lines, primary cultured human airway epithelial cells, and murine models of viral structural protein stimulation and SARS-CoV-2 direct challenge, we demonstrated that SARS-CoV-2 nucleocapsid (N) protein could interact with Smad3, which downregulated cystic fibrosis transmembrane conductance regulator (CFTR) expression via microRNA-145. The intracellular Cl- concentration ([Cl-]i) was raised, resulting in phosphorylation of serum glucocorticoid regulated kinase 1 (SGK1) and robust inflammatory responses. Inhibition or knockout of SGK1 abrogated the N protein-elicited airway inflammation. Moreover, N protein promoted a sustained elevation of [Cl-]i by depleting intracellular cAMP via upregulation of phosphodiesterase 4 (PDE4). Rolipram, a selective PDE4 inhibitor, countered airway inflammation by reducing [Cl-]i. Our findings suggested that Cl- acted as the crucial pathological second messenger mediating the inflammatory responses after SARS-CoV-2 infection. Targeting the Cl- signaling pathway might be a novel therapeutic strategy for COVID-19.


Subject(s)
COVID-19 , Chlorine/metabolism , MicroRNAs , Animals , COVID-19/genetics , Humans , Inflammation/pathology , Mice , MicroRNAs/metabolism , Nucleocapsid Proteins , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , SARS-CoV-2
7.
Int J Mol Sci ; 23(9)2022 May 04.
Article in English | MEDLINE | ID: covidwho-1820296

ABSTRACT

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.


Subject(s)
COVID-19 , Animals , Cilia/metabolism , Cricetinae , Epithelium , Homeostasis , Mesocricetus , Respiratory Mucosa/metabolism , SARS-CoV-2
8.
Int J Mol Sci ; 23(7)2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1785736

ABSTRACT

Lysophosphatidylserine (LysoPS) is an amphipathic lysophospholipid that mediates a broad spectrum of inflammatory responses through a poorly characterized mechanism. Because LysoPS levels can rise in a variety of pathological conditions, we sought to investigate LysoPS's potential role in airway epithelial cells that actively participate in lung homeostasis. Here, we report a previously unappreciated function of LysoPS in production of a mucin component, MUC5AC, in the airway epithelial cells. LysoPS stimulated lung epithelial cells to produce MUC5AC via signaling pathways involving TACE, EGFR, and ERK. Specifically, LysoPS- dependent biphasic activation of ERK resulted in TGF-α secretion and strong EGFR phosphorylation leading to MUC5AC production. Collectively, LysoPS induces the expression of MUC5AC via a feedback loop composed of proligand synthesis and its proteolysis by TACE and following autocrine EGFR activation. To our surprise, we were not able to find a role of GPCRs and TLR2, known LyoPS receptors in LysoPS-induced MUC5AC production in airway epithelial cells, suggesting a potential receptor-independent action of LysoPS during inflammation. This study provides new insight into the potential function and mechanism of LysoPS as an emerging lipid mediator in airway inflammation.


Subject(s)
ErbB Receptors , MAP Kinase Signaling System , Epithelial Cells/metabolism , ErbB Receptors/metabolism , Humans , Inflammation/metabolism , Lysophospholipids/metabolism , Lysophospholipids/pharmacology , Mucin 5AC/metabolism , Respiratory Mucosa/metabolism
9.
J Immunol ; 207(5): 1275-1287, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1771322

ABSTRACT

The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.


Subject(s)
Dinoprostone/metabolism , Inflammation/immunology , Interleukin-6/metabolism , Respiratory Mucosa/metabolism , Adenosine Triphosphate/pharmacokinetics , Alarmins/metabolism , Calcium Release Activated Calcium Channels/metabolism , Cells, Cultured , Humans , Immunomodulation , Interleukin-6/genetics , NFATC Transcription Factors/metabolism , Phospholipases A2/metabolism , Receptors, Purinergic P2X/metabolism , Respiratory Mucosa/pathology , Signal Transduction , Uracil Nucleotides/metabolism
10.
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
11.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1675572

ABSTRACT

BACKGROUND: There are limited effective prophylactic/early treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral entry requires spike protein binding to the angiotensin-converting enzyme-2 receptor and cleavage by transmembrane serine protease 2 (TMPRSS2), a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is in clinical trials in early SARS-CoV-2 infection. METHODS: We used differentiated primary human airway epithelial cells at the air-liquid interface to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. RESULTS: We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-CoV-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, oral camostat is rapidly metabolised in the circulation, with poor airway bioavailability. We therefore modelled local airway administration by applying camostat to the apical surface of differentiated airway cultures. We demonstrated that a brief exposure to topical camostat effectively restricts SARS-CoV-2 infection. CONCLUSION: These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.


Subject(s)
Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/administration & dosage , Administration, Topical , Androgens/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19/prevention & control , COVID-19/virology , Cells, Cultured , Epithelial Cells , Esters/pharmacology , Gene Expression , Goblet Cells/immunology , Goblet Cells/metabolism , Guanidines/pharmacology , Host-Pathogen Interactions/drug effects , Humans , Serine Endopeptidases/genetics , Signal Transduction , Virus Internalization/drug effects , Virus Replication/drug effects
12.
Int J Mol Sci ; 23(3)2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1625612

ABSTRACT

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl- currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl- channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.


Subject(s)
Niclosamide/administration & dosage , Pneumonia/drug therapy , Respiratory System/drug effects , Animals , Asthma/drug therapy , Asthma/metabolism , Asthma/pathology , COVID-19/complications , Cells, Cultured , Disease Models, Animal , Drug Carriers/chemistry , Drug Compounding , Humans , Hydrogels/chemistry , Instillation, Drug , Mice , Microspheres , Mucus/drug effects , Mucus/metabolism , Nanospheres/administration & dosage , Nanospheres/chemistry , Niclosamide/chemistry , Niclosamide/pharmacokinetics , Pneumonia/pathology , Polyethylene Glycols/chemistry , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory System/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Trachea , COVID-19 Drug Treatment
13.
PLoS Biol ; 19(12): e3001065, 2021 12.
Article in English | MEDLINE | ID: covidwho-1594053

ABSTRACT

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.


Subject(s)
Epithelial Cells/immunology , Hot Temperature , Immunity, Innate/immunology , Interferons/immunology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Virus Replication/immunology , Adolescent , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Gene Expression Profiling/methods , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/genetics , Interferons/genetics , Interferons/metabolism , Male , Middle Aged , Models, Biological , RNA-Seq/methods , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Tissue Culture Techniques , Vero Cells , Virus Replication/genetics , Virus Replication/physiology
15.
Front Immunol ; 12: 714027, 2021.
Article in English | MEDLINE | ID: covidwho-1581346

ABSTRACT

In the coronavirus disease 2019 (COVID-19) health crisis, one major challenge is to identify the susceptibility factors of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in order to adapt the recommendations for populations, as well as to reduce the risk of COVID-19 development in the most vulnerable people, especially patients with chronic respiratory diseases such as cystic fibrosis (CF). Airway epithelial cells (AECs) play a critical role in the modulation of both immune responses and COVID-19 severity. SARS-CoV-2 infects the airway through the receptor angiotensin-converting enzyme 2, and a host protease, transmembrane serine protease 2 (TMPRSS2), plays a major role in SARS-CoV-2 infectivity. Here, we show that Pseudomonas aeruginosa increases TMPRSS2 expression, notably in primary AECs with deficiency of the ion channel CF transmembrane conductance regulator (CFTR). Further, we show that the main component of P. aeruginosa flagella, the protein flagellin, increases TMPRSS2 expression in primary AECs and Calu-3 cells, through activation of Toll-like receptor-5 and p38 MAPK. This increase is particularly seen in Calu-3 cells deficient for CFTR and is associated with an intracellular increased level of SARS-CoV-2 infection, however, with no effect on the amount of virus particles released. Considering the urgency of the COVID-19 health crisis, this result may be of clinical significance for CF patients, who are frequently infected with and colonized by P. aeruginosa during the course of CF and might develop COVID-19.


Subject(s)
Cystic Fibrosis , Flagellin/metabolism , Pseudomonas Infections/complications , Respiratory Mucosa/virology , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Bacterial Proteins/metabolism , COVID-19/complications , Cells, Cultured , Humans , Pseudomonas aeruginosa , Respiratory Mucosa/metabolism
16.
Front Immunol ; 12: 743890, 2021.
Article in English | MEDLINE | ID: covidwho-1581344

ABSTRACT

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Asthma , COVID-19 , Imiquimod/pharmacology , Interferon-beta/drug effects , Respiratory Mucosa/drug effects , Adjuvants, Immunologic/pharmacology , Adult , Aged , Bronchi/drug effects , Bronchi/immunology , Bronchi/virology , Cells, Cultured , Female , Humans , Interferon-beta/immunology , Male , Middle Aged , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2
17.
J Immunol ; 208(1): 74-84, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1534334

ABSTRACT

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , Interferon Type I/metabolism , Lung/immunology , Neoplasm Proteins/metabolism , ORAI1 Protein/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/physiology , Stromal Interaction Molecule 1/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , Calcium Signaling , Clustered Regularly Interspaced Short Palindromic Repeats , Disease Resistance , Disease Susceptibility , Gene Expression Profiling , HEK293 Cells , Humans , Lung/virology , MEF2 Transcription Factors/genetics , Neoplasm Proteins/genetics , ORAI1 Protein/genetics , Stromal Interaction Molecule 1/genetics , Transcription Factor AP-1/genetics
18.
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
19.
J Infect Dis ; 224(8): 1357-1361, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1493824

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Receptors, Coronavirus/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/analysis , Biological Variation, Population , Bronchi/cytology , Bronchi/pathology , Bronchi/virology , COVID-19/virology , Epithelial Cells , Female , Humans , Male , Primary Cell Culture , Receptors, Coronavirus/analysis , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Respiratory Mucosa/virology , Sex Factors , Virus Internalization
20.
PLoS One ; 16(9): e0257784, 2021.
Article in English | MEDLINE | ID: covidwho-1440991

ABSTRACT

Drug repurposing has the potential to bring existing de-risked drugs for effective intervention in an ongoing pandemic-COVID-19 that has infected over 131 million, with 2.8 million people succumbing to the illness globally (as of April 04, 2021). We have used a novel `gene signature'-based drug repositioning strategy by applying widely accepted gene ranking algorithms to prioritize the FDA approved or under trial drugs. We mined publically available RNA sequencing (RNA-Seq) data using CLC Genomics Workbench 20 (QIAGEN) and identified 283 differentially expressed genes (FDR<0.05, log2FC>1) after a meta-analysis of three independent studies which were based on severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection in primary human airway epithelial cells. Ingenuity Pathway Analysis (IPA) revealed that SARS-CoV-2 activated key canonical pathways and gene networks that intricately regulate general anti-viral as well as specific inflammatory pathways. Drug database, extracted from the Metacore and IPA, identified 15 drug targets (with information on COVID-19 pathogenesis) with 46 existing drugs as potential-novel candidates for repurposing for COVID-19 treatment. We found 35 novel drugs that inhibit targets (ALPL, CXCL8, and IL6) already in clinical trials for COVID-19. Also, we found 6 existing drugs against 4 potential anti-COVID-19 targets (CCL20, CSF3, CXCL1, CXCL10) that might have novel anti-COVID-19 indications. Finally, these drug targets were computationally prioritized based on gene ranking algorithms, which revealed CXCL10 as the common and strongest candidate with 2 existing drugs. Furthermore, the list of 283 SARS-CoV-2-associated proteins could be valuable not only as anti-COVID-19 targets but also useful for COVID-19 biomarker development.


Subject(s)
COVID-19 Drug Treatment , Drug Repositioning/methods , SARS-CoV-2/genetics , Antiviral Agents/therapeutic use , Drug Evaluation, Preclinical/methods , Epithelial Cells/drug effects , Epithelium/drug effects , Humans , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , Respiratory System/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity
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