SARS‐CoV‐2‐infected human airway epithelial cell cultures uniquely lack interferon and immediate early gene responses caused by other coronaviruses
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC11017760 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The respiratory epithelium is a polarised layer at the interface between the outside environment and deeper lung structures, overlaid by the epithelial lining fluid (ELF). This provides a mechanical and immunological barrier to inhaled particulates, such as viruses. Human respiratory syncytial virus (hRSV) is a major cause of disease in humans, and targets the respiratory epithelium. However, little is known of the disruption of the ELF proteome in the context of virus-driven respiratory illnesses. To address this, a proteomics approach was combined with an ex-vivo human airway epithelial model (HAE) to investigate the apical and basolateral secretome in hRSV-infected cultures. This demonstrated that several apically- and basolaterally-restricted proteins were subsequently secreted in both directions upon infection, while a number of proteins saw their apical/basolateral abundance ratios significantly altered. Furthermore, another 35 proteins were uniquely identified after hRSV treatment. Importantly, some of these changes were correlated in nasal aspirates (NA) from children with and without hRSV. This study showed that hRSV could affect airway secretions, and disrupted the directionality of the respiratory epithelium.
2022-03-01 | PXD004430 | Pride
Project description:Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ?-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (?-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
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Project description:Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
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Project description:Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.
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Project description:We performed RNAseq analysis on primary human airway epithelial cultures either mock infected (PBS) or infected with SARS-CoV-2. Transcriptional profiling studies found that infected pHAE cells had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNFα, CXCL8, and identified NF-κB and ATF4 as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection
2020-07-21 | GSE153970 | GEO
Project description:Airway epithelial cells are crucial for mucosal and adaptive immunity as they produce mucins that trap and clear inhaled particulates by mucociliary activity, and also secrete diverse inflammatory mediators. But whether these cells respond in a memory-dependent manner to a secondary exposure is poorly studied. Our recent studies have highlighted the role of 'memory-based' or ‘trained’ immune responses. In this proposal we would like to probe for the molecular basis underlying the memory-based response in AECs. We will first establish an in-vitro model of memory-based responses using differentiated AECs. Here we will delineate the kinetics of memory-based response in AECs using a microbial ligand, LPS or endotoxin. The cells will be grown in air-liquid interface to study the effect of memory response on mucous phenotype. The RNAseq analyses will be performed on AECs exposed to endotoxin and compared to that of non-exposed AECs to determine the changes in coding and noncoding RNA regions.
2020-12-07 | GSE139370 | GEO
Project description:ObjectivesHuman airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.MethodsAir-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.Main resultsALI cultures were readily infected by HRV. RNA-seq analysis of HRV infected ALI cultures identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial structure and remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1, MUC5AC, CDHR3), and novel ones that were identified for the first time in this study (e.g. CCRL1).ConclusionsALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.
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Project description:Primary Human Airway Epithelial Cultures infected with SARS-CoV-2
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Project description:A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air-liquid interface (HAE-ALI) Basic Protocol 2: Viral inoculation of HAE-ALI Basic Protocol 3: Immunofluorescence (IF)-based detection of infected cells in HAE-ALI.
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Project description:Cultures of primary human airway epithelial cells (HAE cells) were exposed to an MDCK equivalent MOI of 0.01 of several swine- and human-origin influenza viruses and RNA was extracted at the 12, 16, and 24 hours post infection.
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