scholarly journals Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

2020 ◽  
Vol 56 (3) ◽  
pp. 2001123 ◽  
Author(s):  
Jennifer A. Aguiar ◽  
Benjamin J-M. Tremblay ◽  
Michael J. Mansfield ◽  
Owen Woody ◽  
Briallen Lobb ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Healthy Subjects ◽  
Promoter Activity ◽  
Human Lung ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

scholarly journals Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

2020 ◽  
Author(s):  
Jennifer A. Aguiar ◽  
Benjamin J-M. Tremblay ◽  
Michael J. Mansfield ◽  
Owen Woody ◽  
Briallen Lobb ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Healthy Subjects ◽  
Promoter Activity ◽  
Human Lung ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

ABSTRACTIn December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and TMPRSS2 host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147, and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung. We present confirmatory evidence for the presence of TMPRSS2, CD147, and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

scholarly journals AGR3 Regulates Airway Epithelial Junctions in Patients with Frequent Exacerbations of COPD

2021 ◽  
Vol 12 ◽  
Author(s):  
Rui Ye ◽  
Cuihong Wang ◽  
Pengbo Sun ◽  
Shuang Bai ◽  
Li Zhao
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Mrna Expression ◽  
Western Blotting ◽  
Human Lung ◽  
Airway Epithelial Cells ◽  
Copd Exacerbation ◽  
Airway Epithelial ◽  
Copd Exacerbations ◽  
E Cadherin

Background: The mechanisms underlying differences in the susceptibility to chronic obstructive pulmonary disease (COPD) exacerbations between patients are not well understood. Recent studies have shown that the patients with frequent COPD exacerbations is related to specific protein expression in lung tissue. Anterior gradient 3 (AGR3) is expressed in airway epithelial cells in the lung and proteomic analysis revealed that its expression is decreased in patients with frequent COPD exacerbations. Moreover, the loss of epithelial integrity might facilitate trans-epithelial permeability of pathogens in such patients. This study was performed to determine that AGR3 protein play a role in COPD frequency exacerbators.Methods: Human lung tissues were collected from current-smoking patients (Control; n = 15) as well as patients with infrequent COPD exacerbations (IFCOPD; n = 18) and frequent COPD exacerbations (FCOPD; n = 8). While AGR3 protein expression was measured by immunohistochemistry and western blotting, AGR mRNA expression was determined by real time quantitative polymerase chain reaction (RT-qPCR). Furthermore, adherent junctions (AJs) and tight junctions (TJs) protein expression in human lung tissues were measured by immunohistochemistry. The effects of cigarette smoke extract (CSE) on AJ and TJ protein and mRNA expression in BEAS-2B cells were assessed by western blotting and RT-qPCR. In addition, the effect of AGR3 overexpression and knockdown on AJ and TJ protein expression was determined.Results: AGR3 was mainly expressed in the airway epithelium and AGR3-positive products were localized in the cytoplasm. Western blotting and RT-qPCR results showed that AGR3 protein (p = 0.009) and mRNA (p = 0.04) expression in the FCOPD group was significantly lower than that in the IFCOPD group. Moreover, E-cadherin, occludin, and zonula occludens-1 (ZO-1) expression was lower in the FCOPD group than in the IFCOPD group. The protein and mRNA expression of E-cadherin, occludin, and ZO-1 was decreased within 24 h post-CSE exposure. AGR3 overexpression rescued CSE-induced downregulation of E-cadherin, occludin, and ZO-1.Conclusion: Difference in AGR3 expression in the lung tissue might be correlated with increased susceptibility to COPD exacerbation. AGR3 can prevent CSE-induced downregulation of E-cadherin, occludin, and ZO-1 in airway epithelial cells. Loss of AGR3 might promote viral and bacterial infection and induce immune inflammation to increase COPD exacerbation.

scholarly journals The impact of cigarette smoke exposure, and COPD or asthma status on ABC transporter gene expression in human airway epithelial cells

2018 ◽  
Author(s):  
Jennifer A. Aguiar ◽  
Andrea Tamminga ◽  
Briallen Lobb ◽  
Ryan D. Huff ◽  
Jenny Nguyen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cigarette Smoke ◽  
Abc Transporters ◽  
Airway Epithelial Cells ◽  
Cigarette Smoke Exposure ◽  
Airway Epithelial ◽  
Respiratory Mucosa

AbstractRationaleThe respiratory mucosa coordinates responses to infections, allergens, and exposures to air pollution. A relatively unexplored aspect of the respiratory mucosa are the expression and function of ATP Binding Cassette (ABC) transporters. ABC transporters are conserved in prokaryotes and eukaryotes, with humans expressing 48 transporters divided into 7 classes (ABCA, ABCB, ABCC, ABCD, ABDE, ABCF, and ABCG). Throughout the human body, ABC transporters regulate cAMP levels, chloride secretion, lipid transport, and anti-oxidant responses. A deeper exploration of the expression patterns of ABC transporters in the respiratory mucosa is warranted to determine their relevance in lung health and disease.MethodsWe used a bioinformatic approach complemented with in vitro experimental methods for validation of candidate ABC transporters. We analyzed the expression profiles of all 48 human ABC transporters in the respiratory mucosa using bronchial epithelial cell gene expression datasets available in NCBI GEO from well-characterized patient populations of healthy subjects and individuals that smoke cigarettes, or have been diagnosed with COPD or asthma. The Calu-3 airway epithelial cell line was used to interrogate selected results using a cigarette smoke extract exposure model.ResultsUsing 9 distinct gene-expression datasets of primary human airway epithelial cells, we completed a focused analysis on 48 ABC transporters in samples from healthy subjects and individuals that smoke cigarettes, or have been diagnosed with COPD or asthma. In situ gene expression data demonstrate that ABC transporters are i) variably expressed in epithelial cells from different airway generations (top three expression levels - ABCA5, ABCA13, and ABCC5), ii) regulated by cigarette smoke exposure (ABCA13, ABCB6, ABCC1, and ABCC3), and iii) differentially expressed in individuals with COPD and asthma (ABCA13, ABCC1, ABCC2, ABCC9). An in vitro cell culture model of cigarette smoke exposure was able to recapitulate the in situ changes observed in cigarette smokers for ABCA13 and ABCC1.ConclusionsOur in situ human gene expression data analysis reveals that ABC transporters are expressed throughout the airway generations in airway epithelial cells and can be modulated by environmental exposures important in chronic respiratory disease (e.g. cigarette smoking) and in individuals with chronic lung diseases (e.g. COPD or asthma). Our work highlights select ABC transporter candidates of interest and a relevant in vitro model that will enable a deeper understanding of the contribution of ABC transporters in the respiratory mucosa in lung health and disease.

Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L1028-L1035 ◽  
Author(s):  
Yu-Mee Kim ◽  
William Reed ◽  
Weidong Wu ◽  
Philip A. Bromberg ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Human Airway ◽  
Mrna And Protein Expression ◽  
Human Airway Epithelial Cells

Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn2+-induced IL-8 expression. Zn2+ stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn2+ exposure in BEAS-2B cells, indicating that Zn2+-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn2+-induced IL-8 promoter activity. Moreover, Zn2+ exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn2+ exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn2+. In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn2+. Zn2+ treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn2+-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.

scholarly journals Electronic Cigarette Aerosol Is Cytotoxic and Increases ACE2 Expression on Human Airway Epithelial Cells: Implications for SARS-CoV-2 (COVID-19)

2021 ◽  
Vol 10 (5) ◽  
pp. 1028
Author(s):  
Kielan Darcy McAlinden ◽  
Wenying Lu ◽  
Parisa Vahidi Ferdowsi ◽  
Stephen Myers ◽  
James Markos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Cigarette Smoke ◽  
Membrane Integrity ◽  
Cigarette Smoke Extract ◽  
Airway Epithelial Cells ◽  
Electronic Cigarette ◽  
Airway Epithelial ◽  
Ace2 Gene

Tobacco smoking has emerged as a risk factor for increasing the susceptibility to infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via increased expression of angiotensin-converting enzyme-2 (ACE2) in the lung, linked to coronavirus disease 2019 (COVID-19) development. Given the modifiable nature of electronic cigarettes and the delivery of high concentrations of nicotine, we investigate whether electronic cigarette vaping has the potential to increase susceptibility to SARS-CoV-2 infection. We exposed BEAS-2B cells (bronchial epithelium transformed with Ad12-SV40 2B) and primary small airway epithelial cells (SAECs) to electronic cigarette aerosol condensates produced from propylene glycol/vegetable glycerin or commercially bought e-liquid (±added nicotine) and cigarette smoke extract to investigate if electronic cigarette exposure, like cigarette smoke, increases the expression of ACE2 in lung epithelial cells. In BEAS-2B cells, cytotoxicity (CCK-8), membrane integrity (LDH), and ACE2 protein expression (immunofluorescence) were measured for both 4- and 24 h treatments in BEAS-2B cells and 4 h in SAECs; ACE2 gene expression was measured using quantitative polymerase chain reaction (qPCR) for 4 h treatment in BEAS-2B cells. Nicotine-free condensates and higher concentrations of nicotine-containing condensates were cytotoxic to BEAS-2B cells. Higher LDH release and reduced membrane integrity were seen in BEAS-2B cells treated for 24 h with higher concentrations of nicotine-containing condensates. ACE2 protein expression was observably increased in all treatments compared to cell controls, particularly for 24 h exposures. ACE2 gene expression was significantly increased in cells exposed to the locally bought e-liquid condensate with high nicotine concentration and cigarette smoke extract compared with cell controls. Our study suggests that vaping alone and smoking alone can result in an increase in lung ACE2 expression. Vaping and smoking are avoidable risk factors for COVID-19, which, if avoided, could help reduce the number of COVID-19 cases and the severity of the disease. This is the first study to utilize electronic cigarette aerosol condensates, novel and developed in our laboratory, for investigating ACE2 expression in human airway epithelial cells.

scholarly journals Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadzeya Marozkina ◽  
Laura Smith ◽  
Yi Zhao ◽  
Joe Zein ◽  
James F. Chmiel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Nitrogen Oxides ◽  
Airway Epithelium ◽  
Airflow Obstruction ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

Exploring host-pathogen interactions at the epithelial surface: application of transcriptomics in lung biology

2007 ◽  
Vol 292 (2) ◽  
pp. L367-L377 ◽  
Author(s):  
Joost B. Vos ◽  
Nicole A. Datson ◽  
Klaus F. Rabe ◽  
Pieter S. Hiemstra
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Large Scale ◽  
Brain Research ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Host Pathogen Interactions ◽  
Epithelial Surface ◽  
Complex Interactions ◽  
Host Pathogen

The epithelial surface of the airways is the largest barrier-forming interface between the human body and the outside world. It is now well recognized that, at this strategic position, airway epithelial cells play an eminent role in host defense by recognizing and responding to microbial exposure. Conversely, inhaled microorganisms also respond to contact with epithelial cells. Our understanding of this cross talk is limited, requiring sophisticated experimental approaches to analyze these complex interactions. High-throughput technologies, such as DNA microarray analysis and serial analysis of gene expression (SAGE), have been developed to screen for gene expression levels at large scale within single experiments. Since their introduction, these hypothesis-generating technologies have been widely used in diverse areas such as oncology and brain research. Successful application of these genomics-based technologies has also revealed novel insights in host-pathogen interactions in both the host and pathogen. This review aims to provide an overview of the SAGE and microarray technology illustrated by their application in the analysis of host-pathogen interactions. In particular, the interactions between epithelial cells in the human lungs and clinically relevant microorganisms are the central focus of this review.

scholarly journals Recent Advances in Molecular Biology of Human Bocavirus 1 and Its Applications

2021 ◽  
Vol 12 ◽  
Author(s):  
Liting Shao ◽  
Weiran Shen ◽  
Shengqi Wang ◽  
Jianming Qiu
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Structural Proteins ◽  
Human Bocavirus ◽  
Airway Epithelial ◽  
Raav Vector ◽  
Human Airway ◽  
Non Coding Rna

Human bocavirus 1 (HBoV1) was discovered in human nasopharyngeal specimens in 2005. It is an autonomous human parvovirus and causes acute respiratory tract infections in young children. HBoV1 infects well differentiated or polarized human airway epithelial cells in vitro. Unique among all parvoviruses, HBoV1 expresses 6 non-structural proteins, NS1, NS1-70, NS2, NS3, NS4, and NP1, and a viral non-coding RNA (BocaSR), and three structural proteins VP1, VP2, and VP3. The BocaSR is the first identified RNA polymerase III (Pol III) transcribed viral non-coding RNA in small DNA viruses. It plays an important role in regulation of viral gene expression and a direct role in viral DNA replication in the nucleus. HBoV1 genome replication in the polarized/non-dividing airway epithelial cells depends on the DNA damage and DNA repair pathways and involves error-free Y-family DNA repair DNA polymerase (Pol) η and Pol κ. Importantly, HBoV1 is a helper virus for the replication of dependoparvovirus, adeno-associated virus (AAV), in polarized human airway epithelial cells, and HBoV1 gene products support wild-type AAV replication and recombinant AAV (rAAV) production in human embryonic kidney (HEK) 293 cells. More importantly, the HBoV1 capsid is able to pseudopackage an rAAV2 or rHBoV1 genome, producing the rAAV2/HBoV1 or rHBoV1 vector. The HBoV1 capsid based rAAV vector has a high tropism for human airway epithelia. A deeper understanding in HBoV1 replication and gene expression will help find a better way to produce the rAAV vector and to increase the efficacy of gene delivery using the rAAV2/HBoV1 or rHBoV1 vector, in particular, to human airways. This review summarizes the recent advances in gene expression and replication of HBoV1, as well as the use of HBoV1 as a parvoviral vector for gene delivery.

scholarly journals Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria–host interactions

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Ang Su ◽  
Jie Tong ◽  
Yuguang Fu ◽  
Sandy Müller ◽  
Yenehiwot Berhanu Weldearegay ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Layer ◽  
Airway Epithelial Cells ◽  
Infection Model ◽  
Target Cells ◽  
Respiratory Disorder ◽  
Airway Epithelial ◽  
Epithelial Cell Layer ◽  
Well Differentiated

AbstractPasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air–liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

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