Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

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Human Tryptase ε (PRSS22), a New Member of the Chromosome 16p13.3 Family of Human Serine Proteases Expressed in Airway Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m108677200 ◽

2001 ◽

Vol 276 (52) ◽

pp. 49169-49182 ◽

Cited By ~ 31

Author(s):

Guang W. Wong ◽

Shinsuke Yasuda ◽

Mallur S. Madhusudhan ◽

Lixin Li ◽

Yi Yang ◽

...

Keyword(s):

Epithelial Cells ◽

Serine Proteases ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Chromosome 16P13.3

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Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells

Nucleic Acids Research ◽

10.1093/nar/gkaa089 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3513-3524 ◽

Cited By ~ 1

Author(s):

Monali NandyMazumdar ◽

Shiyi Yin ◽

Alekh Paranjapye ◽

Jenny L Kerschner ◽

Hannah Swahn ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Gene Promoter ◽

Regulatory Elements ◽

Airway Epithelial Cells ◽

Control Mechanisms ◽

Airway Epithelial ◽

Cell Type ◽

Long Range Interactions ◽

Cell Type Specific

Abstract The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanisms controlling its transcription are likely divergent between them. Here, we determine how two extragenic CREs that are prominent in epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at −44 and −35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here, we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter, suggesting non-redundant enhancers. The deletions also greatly reduced promoter interactions with the 5′ TAD boundary. We show substantial recruitment of RNAPII to the −35 kb element and identify CEBPβ as a key activator of airway expression of CFTR, likely through occupancy at this CRE and the gene promoter.

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Specific Inhibition of Interferon Signal Transduction Pathways by Adenoviral Infection

Journal of Biological Chemistry ◽

10.1074/jbc.m102030200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47136-47142 ◽

Cited By ~ 31

Author(s):

Theresa D. Joseph ◽

Dwight C. Look

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Transcription Factor ◽

Epithelial Cells ◽

Molecular Mechanisms ◽

Airway Epithelial Cells ◽

Signal Transducer ◽

Airway Epithelial ◽

Complex Assembly ◽

Ifn Γ

Adenoviral evolution has generated strategies to resist host cell antiviral systems, but molecular mechanisms for evasion of interferon (IFN) effects by adenoviruses during late-phase infection are poorly defined. In this study, we examined adenovirus type 5 (AdV) effects on IFN-γ-dependent gene expression and Janus family kinase-signal transducer and activator of transcription signaling components in human tracheobronchial epithelial cells. We found that AdV infection specifically inhibited IFN-γ-dependent gene expression in airway epithelial cells without evidence of epithelial cell injury or generation of a soluble extracellular inhibitor. Furthermore, infection with AdV for 18–24 h blocked phosphorylation/activation of the Stat1 transcription factor that regulates IFN-γ-dependent genes. Although AdV also inhibited IFN-α-dependent phosphorylation of Stat1 and Stat2, interleukin-4-dependent phosphorylation of the related transcription factor Stat6 was not affected, indicating that the virus selectively affected specific signaling pathways. Our results indicate that AdV inhibition of the IFN-γ signal transduction cascade occurs through loss of ligand-induced receptor complex assembly and consequent component phosphorylation and suggest that lack of complex assembly is due to decreased expression of the IFN-γR2 chain of the IFN-γ receptor. IFN-γR2 is required at an early step in Janus family kinase-signal transducer and activator of transcription pathway activation and is expressed at low levels in airway epithelial cells, supporting the concept that adenoviral down-regulation of the level of this IFN-γ receptor component allows for persistent modulation of IFN-γ-dependent gene expression.

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ACE2 Expression is elevated in Airway Epithelial Cells from aged and male donors but reduced in asthma

10.1101/2020.07.26.20162248 ◽

2020 ◽

Author(s):

Peter Wark ◽

Prabuddha Pathinyake ◽

Gerard Kaiko ◽

Kristy Nichol ◽

Ayesha Ali ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Serine Proteases ◽

Airway Epithelial Cells ◽

Lower Airway ◽

Chronic Obstructive ◽

Airway Epithelial ◽

Obstructive Pulmonary Disease ◽

Protein Levels ◽

Asthma Patients

Rationale: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter airway epithelial cells (AECs). Objective: To determine what factors are associated with ACE2 expression particularly in patients with asthma and chronic obstructive pulmonary disease (COPD). Methods: We obtained upper and lower AECs from 145 people from two independent cohorts, aged 2-89, Newcastle (n=115), and from Perth (n= 30) Australia. The Newcastle cohort was enriched with people with asthma (n=37) and COPD (n=38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR, protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AECs. Results: Increased gene expression of ACE2 was associated with older age (p=0.02) and male sex (p=0.03), but not pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma (p=0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in asthma (p=0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface was increased (p=0.02). ACE2 protein levels were lower in endobronchial biopsies from asthma patients. Conclusions: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

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Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response

Journal of Virology ◽

10.1128/jvi.01279-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9618-9631 ◽

Cited By ~ 33

Author(s):

Yashoda M. Hosakote ◽

Allan R. Brasier ◽

Antonella Casola ◽

Roberto P. Garofalo ◽

Alexander Kurosky

Keyword(s):

Epithelial Cells ◽

Respiratory Tract Infections ◽

Airway Disease ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Molecular Pattern ◽

Rsv Infection ◽

Syncytial Virus ◽

Tract Infections

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High-mobility group box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways.IMPORTANCERSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

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Thiazolidinediones induce proliferation of human bronchial epithelial cells through the GPR40 receptor

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90219.2008 ◽

2009 ◽

Vol 296 (6) ◽

pp. L970-L978 ◽

Cited By ~ 29

Author(s):

Delphine Gras ◽

Pascal Chanez ◽

Valérie Urbach ◽

Isabelle Vachier ◽

Philippe Godard ◽

...

Keyword(s):

Cell Growth ◽

Epithelial Cells ◽

Molecular Mechanisms ◽

Receptor Activation ◽

Airway Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Peroxisome Proliferator ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Intracellular Ca

Thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor-γ (PPARγ) ligands that are widely used in type II diabetes treatment. In addition to their ability to improve glucose homeostasis, TZDs possess anti-inflammatory properties and inhibit growth of many cells, particularly cancerous airway epithelial cells. However, the functional effects of PPARγ ligands on nonmalignant human bronchial epithelial cells have never been investigated. In the present study, we questioned whether PPARγ ligands may regulate proliferation of human bronchial epithelial cells, and we studied their potential molecular mechanisms. We found that synthetic PPARγ agonists, rosiglitazone (RGZ) and troglitazone (TGZ), induced proliferation of human bronchial epithelial cells, whereas the endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), inhibited cell growth. RGZ and TGZ (10 μM) induced a rapid and transient intracellular Ca2+ mobilization from thapsigargin-sensitive intracellular stores, whereas 15d-PGJ2 (5 μM) did not induce any Ca2+ signal. The PPARγ antagonist GW-9662 did not inhibit any biological responses, but it reversed the effect of 15d-PGJ2 on cell growth. Using RT-PCR, we detected mRNA expression of the GPR40 receptor, a G protein-coupled receptor recently identified as a receptor for free fatty acids and TZDs, in human bronchial epithelial cells. Downregulation of GPR40 by small-interfering RNA led to a significant inhibition of TZD-induced Ca2+ mobilization and proliferation. This study provides evidence for the proliferative effect of anti-diabetic drug TZDs in nonmalignant human bronchial epithelial cells through GPR40 receptor activation, involving an intracellular Ca2+ signaling pathway.

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Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus

Journal of Innate Immunity ◽

10.1159/000497072 ◽

2019 ◽

Vol 12 (1) ◽

pp. 103-115 ◽

Cited By ~ 4

Author(s):

Azzeddine Dakhama ◽

Reem Al Mubarak ◽

Nicole Pavelka ◽

Dennis Voelker ◽

Max Seibold ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Proinflammatory Response ◽

Type 2 Cytokines ◽

Cytokine Milieu ◽

Human Airways

The negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if Tollip regulates the airway inflammatory response to RV infection in vivo under IL-13 and IL-33 treatment. Following IL-13, IL-33, and RV treatment, Tollip-deficient (vs. -sufficient) HTBE cells produced excessive IL-8, accompanied by decreased sST2 production but increased IRAK1 activation. IL-8 production following IL-13/IL-33/RV exposure was markedly attenuated in IRAK1-deficient HTBE cells, as well as in Tollip KO HTBE cells treated with an IRAK1 inhibitor or a recombinant sST2 protein. Tollip KO (vs. wild-type) mice developed exaggerated airway neutrophilic responses to RV in the context of IL-13 and IL-33 treatment. Collectively, these data demonstrate that Tollip restricts excessive IL-8 production in type 2 cytokine-exposed human airways during RV infection by promoting sST2 production and inhibiting IRAK1 activation. sST2 and IRAK1 may be therapeutic targets for attenuating excessive neutrophilic airway inflammation in asthma, especially during RV infection.

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Human Parvovirus Infection of Human Airway Epithelia Induces Pyroptotic Cell Death by Inhibiting Apoptosis

Journal of Virology ◽

10.1128/jvi.01533-17 ◽

2017 ◽

Vol 91 (24) ◽

Cited By ~ 13

Author(s):

Xuefeng Deng ◽

Wei Zou ◽

Min Xiong ◽

Zekun Wang ◽

John F. Engelhardt ◽

...

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Human Bocavirus ◽

Airway Epithelial ◽

Airway Epithelia ◽

Human Airway ◽

Caspase 1 ◽

Tract Infections ◽

Human Airway Epithelia

ABSTRACT Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study, we confirmed that, when polarized/well-differentiated human airway epithelia are infected with HBoV1 in vitro, they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses, HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA), HBoV1-induced cell death dropped significantly; thus, NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genes BIRC5 and IFI6. When the expression of BIRC5 and/or IFI6 was inhibited by shRNA, the infected cells underwent apoptosis rather than pyroptosis, as indicated by increased cleaved caspase-3 levels and the absence of caspase-1. BIRC5 and/or IFI6 gene inhibition also significantly reduced HBoV1 replication. Thus, HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage, thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus. IMPORTANCE Microbial infection of immune cells often induces pyroptosis, which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes, studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here, we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1), a human parvovirus, causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia.

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