- Open Access
Human primary airway epithelial cells isolated from active smokers have epigenetically impaired antiviral responses
© The Author(s). 2016
- Received: 19 April 2016
- Accepted: 2 September 2016
- Published: 7 September 2016
Cigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses.
We investigated retinoic acid-inducible protein I (RIG-I) and interferon (IFN) induction by influenza A virus (IAV) in human bronchial epithelial cells (HBEC) isolated from smokers or nonsmokers. Subcultured HBEC cells were infected with A/Puerto Rico/8/1934 (PR8) IAV at an MOI of 1. After 24 h of infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, Toll-like receptor3 (TLR3) and IFN expression levels.
IAV exposure induced a vigorous IFN-β, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBEC from nonsmokers and significant induction of RIG-I and TLR3. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87 and 79 % compared to the response from nonsmokers. CS exposure history was associated with inhibition of viral induction of the IFN-β, IFN-λ1 and IFN-λ 2/3 mRNA response by 85, 96 and 95 %, respectively, from that seen in HBEC from nonsmokers. The demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since viral induction of all three IFNs was restored. IFN-β induction of RIG-I and TLR3 was also suppressed in the cells from smokers.
Our results suggest that active smoking reduces expression of antiviral cytokines in primary HBEC cells. This effect likely occurs via downregulation of RIG-I and TLR3 due to smoke-induced epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be a major mechanism contributing to the increased risk and severity of viral respiratory infections in smokers and to viral-mediated acute exacerbations of COPD.
- Innate immunity
Chronic obstructive pulmonary disease (COPD) is a major worldwide cause of morbidity and mortality. According to the World Health Organization, by 2030, COPD will be the third most common global cause of death . Cigarette smoking (CS) is the main risk factor for the development of COPD, which is a chronic inflammatory disease characterized by progressive, partially reversible airflow limitation . CS alters innate and adaptive immune responses, and it has been proposed that many of the deleterious health consequences of CS are due to its adverse immune effects . Respiratory viral infections with influenza virus, rhinoviruses, and respiratory syncytial virus are leading pathogens associated with COPD exacerbations, which are associated with progressive loss of lung function . In fact, 64 % of COPD exacerbations are caused by respiratory infections including influenza and respiratory syncytial virus . Additionally, many studies have established a relationship between CS and the risk of influenza infection [6, 7]. The mechanism of the increased risk and severity of infections in smokers is probably multifactorial, but certainly includes changes in alteration of immunologic host defenses.
The innate immune system responds to influenza A virus (IAV) through three classes of microbial pathogen sensors, called pattern recognition receptors (PRRs). Most cells use the cytosolic sensor, retinoic acid inducible gene I (RIG-I), to detect IAV and trigger antiviral responses . Endosomal based Toll-like receptors (TLRs) are also involved in the recognition of, and response to IAV. TLR3, a double-strand RNA sensor, may be used by some epithelial cells to detect the viral replicative intermediate dsRNA . Plasmacytoid dendritic cells (pDCs) use TLR7 to recognize influenza genomic RNA, upon its release in late endosomes . Finally, the PRR nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRP), including NLRP3 and nucleotide-binding oligomerization domain 2 (NOD2), play multiple roles in regulating the innate immune response during virus infection through modulation of inflammasome activation, antiviral activity, and priming of adaptive immunity [11–14].
Airway epithelial cells are the primary location of replication, and also represent the first line of defense against IAV by eliciting an innate immune response [15, 16]. In airway epithelial cells, RIG-I is the central regulator of IAV-mediated induction of antiviral cytokines . TLR3 also contributes in the response to IAV in epithelial cells . We have demonstrated that RIG-I and TLR3 are the two major PRRs induced by IAV infection in A549 and human primary type II alveolar epithelial cells (AEC) . Either of them may compensate to maintain antiviral immunity when the other signaling mechanism is shut down. One of the major downstream products of RIG-I and TLR3 signaling is the interferon (IFN) cytokine family. IFNs are further divided into type I (mainly IFN-α and β), II (IFN-γ) and III (IFN-λ) subtypes, based in part on the differential use of unique receptors through which they mediate signal transduction to induce antiviral activity. Rapid production of type I and III IFN is a central and essential component of the antiviral response in airway epithelial cells [20, 21]. We have previously shown that RIG-I is crucial for the induction of the early antiviral cytokine response, and cigarette smoke extract (CSE) inhibits the RIG-I initiated innate immune response to IAV [22–24].
Although galectin-3 and the receptor for advanced glycation end products (RAGE) are involved in the innate response to IAV [25–27], there is no evidence that these PRRs recognize IAV and initiate cytokine responses to this virus in human epithelial cells. An extensive immunohistochemical survey of normal human tissues showed RAGE expression in type II alveolar pneumocytes, endothelia and alveolar macrophages . TLR7 and galection-3 mainly recognize pathogens in dendritic cells . In this study, we will focus on the two major PRRs, RIG-I and TLR3, that recognize IAV in epithelial cells and compare antiviral cytokine induction by IAV in primary human bronchial epithelial cells (HBEC) from smokers and nonsmokers.
Isolation of primary human bronchial epithelial cells
Human bronchial epithelial cells (HBEC) were obtained by bronchoscopy and bronchial brushing with the written, informed consent from both smoking and non-smoking, healthy, adult volunteers in accordance with a protocol approved by the Institutional Review Board of the University of Oklahoma Health Sciences Center (IRB # 2197). The smokers had a smoking history of at least 10 pack years with ½ to 1 pack of cigarettes per day. All smoking and nonsmoking participants were gender, age and ethnicity matched. Three or four separate bronchi were brushed and the cells were rinsed from the brush into 10 ml sterile saline until 5 × 106 to 1 × 107 cells total were collected as determined by hemocytometer counts for total and viable cells by trypan blue exclusion. The HBECs were centrifuged at 400 × g for 5 min. Cells were resuspended to 5 × 105 cells/ml in complete Bronchial Epithelial Cell Growth Medium (BEGM; Lonza Group Ltd.); were seeded into collagen coated tissue culture plates (Bio-Coat, BD Biosciences) at a density of 1×105 cells/cm2 and were propagated in an incubator at 37 °C in 5 % CO2. After 24 h the cells were washed with HBSS to remove non-adherent cells and fresh complete BEGM was added. When the cultures were near confluence (7–10 days), the monolayers were lifted with 1x Accutase solution and were subcultured at a 1:5 dilution. After each passage the cells grew to confluence within 4 to 5 days, and when the cultures were split, freezer stocks were prepared in 80 % BEGM + 10 % fetal bovine serum + 10 % DMSO and were stored in liquid nitrogen vapor at −190 °C. Cells were rapidly thawed, washed in BEGM, and cultured till confluence prior to experiments.
Preparation of influenza virus stock
Influenza virus, A/PR/34/8 (PR8), was passaged in Madin–Darby canine kidney (MDCK) cells. Virus was grown in MDCK cells in DMEM/F12 with ITS+ (BD Biosciences, Franklin Lakes, NJ) and trypsin, harvested at 72 h postinfection and titered by plaque assay in MDCK cells. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The lower limit of detection of this assay is 0.1EU/ml or approximately 20 pg/ml LPS.
Preparation of CSE
One (100 mm) cigarette without filter was combusted with a pump. The smoke was bubbled through 25 ml of cell medium at a speed of 50 ml/min. The resulting suspension was filtered through a 0.22-μm pore filter (Lida Manufacturing, Kenosha, WI) to remove bacteria and large particles. This solution, considered to be 100 % CSE, was diluted and applied to cell cultures within 30 minutes of preparation. The nicotine concentration of 100 % CSE was 73.48 ± 1.08 μg/ml.
Measurement of mRNA expression by quantitative real-time PCR (qRT-PCR)
Total RNA from cells was extracted using a modified TRIzol (Invitrogen, Carlsbad, CA) protocol, spectrophometrically quantitated, and the integrity verified by formaldehyde agarose gel electrophoresis. Equal amounts (1 μg) of RNA from each sample were used with oligo (dT) as primers for production of cDNA (SuperScript II First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA). Gene specific primers for the PRRs, cytokines and the β-actin housekeeping genes were used. qRT-PCR was performed using 100 ng sample RNA and SYBR Green (Quanta Biosciences, Gaithersburg, MD) in a Bio-Rad CFX96™ Touch Real-Time PCR Detection System. Results were calculated and graphed from the comparative CT method (ΔΔCT CT method). The primers’ sequences were as follows: RIG-I forward 5′- TCCTTTATGAGTATGTGGGCA -3′; RIG-I reverse 5′- TCGGGCACAGAATATCTTTG -3′; IFN-β forward 5′- GCTCTCCTGTTGTGCTTCTCCAC -3′; IFN-β reverse 5′- CAATAGTCTCATTCCAGCCAGTGC -3′; β-actin forward 5′- GCCAACCGCGAGAAGATGACC-3′; β-actin reverse 5′- CTCCTTAATGTCACGCACGATTTC-3′; TLR3 forward 5′-GTCTGGGAACATTTCTCTTC-3′; TLR3 reverse 5′-GATTTAAACATTCCTCTTCGC-3′; IFN-λ1 forward 5′- CGCCTTGGAAGAGTCACTCA-3′; IFN-λ1 reverse 5′- GAAGCCTCAGGTCCCAATTC-3′; IFN-λ2/3 forward 5′- AGTTCCGGGCCTGTATCCAG-3′; IFN-λ2/3 reverse 5′- GAGCCGGTACAGCCAATGGT-3′; IP-10 forward 5′-TCTAGAACCGTACGCTGTACCTGC-3′; IP-10 reverse 5′-CTGGTTTTAAGGAGATCT-3′; IRF7 forward 5′-CAGATCCAGTCCCAACCAAG-3′; IRF7 reverse 5′- GTCTCTACTGCCCACCCGTA-3′.
ELISA and multiplex immunoassay
ELISAs of IP-10 and IFN-λ2/3 cytokine protein levels in the supernatants were all performed using commercially available kits (R & D system, Minneapolis, MN).
siRNA transfection of HBEC
For siRNA treatment, cells were plated 24 h before treating with siRNAs (Ambion). siRNAs were diluted in 250 μl of Opti-MEM medium and mixed gently. Five μl of Lipofectamine 2000 (Invitrogen) was added in 250 μl Opti-MEM medium and incubated for 5 min. Diluted siRNA and Lipofectamine 2000 were combined and mixed gently and incubated for 20 min at RT. The siRNA-Lipofectamine 2000 complexes were added to each well and mixed gently. siRNA final concentration was 20 nM. The cells were then incubated at 37 °C for 48 h prior to use in experiments.
RIG-I protein determination by immunoblotting
The cells were harvested and homogenized, and then lysed in 500 μl of cold lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 10 mM EDTA, NaF, sodium pyrophosphate, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 10 μg of leupeptin/ml). Cell homogenates were clarified by centrifugation at 10,000 × g, at 4 °C for 10 min, and the clarified lysates were mixed with SDS-PAGE sample buffer (60 mM Tris, pH 6.8, 10 % glycerol, 2.3 % SDS) and heated to 95 °C for 5 min. The samples were separated by 4–15 % gradient gel and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. For the detection of proteins, the membranes were immunoblotted with rabbit polyclonal antibody specific for RIG-I (Abcam, Cambridge, MA) and GAPDH (R & D Systems). The membranes were then treated with horseradish peroxidase-labeled goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) and chemiluminescent reagents (Pierce Biotechnology, Rockford, IL). Blots were developed using the Syngene G:box Bioimaging System and GeneTools software (Syngene, Frederick, MD) and resultant signals were quantified using ImageQuant software (BD/Molecular Dynamics, Bedford, MA).
Where applicable, the data have been expressed as the means ± standard error of the mean (SEM). Statistical significance was determined by one-way ANOVA with Student-Newman-Keuls post hoc correction for multiple comparisons. Significance was considered as P < 0.05.
HBEC isolated from smokers have suppressed antiviral responses during influenza infection
Demethylation reversed the immunosuppressive effects of CS exposure on HBEC
Amplification of RIG-I and TLR3 induction by interferon was suppressed in HBEC from smokers
RIG-I and TLR3-initiated antiviral responses in infected human lung epithelial cells are well known for their ability to reduce viral replication, and both type I and type III IFNs inhibit influenza replication. This suppression was enhanced when both types of IFNs were simultaneously used . We have previously demonstrated that RIG-I and TLR3 are the two major PRRs induced by IAV infection in human A549 and type II AEC . In this report, we demonstrated that influenza-induced antiviral cytokine responses were epigenetically inhibited in primary HBEC from smokers compared with nonsmokers.
CS affects the first lines of defense by increasing airway epithelial permeability, causing tissue disruption, and decreasing mucociliary clearance . Many studies have shown that CS exerts detrimental effects on immunologic host defenses by inhibition of stimulated cytokine production that may interfere with effective and efficient antimicrobial responses [34–36]. Eddleston et al. demonstrated that pretreatment of BEAS-2B cells or normal human bronchial epithelial cells with CSE diminished IP-10 and RANTES mRNA induction by either the viral mimic polyinosine-polycytidylic acid (Poly I:C) or human rhinovirus 16 . Another group showed that CSE-mediated inhibition of poly I:C-induced antiviral innate responses in human peripheral blood mononuclear cells (PBMC) is mainly due to inhibition of IFN-β production . Here, we directly compared the innate response from primary HBEC obtained from smokers with these responses from HBEC from nonsmokers. We demonstrated that HBEC from smokers have a diminished RIG-I and TLR3-initiated antiviral response to influenza infection compared to nonsmokers. It has been demonstrated that active smoking reduces both human lung macrophage expression of TLR3, and dsRNA-induced IP-10 production . However, the expression of mRNA transcripts for nucleic acid receptors by RT-PCR was only measured in resting macrophages without viral stimulation. Our data showed diminished RIG-I and TLR3 mRNA expression in HBEC with IAV stimulation, which is more applicable to actual IAV infection in vivo as these cells are a major site of viral replication. Our research provides further insights into mechanisms by which CS alters epithelial innate immune responses to virus infection. These observations could help to explain the increased susceptibility of cigarette-smoking humans to severe respiratory viral infections.
Our results suggested CS also inhibits autocrine/paracrine amplification of RIG-I and TLR3 induction by IFNs (Fig. 9) in surrounding cells. This may occur through epigenetic modification of the promoters of RIG-I and TLR3 by cigarette smoke, inhibiting binding of activating transcription factors. Epigenetic modifications, mainly DNA methylation and histone modification, regulate gene expression by changing DNA accessibility and chromatin structure without changing the DNA coding sequence. DNA methylation, the attachment of methyl groups to DNA, potentially alters gene expression profiles of CS-exposed target cells [40, 41]. Earlier work from Jaspers et al. found that methylation of IRF7, which is a transcriptional factor in the IFN response, was associated with an impaired antiviral defense response in human NECs from smokers . Our data supports this previous study, and provides further mechanistic information. Using HBEC cells from smokers, our study demonstrated that the demethylation agent 5-Aza reversed the suppressed RIG-I, TLR3, IRF7 and IFN response to IAV. RIG-I and TLR3 play important roles in the recognition of, and response to, IAV in human lung epithelial cells. Since IRF7 is downstream of RIG-I-initiated signaling, epigenetic modifications of RIG-I might provide another mechanism for IRF7 inhibition found by Jaspers. Our work is consisted with previous preliminary data demonstrating that HBEC from subjects with COPD have minimal RIG-I induction and impaired IFN responses to IAV . In our report, we examined both RIG-I and TLR3 expression and revealed that smoke-induced epigenetic changes are responsible for this effect. The immunosuppressive effects of CS exposure are likely due to increased methylation (i.e., hypermethylation) of the promoters of RIG-I and TLR3. It is known that CS alters the methylation pattern of the genome. Recently, many studies have investigated epigenetic changes relating to exposure to cigarette smoke that lead to lung cancer [43–45]. Few studies have, however, reported on the epigenetic changes in the human immune system that may lead to compromised antiviral activity with resultant enhanced viral replication and prolonged recovery. One study found that lung tumors of patients with COPD differ from those of patients without COPD, with differentially methylated and expressed genes being mainly involved in the immune response . Our future studies will focus on whether there are DNA methylation changes in human lung epithelial cells from smokers that alter host defense responses.
Taken together, we have shown CS affects the inductive and amplification phases of the RIG-I and TLR3 mediated antiviral response to IAV in lung airway epithelial cells. We chose IAV PR8 for these studies, as this mouse adapted virus stain is the standard H1N1 strain used in both human and animal studies. However, it is recognized that it is not an unmodified fresh clinical isolate, which might limit the applicability of our conclusions relative to other IAV. Further work needs to be done to study the epigenetic impact of CS exposure and its effect on gene expression of important human innate immune components.
We want to thank Muhammad S. Khan, Bethany Holtslander-Petrone and Laurel Howard for helping to screen subjects or providing support for the procedure. We acknowledge the kind assistance of the nursing and bronchoscopy staff of the Veterans Administration Hospital of Oklahoma City, OK.
The research described in this work was partially supported by a Clinical Innovator Award from the Flight Attendant Medical Research Institute, by the Merit Review Program of the Department of Veterans Affairs, the National Institutes of Health (1 I01 BX001937), and by the National Institutes of Health, projects U19AI62629 and GM103648 (to J.P.M.).
Availability of data and material
The data obtained and/or analysed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.
WW and JPM generated the hypothesis, performed statistical analysis and wrote the manuscript. WZ and XW acquired the data. VLW assisted with acquiring data. JB performed cell culture. DCH and DB enrolled volunteers. HY and CDC performed bronchoscopy. MZ designed the study and contributed to manuscript writing. All authors read and approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
Human bronchial epithelial cells (HBEC) were obtained by bronchoscopy and bronchial brushing with the written, informed consent from both smoking and non-smoking, healthy, adult volunteers in accordance with a protocol approved by the Institutional Review Board of the University of Oklahoma Health Sciences Center (IRB # 2197).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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