Sidestream smoke exposure increases the susceptibility of airway epithelia to adenoviral infection

Abstract

Background: Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR.

Methodology and findings: Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β) is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection.

Conclusions: This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant therapeutics and increase the understanding of potential side effects.

Figure 1. Smoke and air exposure transiently decreases the transepithelial resistance (TER) of polarized CaLu-3 cells.

TER recovers by 18 h post-exposure to mainstream cigarette smoke (MSS), sidestream cigarette smoke (SSS) or filtered air (MSFA or SSFA). Data from six replicates per condition and three independent experiments; mean values expressed as a percentage of control+SE of the mean. *p<0.05 MSS or SSS versus pre- or post-exposure and versus MSFA or SSFA respectively.

Figure 2. Sidestream cigarette smoke (SSS) exposure increases epithelial susceptibility to adenovirus infection (entry and transduction).

Polarized CaLu-3 cells were infected from the apical surface with hAdV5-β-gal (MOI 10 pfu/cell) or mock infected (MOI 0) 18 h post-exposure to air (MSFA or SSFA), mainstream cigarette smoke (MSS), or SSS. Cells were evaluated for A: β-Galactosidase activity (transduction, four to six replicates per condition; three independent experiments) or B: viral genomes (viral entry) 24 h post-infection (four biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments). C: Intracelluar viral genome load 24 h after apical adenovirus infection, at increasing MOI, in SSS- or SSFA-exposed epithelia. Results for MSS, SSS, and FA without virus were identical and were combined for graphical representation (No Virus; four replicates per condition; two independent experiments). Representative experimental results are shown as mean+SE.

Figure 3. CAR expression is increased in polarized CaLu-3 cells 18 h post-sidestream cigarette smoke (SSS) exposure relative to mainstream cigarette smoke (MSS), or filtered air (SSFA or MSFA) exposure.

A: Total CAR mRNA (three to four biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments) and B: CAR^Ex8 mRNA (three to four biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments) quantification using quantitative RT-PCR. Mean values of three independent experiments relative to control+SE of the mean. C: Total CAR and D: CAR^Ex8 protein and corresponding β-actin expression by Western blot (representative) and Multi-Guage image analysis (mean values from three independent experiments expressed as a percentage of control+SE of the mean). *p<0.05.

Figure 4. CAR expression is increased and localization is altered in polarized CaLu-3 cells 18 h post-SSS exposure.

Immunofluorescence staining of A) total endogenous CAR (green) and B) CAR^Ex8 (green), co-stained with antibodies directed against either the tight junction protein ZO-1 (red) or the apical protein ezrin (red), in polarized CaLu-3 cells 18 h after exposure to SSFA or SSS. Nuclei are counterstained with DAPI (blue). X–Z sections representative of three independent experiments are shown. Dotted white line represents the Transwell filter that cells are seeded on. Black line = 10 µm. Confocal microscopy (60× oil immersion).

Figure 5. GSK3β is downregulated 18 h post- sidestream cigarette smoke (SSS) exposure in comparison to air (SSFA or MSFA) or mainstream cigarette smoke (MSS) exposure.

A: Analysis of total GSK3β mRNA levels by quantitative RT-PCR (four to six biological replicates per condition measured in duplicate in each qPCR assay; three independent experiments; mean values from three independent experiments relative to control+SE of the mean). B: GSK3β and C: GSK3β-pS9 protein levels, representative Western blot and densitometric analysis, relative to β-actin (mean values from three independent experiments (duplicate gels per experiment) expressed as a percentage of control+SE of the mean). *p<0.05.

Figure 6. GSK3β is downregulated in polarized CaLu-3 cells 18 h post-GSK3β inhibitor (SB415286) treatment.

A) Total mRNA expression of GSK3β in control (white) or SB415286 (black) treated CaLu-3 epithelia (four biological replicates per condition measured in duplicate in each qPCR assay; mean values from three independent experiments relative to control+SE of the mean). B) Representative Western blot analysis of GSK3β, GSK3β-pS9, and β-actin protein levels. Quantification of C) GSK3β or D) GSK3β-pS9 protein levels, relative to β-actin (mean values from three independent experiments expressed as a percentage of control+SE of the mean). *p<0.05.

Figure 7. CAR expression is upregulated in polarized CaLu-3 cells 18 h post apical treatment with 45 µM of GSK3β inhibitor (SB415286).

A: Total CAR and B: CAR^Ex8 mRNA levels by quantitative RT-PCR (three biological replicates per condition measured in duplicate in each qPCR assay; mean values from three independent experiments relative to control+SE of the mean) and C: total CAR and D: CAR^Ex8 and corresponding β-actin protein expression by Western blot (representative) and quantification using Multi-Guage densitometric analysis (mean values from three independent experiments expressed as a percentage of control+SE of the mean). Apical biotinylation of polarized CaLu-3 cells 18 h post-treatment with 45 µM SB415286 shows increased protein levels of E: total CAR and F: CAR^Ex8 specifically (representative blot shown from three independent experiments). *p<0.05.

Figure 8. Apical entry and transduction of hAdV5-β-gal is increased in polarized CaLu-3 cells 18 h post apical treatment with 45 µM of GSK3β inhibitor (SB415286).

A: TER does not change in polarized CaLu-3 cells treated with GSK3β inhibitor, SB415286, for 18 h. B: A significant increase in adenoviral transduction is observed upon treatment. C: A significant increase in intracellular adenovirus genome copies occurs upon treatment. Representative data shown; mean of four replicates per experiment; three independent experiments. *p<0.05 SB415286 versus control or no virus.

Figure 9. Schematic representation of the proposed mechanism for increased epithelial susceptibility to adenovirus infection upon sidestream cigarette smoke (SSS) exposure.

SSS downregulates GSK3β protein expression, which leads to transcriptional upregulation of CAR^Ex8, leading to increased susceptibility of the epithelium to adenovirus infection from the apical surface. CXADR, CAR gene.