Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD

Abstract

Background: Individuals with respiratory disease are being increasingly exposed to wildfire smoke as populations encroach further into forested regions and climate change continues to bring higher temperatures with lower rainfall. Frequent exposures have significant potential to accelerate conditions such as chronic obstructive pulmonary disease (COPD) which is characterised by an exaggerated inflammatory response to environmental stimuli. Here we employ models of human airway epithelium exposed to wildfire smoke-extract (WFSE) to examine modulation in airway epithelial cell (AEC) survival, fragility and barrier function.

Methods: Submerged cultures of small airway epithelial cells (SAEC) and differentiated air-liquid interface (ALI) cultures of primary bronchial AEC (bAEC) were treated for 1-24 h with 1-10% WFSE generated from plant species found in the Australian bushland. Autophagy (LC3-II and Sequestosome), apoptosis (Poly-(ADP)-Ribose Polymerase (PARP) cleavage) and tight junction proteins were measured using western blot. Barrier function was assessed via permeability of fluorescein tracers and measuring trans-epithelial electrical resistance. The production of IL-6 was assessed using ELISA.

Results: Primary epithelial models exposed to WFSE exhibited a significant blockade in autophagy as evidenced by an increase in LC3-II coupled with a concomitant elevation in Sequestosome abundance. These exposures also induced significant PARP cleavage indicative of apoptotic changes. ALI cultures of bAEC treated with 5% WFSE demonstrated barrier dysfunction with significant increases in paracellular molecular permeability and ionic conductance, and a reduction in the abundance of the tight junction proteins ZO-1 and Claudin-1. These cultures also exhibited increased IL-6 secretion consistent with the aberrant and pro-inflammatory repair response observed in the COPD airways. Further, blocks in autophagy and barrier disruption were significantly elevated in response to WFSE in comparison to similar exposures with cigarette smoke-extract.

Conclusion: WFSE inhibits autophagic flux and induces barrier dysfunction in the airway epithelium. As autophagy is a central regulator of cellular repair, viability, and inflammation, targeting the block in autophagic flux may ameliorate the consequences of wildfire smoke-exposure for individuals with pre-existing respiratory conditions.

Keywords: Airway epithelium; Barrier function; COPD; Cigarette smoke; Environmental health; Exacerbation; Wildfire.

Fig. 1

Necrosis in human airway epithelial cells exposed to WFSE. a Application of 7.5–10% wildfire smoke extract (WFSE) into cultures of the 16HBE14o- airway epithelial cell line elicits a significant cytotoxic response (release of lactate dehydrogenase from compromised cells) vs. control cells, after a 24 h exposure period. Over shorter periods, cytotoxicity was not significantly induced compared to base-line levels of cell turnover. b Primary small airway epithelial cells did not demonstrate a significant cytotoxic response (compared to the control exposure) after 6 h using a similar treatment regimen, and for the 10% cigarette smoke extract (10% CSE) exposure.Intervals are SE. * P ≤ 0.05, **** P ≤ 0.0001. Each time point represents n = 3 experiments

Fig. 2

Inhibition of autophagic flux and apoptotic changes in primary SAEC exposed to WFSE. a Small airway epithelial cells subject to 10% wildfire smoke extract (WFSE) for 6 h undergo apoptotic changes as evidenced by the cleavage of the caspase-3 substrate Poly (ADP-ribose) polymerase (PARP). This may be due to the decrease in the inhibitor of the extrinsic apoptotic pathway Bcl2, and a blockade in normal autophagic flux as determined by the increase in the essential autophagy protein Microtubule-Associated Protein 1A/1B-Light Chain-3-II (LC3-II; lower band), simultaneous with increased Sequestosome, which is degraded with the cargo it shuttles to the autophagosome. While 10% cigarette smoke extract (CSE) also initiated an apoptotic response concomitant with a reduction in Bcl2, this was not accompanied by a block in autophagic flux, as Sequestosome abundance remained relatively low in relation to the elevation in LC3-II. The expression of the pro-inflammatory transcription factor NF-κβ subunit (p65) did not significantly increase in this model. b Histogram analyses of protein expression density scores. Protein expression was baselined to the abundance in the untreated sample, and normalized to the expression of β-actin. Intervals are 95% CI, and significance compared to the control sample can be identified when confidence intervals do not intersect 1 for the Y-axis. *, P ≤ 0.05; **, P ≤ 0.01 ***, P ≤ 0.001 for n = 3 experiments

Fig. 3

Differentiated primary human bAEC exposed to WFSE exhibit a fragile epithelial phenotype Trans-epithelial electrical resistance (TEER; ionic conductance of the paracellular pathway) and passage of sodium fluorescein tracers (paracellular molecular flow) were quantified for primary bronchial airway epithelial cells that were differentiated in a transwell culture system. a A reduction in electrical impedance (vs. the control exposure) is evident for the 2.5–10% wildfire smoke extract (WFSE) treatments after the 6 h exposure interval (left graphic). This relationship is maintained after 24 h (right graphic) when the maximum reduction in electrical resistance is observed for 5–10% WFSE, and the effect of 10% cigarette smoke extract (10% CSE) exceeds 2.5% WFSE. b The passage of sodium fluorescein tracers from the apical to the basal reservoir is evident for the 5 and 10% WFSE exposures at the 6 h interval (left graphic). At the 24 h interval the influence of 5–10% WFSE potentiates a marked increase in paracellular molecular flow, and are followed by 10% CSE treatment. Data is representative of n = 3 cultures from different individuals. Intervals are SE. For clarity “*” were omitted from these graphics, and P values are reported in Additional file 3: Table S1

Fig. 4

Differentiated primary human bAEC exposed to WFSE exhibit a reduction in TJ proteins. a Western analysis of tight junction proteins from primary human airway epithelial cells grown at an air-liquid interface showing a reduction in ZO-1 and Claudin-1 for the 5% wildfire smoke extract (WFSE) exposure after 6 h. While the abundance of Occludin-1 followed a similar trend for 5% WFSE exposure, its downregulation did not reach significance in this model. Apoptosis as evidenced by the cleavage of the caspase-3 substrate Poly (ADP-ribose) polymerase (PARP) was not detected. As significant apoptosis was not anticipated, a doxorubicin exposure (1 μM) was included to produce a caspase-mediated apoptotic outcome as a positive control to identify the products of caspase-3-mediated PARP cleavage in the western blot. b Histogram analyses of protein expression density scores. Protein expression was baselined to the abundance in the untreated sample, and normalized to the expression of β-actin. Intervals are 95% CI, and significance compared to the control sample can be identified when confidence intervals do not intersect 1 for the Y-axis. *, P ≤ 0.05; **, P ≤ 0.01 for n = 3 experiments using cells derived from different participants

Fig. 5

Differentiated primary human bAEC secrete IL-6 in response to WFSE. Differentiated air-liquid interface cultures of primary human bronchial airway epithelial cells were exposed to increasing concentrations of wildfire smoke extract (WFSE) for 6 h. The secretion of IL-6 was significantly increased from bAEC treated with 5% WFSE vs. the control exposure. Intervals are standard error. **, P ≤ 0.01 for n = 3 experiments using cells derived from different participants