Loss of Potassium and Chloride Transport Changes PM-Induced Epithelial Dysfunction

Abstract

Background: Chronic exposure to particulate matter (PM) is recognized as a significant contributor to respiratory health complications, including oxidative stress, inflammatory responses, and compromised epithelial barrier function. In this work, we ask whether the transport of potassium and chloride through the large-conductance calcium-activated potassium (BK_Ca) channel and the cystic fibrosis transmembrane conductance regulator (CFTR) channel may change PM-induced epithelial dysfunction.

Methods: This study aimed to evaluate the impact of PM on cell variability, ROS level, inflammation, mitochondrial function, intracellular calcium homeostasis, and epithelial barrier integrity in three different airway epithelial cell lines: wild-type human bronchial epithelial cells (HBE WT), HBE WT cells with disruption of the KCNMA1 gene encoding the α-subunit of the BK_Ca channel (HBE ΔαBK_Ca) with lost potassium transport, and cystic fibrosis bronchial epithelial cells (CFBE) with dysfunction of the chloride transport.

Results: PM exposure significantly increased ROS synthesis and amplified IL-6 and TNF-α release, particularly in HBE ΔαBK_Ca and CFBE cells. Mitochondrial function was also adversely affected, as evidenced by reduced maximal respiratory capacity in both HBE ΔαBK_Ca and CFBE cells relative to HBE WT. In addition, PM-treated HBE ΔαBK_Ca and CFBE cells showed higher intracellular calcium concentrations. Finally, PM exposure resulted in a pronounced reduction in transepithelial electrical resistance (TEER), with CFBE monolayers exhibiting the most significant susceptibility to barrier disruption.

Conclusion: These findings indicate that impaired potassium and chloride transport through the BK_Ca and CFTR channels exacerbates particulate matter-induced oxidative stress, inflammatory responses, mitochondrial dysfunction, and disturbances in calcium homeostasis in airway epithelial cells. Increased susceptibility of HBE ΔαBK_Ca and CFBE cells to PM exposure, underscores the crucial role of proper ion transport in maintaining airway epithelial integrity.

Keywords: epithelial barrier integrity; inflammation; mitochondrial function; oxidative stress; particulate matter; potassium and chloride transport.

Figure 1

Cell viability and intracellular reactive oxygen species (ROS) level. (A) Effect of PM on HBE WT, HBE ΔαBK_Ca, and CFBE cells in MTT assay. Cells were incubated with PM for 24 hours at concentrations of 10, 50, and 100 µg/mL. Data are presented as mean and SD (n=16). Statistical significance was determined using an unpaired t-test (***p<0.001) compared to each cell line control. (B) The impact of 50 µg/mL PM exposure on intracellular ROS level (in arbitrary units) in HBE WT, HBE ΔαBK_Ca, and CFBE cells was presented after 3 hours of incubation. Data were normalised to the control and expressed as mean ± SEM (n=6) for ROS level analysis (H2DCFDA probe). (C) Data were expressed as a percentage of intracellular ROS level increase after PM exposure compared to control, % ± SEM (n=6). Statistical significance was determined using one-way ANOVA (***p<0.001).

Figure 2

Analysis of the Interleukin-6 (IL-6) presence. IL-6 concentration in HBE WT (A), HBE ΔαBK_Ca (B), and CFBE (C) cell lines after 24 and 48 hours of incubation with 10, 50, 100 µg/mL PM (A–C) and 10 ng/mL TNF-α (D). Absorbance results were converted to concentration values. Error bars represent the mean ± SEM (n=3). Statistical significance was determined using one-way ANOVA (*p<0.05, **p<0.005, ***p<0.001) compared to the controls.

Figure 3

Evaluation of the Tumor Necrosis Factor-α (TNF-α) level. TNF-α concentration in HBE WT (A), HBE ΔαBK_Ca (B), and CFBE (C) cell lines after 24 and 48 hours of incubation with 10, 50, 100 µg/mL PM. Absorbance results were converted to concentration values. Error bars represent the mean ± SEM (n=3). Statistical significance was determined using one-way ANOVA (*p<0.05, ***p<0.001) compared to the controls.

Figure 4

Comparison of mitochondrial oxygen consumption rate in HBE WT, HBE Δα BK_Ca, and CFBE cells. (A) Representative recording of changes in oxygen concentration in HBE WT cells treated with oligomycin (4 µg/mL), FCCP (3 µM), rotenone (1 µM) and antimycin (1 µM). (B) Oxygen consumption rate in untreated cells (control). (C) Oxygen consumption rate in treated cells after 24 hours of incubation with 10 ng/mL TNF-α, cells were exposed to oligomycin (4 µg/mL), FCCP (3 µM), rotenone (1 µM), and antimycin A (1 µM). Results were obtained using a probe measuring the OCR, and shown as mean ± SEM (n=3). Statistical significance was determined using one-way ANOVA (*p<0.05) compared to each cell line.

Figure 5

Analysis of the calcium intracellular content. (A) Representation of the intracellular Ca²⁺ levels in HBE WT, HBE Δα BK_Ca, and CFBE cell lines in HBSS buffer. (B) Representation of the intracellular Ca²⁺ levels in HBE WT, HBE Δα BK_Ca, and CFBE cell lines in Ringer solution deprived of Ca²⁺ and EGTA addition. Cell lines were treated with 10, 50, and 100 µg/mL PM and 5 µM ionomycin (positive control). Results were calculated as the fluorescent ratio of F₃₄₀/F₃₈₀ and presented as mean ± SD (n=7). Statistical significance was determined using an unpaired t-test (*p<0.05, ***p<0.001) compared to each cell line.

Figure 6

Changes in transepithelial electrical resistance (TEER) on airway epithelium in the presence of particulate matter. (A) Impact of PM on TEER in HBE WT cell monolayers. (B) Effect of PM on HBE ΔαBK_Ca cell monolayers. (C) Impact of particulate matter on transepithelial electrical resistance in CFBE cell monolayers. Cell monolayers were treated with 50 µg/mL PM, and TEER was measured after 30, 60, 90, and 180 minutes of incubation. Results were normalised to the TEER value measured before treatment (time 0 min) and were shown as mean values ± SD (n=6). Statistical significance was determined using a paired t-test (*p<0.05, **p<0.01, ***p<0.001) compared to each cell line.