Secondhand vape exposure regulation of CFTR and immune function in cystic fibrosis

Abstract

Secondhand smoke exposure (SHSe) is a public health threat for people with cystic fibrosis (CF) and other lung diseases. Primary smoking reduces CF transmembrane conductance regulator (CFTR) channel function, the causative defect in CF. We reported that SHSe worsens respiratory and nutritional outcomes in CF by disrupting immune responses and metabolic signaling. Recently, electronic cigarette (e-cigs) usage by caregivers and peers has increased rapidly, causing new secondhand e-cig vape exposures. Primary vaping is associated with immunologic deficits in healthy people, but it is unknown whether e-cigs similarly impacts CF immune function or how it differs from SHSe. Human CF and non-CF blood monocyte-derived macrophages (MDMs) and bronchial epithelial cells (HBECs) were exposed to flavored and unflavored e-cigs. The effect of e-cigs on CFTR expression and function, bacterial killing, cytokine signaling, lipid mediators, and metabolism was measured during treatment with CFTR modulators. E-cigs decreased CFTR expression and function in CF and non-CF MDMs and negated CFTR functional restoration by elexacaftor/tezacaftor/ivacaftor (ETI). E-cigs also negated the restoration of anti-inflammatory PGD₂ expression in CF MDMs treated with ETI compared with controls. Flavored but not unflavored e-cigs increased proinflammatory cytokine expression in CF MDMs and e-cigs promoted glycolytic metabolism. E-cigs did not impact bacterial killing. Overall, HBECs were less impacted by e-cigs compared with MDMs. E-cigs reduced macrophage CFTR expression and hindered functional CFTR restoration by CFTR modulators, promoting a glycolytic, proinflammatory state. E-cigs are an emerging public health threat that may limit the efficacy of CFTR modulators in people with CF.NEW & NOTEWORTHY New research reveals that e-cigarettes pose a serious health risk for individuals with cystic fibrosis (CF). Exposure to electronic cigarette (e-cig) vapors decreases CF transmembrane conductance regulator (CFTR) function and undermines the effectiveness of CFTR modulators, potentially worsening inflammation and metabolic responses. This highlights an urgent need for awareness around e-cig use, especially among caregivers and peers of those with CF. E-cigarettes may further complicate the management of this chronic lung disease.

Keywords: cystic fibrosis; inflammation; modulators; omics; vaping.

Figure 1:. E-cigs alter cellular stability.

A) Apoptosis was measured via flow cytometry detection of Annexin V+ PI+ non-CF and CF human MDMs exposed to vehicle (NT) or increasing concentrations of cigarette extracts or ETI, n=4–5. B) Apoptosis was measured via flow cytometry detection of Annexin V+ PI+ non-CF and CF human MDMs exposed to increasing concentrations of e-cig extracts or ETI, n=3–5. C) Change in trans-epithelial electrical resistance (TEER) measurements over 5 days (Ohms × cm²) in CF and non-CF well-differentiated HBECs during 5-day intermittent apical exposures to 1% unflavored and flavored e-cigs, n=8 biologic replicates. “*” = p value <0.05, “**” = p value < 0.01, “***” = p value< 0.001. Statistics via one-way ANOVA for 1A, 1B with post-hoc Dunnett’s F multiple comparison test and unpaired t-test for 1C.

Figure 2:. E-cigs decrease MDM CFTR function and expression.

CFTR expression was measured in CF and non-CF MDMs via flow cytometry with or without exposure to vehicle (NT) or A) acute (1-time) or B) repeated (3-day) ETI, 1% e-cig, nicotine alone (nico), or combinations, n=3–6. CFTR function measured via halide efflux in non-CF and CF MDMs with or without C) acute or D) repeated exposures to ETI, 1% e-cig, nicotine, or combinations. Repeated exposure also with 1% cigarettes (cig). n=3–6 for A-D, “*” = p value <0.05, “**” = p value < 0.01, “***” = p value< 0.001 via one-way ANOVA with Dunnett’s F multiple comparison test for CF compared to non-CF NT control. Additional within exposure one-way ANOVA with Tukey multiple comparison test denoted with bracketed individual comparisons of significance for A-D. E) CFTR expression measured via Western blot in CF and non-CF HBECs with repeated e-cig exposure, representative blots shown with GAPDH as loading control. HBECs additionally exposed to cinnamon-flavored e-cig extracts.

Figure 3:. E-cigs and bacterial killing.

A) MRSA, B) Pseudomonas aeruginosa (Pa), and C) Burkholderia cenocepacia (Bc) bacterial killing via CFU assay in response to repeated e-cig or cigarettes (cig) exposure, treatment with ETI, or combinations. N=3–6, p value via one-way ANOVA with Dunnett’s F multiple comparison test, “*” = p value <0.05, “***” = p value <0.01.

Figure 4:. E-cigs and immunometabolites.

A) MDM production of eicosanoid metabolites in response to ecigs, ETI, nicotine, or combinations. B) HBEC production of eicosanoid metabolites in response to flavored or non-flavored ecigs, ETI, nicotine, or combinations. N=3–4, significance via one-way ANOVA with Dunnett’s F multiple comparison test.

Figure 5:. E-cigs and cytokine pathways.

Luminex cytokine results for 44 cytokines in A) CF and non-CF MDMs (38 detected) and B) HBECs (34 detected) during exposure to flavored and unflavored e-cigs, nicotine, ETI or combinations. Columns represent summed exposures (n=4) grouped by hierarchical clustering. A color-coded heatmap is displayed for expression values (red=high, blue=low). “*” indicates cytokines altered by flavored e-cigs. C) Voronoi pathway visualization from Reactome analysis of e-cig exposure in CF MDMs from 5A. The size of the component indicates a higher amount of related cytokine pathways, with the most significantly upregulated pathways in yellow.

Figure 6:. E-cigs and immunometabolism.

CF and non-CF MDM metabolism during e-cig exposure alone or in combination with ETI. Outputs measured were oxygen consumption rate (OCR), proton efflux rate (PER) and extracellular acidification rate (ECAR). Mean results for n=3 over 60 minutes. A) Schematic of seahorse OCR. B) CF and C) Non-CF MDM OCR basally, in response to e-cigs, ETI, or combinations. D) Percent glycolytic and oxidative phosphorylation (OXPHOR) rates in CF and non-CF MDMs in response to e-cigs, ETI, or combinations. ECAR in E) CF and F) Non-CF MDMs in response to e-cigs, ETI, or combinations. “*” = p value < 0.05, one-way ANOVA with Dunnett’s F multiple comparison test.