IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels

Abstract

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

Keywords: COPD; IL-33; basal cell; cigarette smoke; gene expression.

Figure 1.

Flow chart of the present study methods. One longitudinal and four cross-sectional in vivo studies were analyzed to assess the gene expression profile of IL-33 and IL-33 pathway related genes. Cellular deconvolution was performed on the studies in which bulk RNA sequencing was performed, as the method is not suitable for microarray study. One in vivo and two in vitro single-cell RNA sequencing studies were analyzed to assess the IL-33 expression at the single-cell level and cell trajectory (–26, 27, 28). ALI = air-liquid interface; COPD = chronic obstructive pulmonary disease; CRUKPAP = Cancer Research UK Papworth Hospital; GLUCOLD = Groningen and Leiden Universities Study of Corticosteroids in Obstructive Lung Disease; NORM = Study to Obtain Normal Values of Inflammatory Variables From Healthy Subjects; STOP = Stop Smoking Study.

Figure 2.

Expression of IL-33 and IL1RL1 in bronchial datasets. (A) IL-33 and (B) IL1RL1 expression levels from the STOP study of bronchial biopsies (N = 16) before and 12 months after smoking cessation. (C) IL-33 and (G) IL1RL1 expression levels of bronchial biopsies from the GLUCOLD (microarray) study of ex-smokers (n = 33) and current smokers (n = 46) with chronic obstructive pulmonary disease (COPD). (D) IL-33 and (H) IL1RL1 expression levels of bronchial brushing samples from the COPD microarray study of ex-smokers (n = 57) and current smokers (n = 30) with COPD (GSE37147). (E) IL-33 and (I) IL1RL1 expression of bronchial biopsies from the NORM study of never-smokers (n = 40) and healthy current smokers (n = 37). (F) IL-33 and (J) IL1RL1 expression of bronchial biopsies from the CRUKPAP study of never-smokers (n = 8), ex-smokers (n = 151), and current smokers (n = 77). All analyses were corrected for age and sex. The numbers shown are P values taken from the results of differential expression analyses conducted in edgeR, corrected for age and sex. Error bars represent the SD. P values shown are false discovery rate–corrected for the number of genes tested. CRUKPAP = Cancer Research UK Papworth Hospital; GLUCOLD = Groningen and Leiden Universities study of Corticosteroids in Obstructive Lung Disease; NORM = Study to Obtain Normal Values of Inflammatory Variables From Healthy Subjects; STOP = Stop Smoking Study.

Figure 3.

The influence of smoking status on the IL-33 pathway and protein levels. IL-33 activation pathway analysis from (A) the STOP study of bronchial biopsies (n = 16) during current smoking activity and 1 year after cessation, (B) the GLUCOLD (RNA-Seq) study of ex-smokers (n = 18) and current smokers (n = 38) with chronic obstructive pulmonary disease (COPD), (C) the COPD microarray study of bronchial brushing samples from ex-smokers (n = 57) and current smokers (n = 30), (D) the NORM study of bronchial biopsies from healthy never-smokers (n = 40) and current smokers (n = 37), and (E) the CRUKPAP study of bronchial biopsies from never-smokers (n = 8), ex-smokers (n = 151), and current smokers (n = 77). (F) Western blot of whole tissue lysates from current (n = 10) and former (n = 9) smokers with COPD for IL-33 and β-actin. Quantification of the (G) 31-kD (full-length protein), (H) 20-kD (processed active form), and (I) 16-kD (small IL-33 isoform) bands corrected by β-actin. P values were acquired from Wilcoxon signed rank tests for paired analyses and Mann-Whitney tests for unpaired analyses. Error bars represent the SD. CRUKPAP = Cancer Research UK Papworth Hospital; GLUCOLD = Groningen and Leiden Universities Study of Corticosteroids in Obstructive Lung Disease, NORM = Study to Obtain Normal Values of Inflammatory Variables From Healthy Subjects; STOP = Stop Smoking Study.

Figure 4.

High expression of IL-33 in basal cells. (A and B) Uniform Manifold Approximation and Projection (UMAP) of IL-33 and IL1RL1 expression of samples taken from the nasal, airways and lungs from the Human Lung Cell Atlas V1.0 Predicted basal cell percentages based on cellular deconvolution from (C) the STOP study of bronchial biopsy RNA sequencing data before and after 1 year of smoking cessation (n = 16), (D) GLUCOLD (RNA-Seq) study of bronchial biopsies of ex-smokers (n = 18) and current smokers (n = 38) with chronic obstructive pulmonary disease, (E) NORM study of never-smokers (n = 40) and respiratorily healthy current smokers (n = 37), and (F) CRUKPAP study of current smokers (n = 77), former smokers (n = 151), and never-smokers (n = 8). P values were obtained by Mann-Whitney nonparametric unpaired t test. (G) Correlation between change in basal cell percentage and change in IL-33 expression from the STOP study comparing before versus after smoking cessation. Correlation between basal cell percentage and IL-33 expression levels in bronchial biopsies from (H) the GLUCOLD (RNA-Seq) study of current smokers (n = 46) and former smokers (n = 33), (I) the NORM study of never-smokers (n = 40) and respiratorily healthy current smokers (n = 37), and (J) the CRUKPAP study of never-smokers (n = 8), ex-smokers (n = 151), and current smokers (n = 77). P values were obtained by nonparametric Spearman correlation analysis. A significant difference was noted at P < 0.05. Error bars represent the SD. CRUKPAP = Cancer Research UK Papworth Hospital; EC = endothelial cell; GLUCOLD = Groningen and Leiden Universities Study of Corticosteroids in Obstructive Lung Disease; NORM = Study to Obtain Normal Values of Inflammatory Variables From Healthy Subjects; STOP = Stop Smoking Study.

Figure 5.

Expression of IL-33 in current smokers compared with never-smokers in single-cell RNA sequencing and at the protein level. (A) Uniform Manifold Approximation and Projection (UMAP) of cell type clusters of bronchial brushing samples from never-smokers (n = 6) and current smokers (n = 6) in the single-cell RNA sequencing dataset (GSE131391). UMAP of IL-33 expression in different cell clusters of (B) never-smokers and (C) current smokers. (D) Violin plots of IL-33 expression across cell types separated based on smoking status. (E) Proportion of resting basal cells (%) in never-smokers and current smokers. (F) Expression of IL-33 in the resting basal cell population. Mann-Whitney nonparametric unpaired t test was performed to assess significance. Error bars represent the SD. Immunofluorescent staining for IL-33 and p40 were performed in lung tissue sections. Representative images of (G) DAPI, (H) p40 (polyclonal rabbit, anti-human, ab 167612, 1:100), (I) IL-33 antibody (monoclonal mouse, anti-human, ALX-804–840-C100, 1:800), and (J) merged lung tissue section. (K) Staining quantification of the percentage of IL-33–positive p40⁺ cells in current smokers (n = 9), ex-/never-smokers (n = 26), and ex-smokers with chronic obstructive pulmonary disease (COPD) (n = 25). (L) Secondary cohort staining quantification of the percentage of p40⁺ cells positive for IL-33 in current smokers with COPD (n = 9), ex-smokers with COPD (n = 7), and ex-smokers without COPD (n = 7). One-way ANOVA was conducted with a Dunnett P value correction for multiple testing. Error bars represent the SD.

Figure 6.

Expression of IL-33 during basal cell differentiation in single-cell RNA sequencing in vitro studies. Two in vitro single-cell RNA sequencing studies of airway epithelial cells differentiated at an air–liquid interface were analyzed. The first was performed on the bronchial epithelial cells collected from healthy never-smokers (n = 3) and former and current smokers with chronic obstructive pulmonary disease (COPD) (n = 4), and the second was performed on small airway epithelial cells collected from healthy never-smokers (n = 3) and current smokers with COPD (n = 3). Uniform Manifold Approximation and Projection (UMAP) and IL-33 expression of the first (A and B) and the second (C and D) study. Violin plot of the expression of IL-33 is shown for the first (E) and the second (F) single-cell study. The cell trajectories of different cellular subpopulations along with IL-33 expression for the first (G and H) and the second (I and J) in vitro study. The arrow represents the direction of the cell trajectory. Percentages of (K) resting basal cells and (L) suprabasal cells from the second single-cell study whereby the airway epithelial cells were exposed to acute (4 d) and chronic (28 d) cigarette smoke. (M) Delta heat map of the IL-33 expression in smoke exposure compared with air shown for different cell types. The color scale indicates the change in expression of IL-33 in smoke exposure compared with air. There are gray blocks in the heat map that are missing values, as there was a missing value for smoke or air for the respective sample. (N) A dot plot representing the IL-33 expression in resting basal cells for acute (4 d) and chronic (28 d) exposure in the second single-cell study.