Epigenetic reprogramming of airway macrophages promotes polarization and inflammation in muco-obstructive lung disease

Abstract

Lung diseases, such as cystic fibrosis and COPD, are characterized by mucus obstruction and chronic airway inflammation, but their mechanistic link remains poorly understood. Here, we focus on the function of the mucostatic airway microenvironment on epigenetic reprogramming of airway macrophages (AM) and resulting transcriptomic and phenotypical changes. Using a mouse model of muco-obstructive lung disease (Scnn1b-transgenic), we identify epigenetically controlled, differentially regulated pathways and transcription factors involved in inflammatory responses and macrophage polarization. Functionally, AMs from Scnn1b-transgenic mice have reduced efferocytosis and phagocytosis, and excessive inflammatory responses upon lipopolysaccharide challenge, mediated through enhanced Irf1 function and expression. Ex vivo stimulation of wild-type AMs with native mucus impairs efferocytosis and phagocytosis capacities. In addition, mucus induces gene expression changes, comparable with those observed in AMs from Scnn1b-transgenic mice. Our data show that mucostasis induces epigenetic reprogramming of AMs, leading to changes favoring tissue damage and disease progression. Targeting these altered AMs may support therapeutic approaches in patients with muco-obstructive lung diseases.

Fig. 1. AMs from mice with muco-obstructive lung disease are epigenetically distinct from WT AMs.

a Graphical representation of the experimental workflow, created with www.biorender.com. b Distribution of Scnn1b-transgenic (Tg) vs. wild-type (WT) airway macrophage (AM) DNA methylation differences of differentially methylated regions (DMR), defined by DSS. DMRs are characterized by at least three CpGs with an adjusted (adj.) P value < 0.05, a width of >50 bp, and an average change of methylation >0.1. Hierarchical clustering of c DMRs methylation levels and f differentially accessible regions (DAR) accessibility. e Volcano plot of the chromatin accessibility analysis, performed with DiffBind. DARs: adj. P value < 0.05, absolute log2 fold change >1. Red dots: increased accessibility in Scnn1b-Tg AMs; blue dots reduced accessibility in Scnn1b-Tg AMs. Annotation and enrichment of d DMRs and g DARs to gene regulatory regions, as determined by the LOLA method. h Integrated analysis of overlapping DARs and DMRs. The gray diagonal represents the linear regression. Shaded areas are confidence intervals of the correlation coefficient at 95%. Correlation coefficients and P values were calculated by the Pearson correlation method. i Locus plot of selected DMRs and DARs showing average methylation and chromatin accessibility of Scnn1b-Tg AMs and WT AMs. Shaded areas indicate 95% confidence intervals. Motif enrichment of j DMRs and k DARs (adj. P value < 0.05) stratified in hypo- and hypermethylated DMRs and open and closed DARs, respectively. l Predicted upstream regulator analysis of chromatin accessibility changes. Tagmentation-based whole-genome bisulfite sequencing (tWGBS), Scnn1b-Tg (n = 3) vs. WT (n = 4); Assay for transposase-accessible chromatin sequencing (ATACseq), Scnn1b-Tg (n = 4) vs. WT (n = 3). Source data are provided as a Source Data file.

Fig. 2. Transcriptional activation of Scnn1b-Tg AMs coincides with epigenetic patterns of reduced methylation and increased chromatin accessibility.

a Unsupervised hierarchical clustering of scaled gene expression in Scnn1b-transgenic (Tg) and wild-type (WT) airway macrophages (AM). b Volcano plot of differential gene expression analysis. Differentially expressed genes (DEG), are defined by DESeq2: adjusted (adj.) P value < 0.1, absolute log2 fold change >0.5. Red dots: increased expression in Scnn1b-Tg AMs; blue dots reduced expression in Scnn1b-Tg AMs. c Integrated analysis of gene expression and promoter DNA methylation changes. Methylation differences of promoter differentially methylated regions (DMR) (<5 kbs from transcriptional start sites) vs. expression changes of the corresponding DEGs (adj. P value < 0.1). The gray diagonal represents the linear regression. Shaded areas are confidence intervals of the correlation coefficient at 95%. Correlation coefficients and P values were calculated by the Pearson correlation method. d Integrated analysis of gene expression and chromatin accessibility changes. Log2 fold change of differentially accessible regions (DARs) (adj. P value < 0.05) and DEGs (adj. P value < 0.1). The gray diagonal represents the linear regression. Shaded areas are the confidence intervals of the correlation coefficient at 95%. Correlation coefficients and P values were calculated by the Pearson correlation method. e Gene set enrichment analysis using custom gene sets relevant to lung diseases and cellular physiology. f Predicted upstream regulator analysis of gene expression changes. g Gene expression for Ccl17, Ccl22, Cxcr1, Trem2, Ptgir, Mmp12, Arg1, Ptgs1, Anpep, Cd86, Igf1, and Igf2bp3 was measured by qPCR. Box plots indicate the largest value within the 1.5 times interquartile range above 75th percentile, 75th percentile, median, 25th percentile, and smallest value within the 1.5 times interquartile range below 25th percentile of g n = 20 per group (Ccl17, Ccl22, Trem2), n = 19 per group (Ptgir, Mmp12, Arg1, Ptgs1, Anpep, Igf1, Igf2bp3), n = 18 WT, n = 17 Scnn1b-Tg (Cxcr1), n = 19 WT, n = 20 Scnn1b-Tg (Cd86). *P value < 0.05; **P value < 0.01; ***P value < 0.001 by Mann–Whitney U-test. RNA sequencing (RNAseq), n = 6 per group. Source data are provided as a Source Data file.

Fig. 3. Single-cell analysis of macrophage surface marker expression validates enhanced activation of Scnn1b-Tg AMs.

a Frequency of indicated surface markers expressed by airway macrophages (AM). b Differential surface marker expression of Scnn1b-transgenic (Tg) vs. wild-type (WT) AMs, defined by cluster analysis. Red dots: surface markers for classical macrophage polarization (M1); blue dots: surface markers for alternative macrophage polarization (M2). c Uniform Manifold Approximation and Projection (UMAP) of 50,000 randomly sampled Scnn1b-Tg and WT AMs. d Scaled M1 and M2 surface marker expression of AM clusters. Box plots indicate the largest value within the 1.5× interquartile range above 75th percentile, 75th percentile, median, 25th percentile, and smallest value within the 1.5× interquartile range below 25th percentile of a n = 10 per group. *P value < 0.05; **P value < 0.01; ***P value < 0.001 by Mann–Whitney U-test. CD11b, P < 0.001; MHCII, P = 0.0185. Source data are provided as a Source Data file.

Fig. 4. AM-specific functions are impaired in Scnn1b-Tg mice.

Representative flow cytometry plots and percentage of pHRodo⁺ airway macrophages (AM), depicting a efferocytosis and b phagocytosis capacities. c Gene expression of Nos2, Il6, Il1b, Il12b, and Mmp12, assessed by qPCR and d protein expression of IL-1α, IL-6, IL-23p40/p19, CCL2, and CXCL1, assessed by cytokine bead array, at indicated time points post lipopolysaccharide (LPS) treatment. PCA of e RNA sequencing (RNAseq) and f Assay for transposase-accessible chromatin sequencing (ATACseq) data from Scnn1b-transgenic (Tg) vs. wild-type (WT) AMs treated with lipopolysaccharide (LPS) for 12 h (h). g Functional enrichment analysis of the top 100 genes explaining PC1 (left panel) and PC2 (right panel). h Volcano plot visualizing the treatment response (LPS vs. medium) on chromatin accessibility level. differentially accessible regions (DARs): adjusted (adj.) P value < 0.05; absolute log2 fold change >2. Red dots: increased accessibility in LPS treated AMs; blue dots reduced accessibility in LPS treated AMs. i Profile plot of all identified LPS responsive DARs, visualized for LPS treated Scnn1-Tg and WT AMs (top panel) and primary uncultured Scnn1-Tg and WT AMs at baseline (lower panel). Box plots indicate the largest value within the 1.5× interquartile range above 75th percentile, 75th percentile, median, 25th percentile, and smallest value within the 1.5× interquartile range below 25th percentile of a n = 17 WT, n = 19 Scnn1b-Tg, and b n = 10 per group. *P value < 0.05; **P value < 0.01; ***P value < 0.001 per group by Mann–Whitney U-test. a P < 0.001, b P = 0.0011. Bar plots show mean ± SEM of c n = 10 per group (6, 12 h) and n = 9 per group (24 h). *P value < 0.05; **P value < 0.01; ***P value < 0.001 per group by Mann–Whitney U-test. Nos2: 6 h P = 0.0021, 12 h P < 0.001, 24 h P = 0.178; Il6: 12 h P = 0.0052, 24 h P = 0.0078; Il1b: 12 h P = 0.0048; Il12b: 12 h P = 0.0089, 24 h P = 0.0314; Mmp12: 6 h P = 0.0021, 12 h P = 0.0887, 24 h P < 0.001. d Lipopolysaccharide (LPS) treatment: IL-1α, IL-23p40/p19, IL-6, CXCL1: n = 10 per group (6, 12 h), n = 9 per group (24 h); CCL2: n = 10 wt (6, 12 h), n = 9 wt (24 h), n = 9 Scnn1b-Tg (6, 12, 24 h). Medium control: IL-1α, IL-23p40/19, CCL2, CXCL1: n = 8 WT (6 h), n = 10 WT (12 h), n = 9 WT (24 h), n = 9 Scnn1b-Tg (6, 12, 24 h), IL-6: n = 9 WT (6 h), n = 10 WT (12 h), n = 9 WT (24 h), n = 9 Scnn1b-Tg (6, 12, 24 h). *P value < 0.05; **P value < 0.01; ***P value < 0.001 by One-Way ANOVA followed by Tukey´s post hoc test. e–i n = 3 per group. ND not detectable. Source data are provided as a Source Data file.

Fig. 5. Differential transcription factor activity orchestrates LPS responses in Scnn1b-Tg AMs.

a Differential transcription factor (TF) activity analysis of lipopolysaccharide (LPS) treated Scnn1b-transgenic (Tg) vs. wild-type (WT) airway macrophages (AM). A positive weighted mean difference indicates increased TF activity in Scnn1b-Tg AMs. Size indicates the number of TF binding sites (TFBS). Green labeled TFs are predicted activators, red labeled TFs are predicted repressors, and black labeled TFs have no direction assigned. b Pearson correlation of TF activity of significantly enriched TFs (adjusted (adj.) P value < 0.001) in Scnn1b-Tg vs. WT AM treated with LPS on the y-axis, with mean target gene expression change on the x-axis. The gray diagonal represents the linear regression. c Clustering of differentially activated TFs (adj. P value <10e−6) based on the similarity of their position weight matrixes. Coloring of tree leaves based on TF activity. Coloring of labels defined by TF class. d Normalized Irf1 gene counts obtained by RNA sequencing (RNAseq) (Scnn1b-Tg LPS vs. WT LPS) in treated and untreated Scnn1b-Tg and WT AMs. Adj. P values were determined by DESeq2. The data show mean ± SEM of n = 3. Source data are provided as a Source Data file.

Fig. 6. Mucus triggers AM immune responses and functions.

a Gene expression levels of Ccl22, Cccl17, Arg1, Mmp12, Il1a, and Il1b, and b protein expression levels of CCL2, MMP12, and IL-1α in the supernatant of wild-type (WT) airway macrophages (AM) treated with increasing mucus concentrations or medium (med) for 6, 12, and 24 h (hrs). Representative flow cytometry plots and percentage of pHRodo⁺ AMs, depicting c phagocytosis and d efferocytosis capacities of AMs treated with 2% mucus or medium. Bar plots show mean ± SEM of a 6, 24 h: n = 10 per group. 12 h: n = 9 (med), n = 7 (0.1, 1%), n = 10 Ccl22, Ccl17, Mmp12, Arg1, Il1b (2, 5, 10%), n = 9 Il1a (2, 5, 10%), and b 6 h: n = 8 (CCL22, MMP12, med), n = 7 (IL-1α, med), n = 9 (0.1, 1%), n = 9 (CCL22, IL-1α, 2%), n = 8 (MMP12, 2%), n = 9 (CCL22, IL-1α, 5%), n = 6 (MMP12, 5%), n = 9 (CCL22, IL-1α, 10%), n = 7 (MMP12, 10%). 12 h: n = 9 (med), n = 7 (CCL22, IL-1α, 0.1%), n = 6 (MMP12, 0.1%), n = 7 (1%), n = 10 (2, 5, 10%). 24 h: n = 9 (CCL22, IL-1α, med), n = 7 (MMP12, med), n = 9 (CCL22, IL-1α, 0.1%), n = 7 (MMP12, 0.1%), n = 9 (1%), n = 8 (CCL2, MMP12, 2%), n = 9 (IL-1α, 2%), n = 8 (CCL2, IL-1α, 5%), n = 6 (MMP12, 5%), n = 9 (CCL22, IL-1α, 10%), n = 6 (MMP12, 10%). *P value < 0.05; **P value < 0.01; ***P value < 0.001 by one-way ANOVA following Bonferroni´s post hoc test. Box plots indicate the largest value within the 1.5× interquartile range above 75th percentile, 75th percentile, median, 25th percentile, and smallest value within the 1.5× interquartile range below 25th percentile of c, d n = 9 per group. *P value < 0.05; **P value < 0.01; ***P value < 0.001 by Mann–Whitney U-test. Source data are provided as a Source Data file.