Hypercapnia Alters Expression of Immune Response, Nucleosome Assembly and Lipid Metabolism Genes in Differentiated Human Bronchial Epithelial Cells

Abstract

Hypercapnia, the elevation of CO₂ in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases, and is associated with increased risk of mortality. Recent studies have shown that hypercapnia adversely affects innate immunity, host defense, lung edema clearance and cell proliferation. Airway epithelial dysfunction is a feature of advanced lung disease, but the effect of hypercapnia on airway epithelium is unknown. Thus, in the current study we examined the effect of normoxic hypercapnia (20% CO₂ for 24 h) vs normocapnia (5% CO₂), on global gene expression in differentiated normal human airway epithelial cells. Gene expression was assessed on Affymetrix microarrays, and subjected to gene ontology analysis for biological process and cluster-network representation. We found that hypercapnia downregulated the expression of 183 genes and upregulated 126. Among these, major gene clusters linked to immune responses and nucleosome assembly were largely downregulated, while lipid metabolism genes were largely upregulated. The overwhelming majority of these genes were not previously known to be regulated by CO₂. These changes in gene expression indicate the potential for hypercapnia to impact bronchial epithelial cell function in ways that may contribute to poor clinical outcomes in patients with severe acute or advanced chronic lung diseases.

Figure 1

Hypercapnia induces transcriptional changes in NHBE cells. Global gene expression was assessed in ALI-differentiated NHBE cells after exposure to 5% CO₂ (normocapnia) or 20% CO₂ (hypercapnia) for 24 h. (a) Pie chart indicating proportion of genes downregulated or upregulated by hypercapnia. (b) Volcano plot showing statistical significance (−log₁₀ [P value]) plotted against log₂ fold change for hypercapnia vs normocapnia. Plot indicates significantly upregulated genes (log₂ [fold change]≥+0.5, adjusted P value ≤ 0.05) in red and downregulated genes (log₂ [fold change]≤−0.5, adjusted P value ≤ 0.05) in blue. (c) Bars represent the top 10 GO biological processes downregulated (blue) and upregulated (red) by high CO₂.

Figure 2

Networks of GO biological processes downregulated by hypercapnia. Gene clusters associated with GO biological processes containing five or more hypercapnia-downregulated genes and their intra- and inter-cluster connections, as determined by unbiased analysis using Mathematica® v11.2.

Figure 3

Networks of GO biological processes upregulated by hypercapnia. Gene clusters associated with GO biological processes containing five or more hypercapnia-upregulated genes and their intra- and inter-cluster connections, as determined by unbiased analysis using Mathematica® v11.2.

Figure 4

Hypercapnia alters expression of genes involved in innate immunity and host defense. ALI-differentiated NHBE cells were exposed to normocapnia (NC) or hypercapnia (HC) for 24 h prior to analysis. (a) Cluster A genes altered by hypercapnia and their associated GO biological processes. (b) Heatmap and hierarchical clustering of gene expression profiles in normocapnia and hypercapnia. (c) CXCL1, CXCL14, CCL28, IL6R, and TLR4 mRNA expression levels were assessed by qPCR and expression in hypercapnia was expressed as fold change relative to normocapnia. Results shown are means ± SE; n = 3. (d) Representative fluorescence micrographs of NHBE cells double-stained for TLR4 (red) and the cilia marker acetylated tubulin (green), and counter stained with Hoechst (blue). Non immune control (NIC) cells stained without primary antibodies. (e) Immunoblotting of whole cell lysates for TLR4. Histogram shows densitometry of TLR4 normalized to β-actin (loading control). Results shown are means ± SE; n = 3.

Figure 5

Hypercapnia alters expression of genes involved in nucleosome assembly. ALI-differentiated NHBE cells were exposed to normocapnia (NC) or hypercapnia (HC) for 24 h prior to analysis. (a) Cluster B genes altered by hypercapnia and their associated GO biologic processes. (b) Hierarchical clustering of the gene expression profiles in normocapnia and hypercapnia. (c) HIST1H2AC, HIST1H2BD and HIST1H2BK mRNA expression assessed by qPCR and hypercapnia was expressed as fold change relative to normocapnia. Results shown are means ± SE; n = 3.