Hypercapnia selectively modulates LPS-induced changes in innate immune and DNA replication-related gene transcription in the macrophage

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 death. Recent studies have shown that hypercapnia inhibits expression of select innate immune genes and suppresses host defence against bacterial and viral pneumonia in mice. In the current study, we evaluated the effect of culture under conditions of hypercapnia (20% CO₂) versus normocapnia (5% CO₂), both with normoxia, on global gene transcription in human THP-1 and mouse RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We found that hypercapnia selectively downregulated transcription of LPS-induced genes associated with innate immunity, antiviral response, type I interferon signalling, cytokine signalling and other inflammatory pathways in both human and mouse macrophages. Simultaneously, hypercapnia increased expression of LPS-downregulated genes associated with mitosis, DNA replication and DNA repair. These CO₂-induced changes in macrophage gene expression help explain hypercapnic suppression of antibacterial and antiviral host defence in mice and reveal a mechanism that may underlie, at least in part, the high mortality of patients with severe lung disease and hypercapnia.

Keywords: CO2; gene expression; hypercapnia; innate immunity; lipopolysaccharide; macrophage.

Figure 1.

LPS induces transcriptional changes in human and mouse macrophages. Global gene expression was assessed on Illumina microarrays in human THP-1 and mouse RAW 264.7 cells stimulated with LPS (1 ng ml⁻¹) in normocapnia (5% CO₂, PCO₂ 44 mmHg) for 0.5, 1.5 or 3 h. Transcripts downregulated and upregulated by greater than or equal to 1.35-fold at 0.5, 1.5 or 3 h after LPS treatment (a). Bars represent the top GO biological processes of downregulated (blue) and upregulated (red) genes induced by LPS after 3 h in THP-1 cells (b). Venn diagrams of human and mouse common genes changed by LPS at 0.5, 1.5 or 3 h (c). Heat map of common genes changed by LPS over time in human macrophages (d). Networks of GO biological processes upregulated (e) and downregulated (f) by LPS after 3 h of treatment.

Figure 2.

Hypercapnia alters expression of LPS-upregulated and -downregulated genes in human and mouse macrophages. Global gene expression was assessed on Illumina microarrays in human THP-1 and mouse RAW 264.7 cells stimulated with LPS (1 ng ml⁻¹) in normocapnia (5% CO₂, PCO₂ 44 mmHg) or hypercapnia (20% CO₂, PCO₂ 112 mmHg) for 0.5, 1.5 or 3 h. K-means clustering of common differentially expressed genes changed by LPS and then by hypercapnia in human THP-1 (i) and mouse RAW 264.7 (ii) macrophages are presented as a heatmap (a). K-means generate three clusters C1, C2 and C3. Bars show the number of transcripts upregulated by LPS in normocapnia that are uniquely and commonly downregulated (C1) or further upregulated (C2) by hypercapnia (i), and transcripts downregulated by LPS in normocapnia that are uniquely and commonly upregulated (C3) by hypercapnia (ii) in human and mouse (b). GO biological processes of C1 and C3 (c). Global network analysis of C1 (d) and C3 (e).

Figure 3.

qPCR confirmation of selected immunoregulatory genes. THP-1 cells stimulated with or without LPS (1 ng ml⁻¹) in normocapnia (5% CO₂; NC) or hypercapnia (15% CO₂; HC) for 3 h. CCL2, IL-6, ICAM1, NFKB1, EBI1 and EGR1 mRNA expression assessed by qPCR. Means ± s.e.m., n = 4–6 from at least four independent experiments; *p < 0.01 versus control in NC, **p < 0.05 versus LPS in NC.