Hypercapnia alters mitochondrial gene expression and acylcarnitine production in monocytes

Abstract

CO2 is produced during aerobic respiration. Normally, levels of CO2 in the blood are tightly regulated but pCO2 can rise (hypercapnia, pCO2 > 45 mmHg) in patients with lung diseases, for example, chronic obstructive pulmonary disease (COPD). Hypercapnia is a risk factor in COPD but may be of benefit in the context of destructive inflammation. The effects of CO2 per se, on transcription, independent of pH change are poorly understood and warrant further investigation. Here we elucidate the influence of hypercapnia on monocytes and macrophages through integration of state‐of‐the‐art RNA‐sequencing, metabolic and metabolomic approaches. THP‐1 monocytes and interleukin 4–polarized primary murine macrophages were exposed to 5% CO2 versus 10% CO2 for up to 24 h in pH‐buffered conditions. In hypercapnia, we identified around 370 differentially expressed genes (DEGs) under basal and about 1889 DEGs under lipopolysaccharide‐stimulated conditions in monocytes. Transcripts relating to both mitochondrial and nuclear‐encoded gene expression were enhanced in hypercapnia in basal and lipopolysaccharide‐stimulated cells. Mitochondrial DNA content was not enhanced, but acylcarnitine species and genes associated with fatty acid metabolism were increased in hypercapnia. Primary macrophages exposed to hypercapnia also increased activation of genes associated with fatty acid metabolism and reduced activation of genes associated with glycolysis. Thus, hypercapnia elicits metabolic shifts in lipid metabolism in monocytes and macrophages under pH‐buffered conditions. These data indicate that CO2 is an important modulator of monocyte transcription that can influence immunometabolic signaling in immune cells in hypercapnia. These immunometabolic insights may be of benefit in the treatment of patients experiencing hypercapnia.

Orphan Nuclear Receptor Family 4A (NR4A) Members NR4A2 and NR4A3 Selectively Modulate Elements of the Monocyte Response to Buffered Hypercapnia

“…Buffered hypercapnia (10% CO 2 ) for 4 h was a modest stimulus in these cells, with 52 transcripts differentially expressed in the basal state ( Figure 2 A, Supplementary Table S1 ) and 646 transcripts in the LPS-stimulated cells ( Figure 2 B, Supplementary Table S2 ). Interestingly, there were markedly fewer transcripts in shNR4A2 cells compared to non-target short hairpin RNA (sh)-controls ((380 genes (basal) and 1889 genes (LPS-stimulated)) [ 26 ], suggesting that the depletion of NR4A2 attenuated the cellular response to hypercapnia. Of the 52 genes affected by hypercapnia in the basal state, 42 (or 80%) were also affected by hypercapnia in sh-control cells ( Figure 2 C).…”

“…We have previously published a strong signature for increased mitochondrial transcription in response to acute hypercapnia in monocytes [ 26 ]. When comparing the CO 2 sensitivity of shNR4A2 cells to shNT cells, this mitochondrial signature was preserved, although there were much fewer DEGs identified.…”

“…Hypercapnia exposures were performed as described previously [ 26 ]. Briefly, hypercapnic exposures were performed in humidified environmental chambers (Coy Laboratories, Grass Lake, MI, USA) at 37 °C, at 5% or 10% CO 2 in pH-buffered, pre-equilibrated Dulbecco’s Modified Eagle Medium (DMEM) media [ 22 ].…”

“…For most THP-1 experiments, cells were used at a density of 7.5 × 10 5 cells/mL. The maintenance of intracellular and extracellular pH under these conditions has recently been described [ 26 ].…”

“…The RNA-seq protocol was performed as described previously [ 15 , 26 ]. Briefly, total RNA was extracted from cultured cells using the E.Z.N.A.…”