Modulation of Nuclear Receptor Family 4A (NR4A) Expression by CO2
Authors: Phelan David P. , Crean Daniel , Cummins Eoin P. 
Published: June 2021

Respiratory disorders such as Chronic Obstructive Pulmonary Disease and Obesity Hypoventilation Syndrome can lead to hypercapnia due to insufficient expiration of CO2. Hypercapnia has been linked to worse outcomes in infection due to immunosuppression. Previous work has shown that hypercapnia suppresses genes associated with immune signalling in mice, Drosophila and nematodes. In mice, increased CO2 leads to a decrease in LPS stimulated cytokine release from macrophages. A further immunosuppressive effect of hypercapnia in these cells is the reduction in NF‐κB signalling and phagocytic activity.

The NR4As (1–3) are a family of immediate early response genes in inflammation which are stimulated by many proinflammatory factors. These genes encode transcription factors that primarily act to suppress the inflammatory response, reducing the expression of proinflammatory cytokines and chemokines. Furthermore, NR4A1 has been shown to reduce the inflammatory response by preventing the translocation of NF‐κB to the nucleus. Evolutionary conservation of the NR4As indicates that they play an important role in the regulation of gene expression. The expression of nuclear receptors has been shown to be altered in hypercapnia in C. elegans.

Due to the effects of increased CO2 on nuclear receptor expression, and the role of the NR4As in NF‐κB signalling, we hypothesise that the immunosuppressive effects of hypercapnia are due, in part, to altered expression of the NR4As. The aim of this study was to investigate whether changes in CO2 concentration affects the expression of the NR4As.

Human undifferentiated monocytic THP‐1 cells were incubated for 2 hours in pH buffered media at 0.04/5/10% CO2 for 2 hours and subsequently stimulated with 2.5μg/ml lipopolysaccharide (LPS) for 0.5/1/2/4/6 hours. Untreated controls at 0 and 6 hours were used to account for any LPS independent changes in NR4A expression. mRNA and protein were extracted from all samples. RNA expression was analysed by qPCR and protein expression was analysed by Western Blot.

In normocapnia, stimulation with LPS led to a transient induction of NR4A1/2/3 mRNA expression peaking between 2 and 4 hours and returning to approximate control levels by 6 hours. For NR4A1 and NR4A2, the maximum expression was seen at 2 hours, while for NR4A3 the maximum expression was seen at 4 hours in normocapnia. 10% CO2 significantly increased the expression of NR4A1 at the peak expression time of 2 hours. 10% CO2 also significantly increased NR4A3 expression, shifting the peak expression time from 4 to 2 hours. Conversely, 10% CO2 reduced the expression of NR4A2 both at 2 and 4 hours. This reduction at 4 hours was also seen at the protein level. Taken together, these results indicate that the kinetics of LPSinduced NR4A expression is markedly affected by CO2 levels.

LPS stimulated NR4A expression is affected by changes in CO2 concentration. The expression of NR4A3 mRNA is induced earlier in hypercapnia. NR4A3 is known to suppress proinflammatory cytokines in monocytes and this novel transcriptional consequence of elevated CO2 may give mechanistic insight into CO2 – dependant immune regulation.

Support or Funding Information

This research is supported by Science Foundation Ireland (Grant 15/CDA/3490)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.