## 3 Discussion  
The primary aim of this study was to investigate the role of NR4A2 and NR4A3 in CO\({}_{2}\)-dependent signalling in monocytes. As discussed in the introduction, we hypothesised that these members of the nuclear receptor family may represent master regulators of the cellular response to CO\({}_{2}\). Our initial experiments demonstrated a temporal response of NR4A family members to LPS (Figure 1). All three NR4A family members were rapidly induced at the transcript level in response to LPS, with _NR4A3_ showing the highest level of induction relative to controls. The rapid induction of NR4A mRNA has previously been demonstrated in THP-1 monocytes, with similar observations regarding _NR4A3_[27; 28]. We noted various sensitivities of the different NR4A family members to CO\({}_{2}\) concentrations in THP-1 cells. _NR4A1_ showed the least sensitivity. _NR4A2_ expression was reduced in hypercapnia, with lower expression at both 2 h and 4 h of LPS stimulation in 10% CO\({}_{2}\) compared to 5% CO\({}_{2}\). For _NR4A3_, the maximal increase in NR4A3 mRNA expression in response to LPS shifted from 4 h at 5% CO\({}_{2}\) to 2 h at 10% CO\({}_{2}\). Taken together, these data suggest a CO\({}_{2}\)-dependent shift in the temporal response of NR4A family members _NR4A2_ and _NR4A3_ to LPS (Figure 1). This is associated with a reduction in NR4A2 protein levels in hypercapnia (Supplementary Figure S1). Interestingly, microarray data from human macrophages stimulated with LPS found _NR4A2_ to be the most highly upregulated gene following a 90-minute exposure to hypercapnia-acidosis (20% CO\({}_{2}\)) [20]. While _NR4A2_ was not the focus of the Casalino-Matsuda study, it further supports the idea that environmental CO\({}_{2}\) conditions shape the cellular response to inflammatory stimuli. Figure 5: ETS-1 nuclear expression is increased in hypercapnia. Western blot analysis of nuclear lysates from THP-1 monocytes exposed to 5% or 10% CO\({}_{2}\) for 24 h. Lysates were probed using a revert total protein stain and imaged in the 700 nm (red) channel or incubated with an (**A**) ETS-1 or (**C**) Lamin primary antibody followed by a fluorescent secondary mouse antibody and imaged in the 700 nm (red) or 800 nm (green) channel on an Li-COR imaging system. The image is representative of 3 independent experiments. Protein quantification was performed using EmpiriaStudio and plotted as fold change (Hypercapnia/normocapnia) for protein expression of (**B**) ETS-1 and (**D**) Lamin normalised to total revert stain intensity. Arrows indicate the bands on the western blot that were quantified for protein expression. Given the CO\({}_{2}\) sensitivity of _NR4A2_ and _NR4A3_ to CO\({}_{2}\) in this model, we investigated the impact of depletion of these genes in our model of buffered hypercapnia. In the basal state, depletion of _NR4A2_ decreased the number of genes significantly differentially expressed in hypercapnia compared to shNT cells (Figure 2A,C), while depletion of _NR4A3_ increased this number in the basal state (Figure 3A,C). This could suggest that in the basal state, _NR4A2_ has a role in the regulation of CO\({}_{2}\)-sensitive genes, while _NR4A3_ may act to attenuate the effect of hypercapnia on gene expression. The difference in CO\({}_{2}\) sensitivity seen in shNR4A2 and shNR4A3 cells in the absence of LPS is somewhat surprising, as the NR4 As are expressed at low levels in the basal state. In the presence of LPS, both shNR4A2 and shNR4A3 cells had fewer significant DEGs in response to hypercapnia compared to shNT cells (Figures 2B,D and 3B,D). It is possible that the different pattern of CO\({}_{2}\) sensitivity observed between the basal and LPS states may be due to the nature of the knockdown. While _NR4A2_ and _NR4A3_ mRNA expression were clearly depleted in shNR4A2 and shNR4A3 cells, respectively, they were still capable of being increased by LPS exposure (Supplementary Figure S9).