## 3 Discussion
Taken together, these data indicate that the level of _NR4A2_ and _NR4A3_ in monocytes affects the cellular response to CO\({}_{2}\). In the case of shNR4A2 cells in the basal state, it is interesting to note that _TNF_, a known CO\({}_{2}\)-sensitive transcript in lung cells [29], is only significantly affected in shNR4A2 cells. NR4A2 is known to act as a suppressor of NF\(\kappa\)B and inflammatory signalling [25]. The change in the sensitivity of _TNF_ under these particular conditions may reflect a more pro-inflammatory milieu in shNR4A2 cells. Thus, the cellular microenvironment, which can be influenced by inflammatory signalling and metabolic changes, is likely a key determinant in shaping the cellular response to CO\({}_{2}\). This concept is further supported by our RNA-seq data indicating that (increased) _NR4A3_ is only sensitive to CO\({}_{2}\) in shNR4A2 cells in response to LPS (Figure 2D, Supplementary Figure S9C) and that (decreased) _NR4A1_ is only sensitive to CO\({}_{2}\) in shNR4A3 cells in the basal state (Figure 3C, Supplementary Figure S9A). Therefore, the summation of CO\({}_{2}\) signalling, and the cellular microenvironment of inflammation/ metabolism ultimately dictate the transcriptional response. 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. This mitochondrial signature is reflected both in the significant differential expression of mitochondrial OXPHOS genes, as well as in the GO terms associated with hypercapnia in shNR4A2 cells (Supplementary Figure S8). Interestingly, when analysing the CO\({}_{2}\) sensitivity of shNR4A3 cells, the mitochondrial signature seems to be much reduced (Supplementary Figure S8). While there were more significantly differentially expressed genes in hypercapnia in shNR4A3 cells in the basal state, none of the mitochondrial genes reached the threshold for significance (Supplementary Figure S6). Furthermore, Mitochondrial Transcription Factor 1 (TFAM) interacted with shNT and shNR4A2 cells but not shNR4A3 cells in hypercapnia (+LPS) (Supplementary Table S5), further supporting the concept that the mitochondrial response to CO\({}_{2}\) is blunted in shNR4A3 cells. The NR4 As have been shown to regulate genes associated with fuel utilisation [5; 30; 31]. In addition, _NR4A3_ knockdown in C2C12 myotubes reduces mitochondrial health and mRNA levels of key mitochondrial genes, e.g., Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (_PGC1a_) and _TFAM_, as well as the protein levels of mitochondrial complexes I-IV [32]. Thus, a specific role for NR4A3 in the regulation of mitochondrial and metabolic pathway genes may account for the lack of sensitivity of these pathways to hypercapnia in shNR4A3 monocytes. shNR4A2 cells appear to differ in their response to hypercapnia with respect to heat shock proteins (Hsps). Hsps are induced in response to cellular stress and protect the cell through various functions, including maintaining proteostasis by chaperoning misfolded proteins and assisting with antigen presentation [33; 34]. Hsp induction is highly conserved and is regulated by the transcription factor heat shock factor 1 (HSF1). Hypercapnia has previously been reported to alter the activity of HSF1 in immune cells, with increased HSF1 protein expression and nuclear localisation observed in murine alveolar macrophages after exposure to 15% CO\({}_{2}\) for 16 h [35]. Furthermore, mass spectrometry has identified Hsp70 as a major NR4A-interacting protein in human embryonic kidney cells (HEK2993) [36].