Redistribution of SOD3 expression due to R213G polymorphism affects pulmonary interstitial macrophage reprogramming in response to hypoxia

The extracellular isoform of superoxide dismutase (SOD3) is decreased in patients and animals with pulmonary hypertension (PH). The human R213G single nucleotide polymorphism (SNP) in SOD3 causes its release from tissue extracellular matrix (ECM) into extracellular fluids, without modulating enzyme activity, and increases cardiovascular disease risk in humans and chronic hypoxic PH in mice. Given the importance of interstitial macrophages (IM) to PH pathogenesis, this study aimed to determine whether R213G SOD3 worsens PH via increased IM accumulation and altered reprogramming. R213G mice and wild-type (WT) controls were exposed to hypobaric hypoxia for 4 or 14 days compared to normoxia. Flow cytometry demonstrated a transient increase in IMs at day 4 in both strains. Contrary to our hypothesis, the R213G SNP did not augment IM accumulation. To determine strain differences in the IM reprogramming response to hypoxia, we performed RNAsequencing on IMs isolated at each time point. We found that IMs from R213G mice exposed to hypoxia activated ECM-related pathways and a combination of alternative macrophage and proinflammatory signaling. Furthermore, when compared to WT responses, IMs from R213G mice lacked metabolic remodeling and demonstrated a blunted anti-inflammatory response between the early (day 4) and later (day 14) time points. We confirmed metabolic responses using Agilent Seahorse assays whereby WT, but not R213G, IMs upregulated glycolysis at day 4 that returned to baseline at day 14. Finally, we identify differential regulation of several redox-sensitive upstream regulators that could be investigated in future studies. Overall design: To investigate the effect of the R213G SOD3 polymorphism on pulmonary interstitial macrophage (IM) transcriptional responses to hypoxia, we exposed R213G and C57BL/6J (WT) mice to 4 days (peak inflammation) or 14 (resolution) days of hypobaric hypoxia. IMs were isolated from mouse lungs by FACS and RNAseq performed on each population. 4 mice (2 male and 2 female) were pooled for each sample and n=3-4 samples analyzed per group.