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, increasing cardiovascular disease risk in humans and exacerbating chronic hypoxic PH in mice. Given the importance of interstitial macrophages (IMs) to PH pathogenesis, this study aimed to determine whether R213G SOD3 increases IM accumulation and alters IM reprogramming in response to hypoxia. R213G mice and wild-type (WT) controls were exposed to hypobaric hypoxia for 4 or 14 days compared with normoxia. Flow cytometry demonstrated a transient increase in IMs at <i>day 4</i> 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 timepoint. 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 with WT responses, IMs from R213G mice lacked metabolic remodeling and demonstrated a blunted anti-inflammatory response between the early (<i>day 4</i>) and later (<i>day 14</i>) timepoints. We confirmed metabolic responses using Agilent Seahorse assays, whereby WT, but not R213G, IMs upregulated glycolysis at <i>day 4</i> that returned to baseline at <i>day 14</i>. Finally, we identify differential regulation of several redox-sensitive upstream regulators that could be investigated in future studies.<b>NEW & NOTEWORTHY</b> Redistributed expression of SOD3 out of tissue ECM due to the human R213G SNP exacerbates chronic hypoxic PH. Highlighting the importance of macrophage phenotype, our findings reveal that the R213G SNP does not exacerbate pulmonary macrophage accumulation in response to hypoxia but influences their metabolic and phenotypic reprogramming. We demonstrate a deficiency in the metabolic response to hypoxic stress in R213G macrophages, associated with weakened inflammatory resolution and activation of profibrotic pathways implicated in PH.