Nitro‐Oleic Fatty Acids Reduce Cellular Metabolism in Activated Macrophages Leading to Reduced Inflammatory Potential

Abstract

Nitro‐oleic fatty acid (OA‐NO2), a potent electrophile, is endogenously found in humans and modifies cysteine residues via Michael addition to alter protein function. It reduces inflammatory activation in the cardiovascular system, and potentially the lung. Intratracheal bleomycin (ITB) is a model of pulmonary inflammation, resolution, and fibrosis. Earlier work showed a loss of resident alveolar macrophages (AMs) (CD45+, SiglecF+, F4/80+, CD11c+) with ITB administration (95 ± 3.9% vs 49 ± 3.9%*) which was abrogated by OA‐NO2 at 7 (69 ± 3.9%#) and 17 days ((73 ±3.8%*). Activation of mature interstitial macrophages (IMs) (CD45+, SiglecF‐, F4/80+, CD11c+) was observed in the early phase of the response to ITB (7 days) that was reduced by OANO2, as measured by expression of Ly6C and mannose receptor (MR). At 7 days, the proportion of mature IMs expressing both Ly6C (43 ± 3.7% vs 13 ± 3.4%*) and MR (28 ± 3.6% vs 5 ± 3.4%*) were increased in ITB mice with levels returning closer to control at 17 days (Ly6C+ 16 ±2.4%; and MR+ 9 ± 1.2%). This activation was ameliorated with OA‐NO2 administration at 7 days (Ly6C+ 27 ± 2.4%*; and MR+ 13 ± 3.9%*), but perpetuates activation at 17 days (Ly6C+ 28 ± 5.1%*; and MR+ 10 ± 2.3%). We proposed that OA‐NO2 was altering macrophage activation via inducing a metabolic shift. Therefore, utilizing RAW 264.7 murine macrophage cells we assessed the metabolic effects of LPS activation (1000 ng/mL), with and without OA‐NO2 (10mM) administration, using a Seahorse Metabolic Analyzer (XFe96). Through analysis of mitochondrial respiration, cells exposed to LPS had decreased basal mitochondrial oxygen consumption compared to controls (224±9.2 vs 117‐±9.2pmol/min*). This was further decreased with OA‐NO2 (17pmol/min*). Baseline glycolysis rates were not altered by LPS exposure (64±6.8 vs 73±5.9pmol/min) but were greatly reduced by OA‐NO2 (17±5.9pmol/min*). Based on these metabolic changes, the glycolytic capacity and spare respiratory capacity were assessed. With LPS administration, the glycolytic capacity is increased compared to controls (98‐±9.2 vs 100±8.0pmol/min*). The glycolytic capacity was significantly decreased with OA‐NO2 administration (24‐±8.0*). The spare respiratory capacity was not significantly altered with LPS compared to control (218±9.5 vs 172±10.0%pmol/min). This was significantly increased with OA‐NO2administration (335±10.0%pmol/min*). Based on this analysis, it can be determined that OA‐NO2 administration results in a metabolically quiescent state in macrophages, regardless of LPS stimulation. Additionally, OA‐NO2 does not change the balance of metabolic pathways, rather, it results in an overall decrease in their use. This is not true of LPS stimulated cells, where metabolism is shifted in favor of glycolysis. Overall, OA‐NO2 administration reduces macrophage activation, potentially reducing their inflammatory and fibrotic potential. This change may be attributed to a reduction in overall metabolic rate by OA‐NO2, which may increase quiescence. (* p<0.05 vs control; p<0.5 vs ITB).Support or Funding InformationSupported by NIH Grants: HL087761, AR055073, ES004738, ES00502, HL086621