Nitroalkene regulation of macrophage activation in lung injury and fibrosis
Creators: name: Wilkinson, Melissa Leigh
Publisher: name: No Publisher Supplied
Year: 2022

description: Fatty acid nitroalkenes, such as nitro-oleic acid (OA-NO2), are reversibly-reactive electrophiles, endogenously detectable at nM concentrations. They have demonstrated anti-inflammatory properties but have not been studied extensively in lung injury and diseases. We hypothesize that OA-NO2 regulates macrophage activation and consequent fibrosis in acute lung injury (ALI) and interstitial lung disease (ILD). This research explores the mechanisms whereby OA-NO2 alters macrophage activation with a view to its potential as a therapeutic for ALI and ILD and a potential mechanism by which these changes occur. To test this hypothesis, OA-NO2 was administered as a treatment in a bleomycin-induced model of ALI and ILD. Additionally, signaling mechanisms were assessed in an acutely activated macrophage cell line. To assess effects on ALI, C57BL6- J mice were administered bleomycin (3 U/kg) intratracheally (ITB) to induce pulmonary inflammation and acute injury, or saline and were treated with 50 μL OA-NO2 (50 μg) or vehicle in the same instillation and 72 h post-exposure to assess anti-inflammatory properties. OA-NO2 significantly improved weight loss, cellular infiltration, proteinaceous debris deposition, and airway epithelial injury compared to ITB alone. Additionally, OA-NO2 administration preserved the resident alveolar macrophage population and prevented pro-inflammatory, pro-fibrotic phenotypes in interstitial macrophages. OA-NO2 also reduced the fibrogenic capacity of mesenchymal stem cells as measured by reductions in CD44 expression. To develop a bleomycin-induced model of ILD, C57BL6-J mice were injected IP with 0.8U bleomycin or PBS control every 3d up to 15d and were sacrificed at 21 or 40d. This model was utilized to assess the therapeutic potential of OA-NO2. 50μg of OA-NO2 was administered intratracheally four times over a 15d period, and the responses of control and treated was measured at 40d. OA-NO2 allowed higher tolerance for bleomycin dosing without experiencing disqualifying side-effects. Additionally, OA-NO2 improved lung histopathology when compared to IPB alone. Despite this, OA-NO2 had little effect on alveolar or interstitial macrophage phenotype. Based on phenotypic changes seen in macrophages during ALI, mechanisms were assessed in LPS-activated RAW 264.7 cells. OA-NO2 inhibited TLR-4 mediated pro-inflammatory signaling and inhibited NOS2 expression and iNOS translation and activity. Further, OA-NO2 reduced NF-kB activity through direct adduction to the p50 and p65 subunits. The therapeutic potential of fatty acid nitroalkenes in ALI treatment is further supported by oxygen consumption and metabolomic analyses showing that OA-NO2 shifted macrophages to a quiescent state following LPS administration. This work has demonstrated the therapeutic potential of OA-NO2 in ALI inhibition of pro-inflammatory macrophage phenotypes. In vitro analysis of activated macrophages confirmed changes in signal transduction through OA-NO2 inhibition of TLR-4 signaling, NF-kB and iNOS activity, and reduced metabolic demand. Additionally, this work demonstrates the critical role of macrophage phenotypic switching in ALI and better defines a potential therapeutic agent. In ILD, OA-NO2 changes the response to bleomycin, but this change does not correlate with macrophage activation. Overall, OA-NO2 is a pleiotropic signaling molecule that modifies macrophage phenotype and inflammatory signaling acutely, while improving lung histology chronically. Therefore, OA-NO2 shows potential as an anti-inflammatory, anti-fibrotic agent acutely, and more research is needed to assess its effects on chronic pulmonary diseases.