Fatty acid nitroalkenes inhibit the inflammatory response to bleomycin-mediated lung injury

Abstract

Fatty acid nitroalkenes are reversibly-reactive electrophiles, endogenously detectable at nM concentrations, displaying anti-inflammatory actions. Nitroalkenes like 9- or 10-nitro-octadec-9-enoic acid (e.g. nitro-oleic acid, OA-NO₂) pleiotropically suppress cardiovascular inflammatory responses, with pulmonary responses less well defined. C57BL/6 J male mice were intratracheally administered bleomycin (3 U/kg, 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. Bronchoalveolar lavage (BAL) and lung tissue were collected 7d later. ITB mice lost body weight, with OA-NO₂ mitigating this loss (-2.3 ± 0.94 vs -0.4 ± 0.83 g). Histology revealed ITB induced cellular infiltration, proteinaceous debris deposition, and tissue injury, all significantly reduced by OA-NO₂. Flow cytometry analysis of BAL demonstrated loss of Siglec F⁺/F4/80⁺/CD45⁺ alveolar macrophages with ITB (89 ± 3.5 vs 30 ± 3.7%). Analysis of CD11b/CD11c expressing cells showed ITB-induced non-resident macrophage infiltration (4 ± 2.3 vs 43 ± 2.4%) was decreased by OA-NO₂ (24 ± 2.4%). Additionally, OA-NO₂ attenuated increases in mature, activated interstitial macrophages (23 ± 4.8 vs. 43 ± 5.4%) in lung tissue digests. Flow analysis of CD31^-/CD45^-/Sca-1⁺ mesenchymal cells revealed ITB increased CD44⁺ populations (1 ± 0.4 vs 4 ± 0.4MFI), significantly reduced by OA-NO₂ (3 ± 0.4MFI). Single cell analysis of mesenchymal cells by western blotting showed profibrotic ZEB1 protein expression induced by ITB. Lung digest CD45⁺ cells revealed ITB increased HMGB1⁺ cells, with OA-NO₂ suppressing this response. Inhibition of HMGB1 expression correlated with increased basal phospholipid production and SP-B expression in the lung lining. These findings indicate OA-NO₂ inhibits ITB-induced pro-inflammatory responses by modulating resident cell function.

Keywords: Acute Lung Injury; Bleomycin; Inflammation; Macrophage; Nitro-Oleic Acid; Nitroalkene.