Nitrated Fatty Acids Reduce Inflammatory Cell Recruitment in Bleomycin‐Induced Lung Injury

Abstract

Acute lung injury (ALI) leads to infiltration of inflammatory cells and impaired lung function. Matrix Metalloproteinase 9 (MMP9) is a zinc‐dependent endopeptidase that degrades the extra cellular matrix and increases the porosity of the lung. CD11b is a marker for macrophage activation and migration. Nitro‐oleic fatty acid (OANO2), an electrophile that reduces cardiovascular inflammation has not been examined in lung injury. OANO2 modifies cysteine residues via Michael addition altering protein function. The goal of this study were to determine if OANO2 administration would reduce MMP9 expression and cellular infiltration following ALI. An intratracheal bleomycin (ITB) ALI model was used. Two groups of 6 C57BL/6‐J mice were treated intratracheally with bleomycin (3U/kg) or PBS (50uL). Half of the animals received OANO2 (50μg) in the same instillation and 72 hours later. The lungs were collected 7 days post ITB treatment. As previously seen, mice treated with ITB lost a significant amount of body weight; however, the addition of OANO2 mitigated this loss (−2.3 +/− 0.94 vs −0.4 +/− 0.83 g). Histology revealed cellular infiltration, deposition of proteinaceous debris, and airway epithelial damage in ITB mice that was reduced with OANO2 administration. The number of CD11b+ cells (marker for migratory macrophages) and MMP9+ cells was determined by immunohistochemistry. ITB increased number of CD11b+ (69 +/− 17.1 vs 195 +/− 17.1 #cells/HPF). OANO2 treatment reduced CD11b+ cells most in ITB animals (121 +/− 17.1#cells/HPF). ITB increased MMP9+ cell numbers compared to controls (71 +/− 15.7 vs 124 +/− 15.7 #cells/HPF). However, MMP9 expression was increased with OANO2 administration in PBS animals (304 +/− 15.7; 166 +/− 15.7 #cells/HPF), though this increase was not significant in ITB mice. Increased MMP9 expression was confirmed by western blot of whole lung tissue, where both ITB OANO2 and control OANO2 animals displayed high expression. Increased MMP9 expression may result from reduced self‐degradation as a consequence of OANO2 mediated enzyme inhibition. Overall, OANO2 increased MMP9+ cells and decreased CD11b+ cell populations. These findings suggest that treatment with OANO2 may oppose ITB‐mediated pro‐inflammatory cellular activation possibly by inhibition of extracellular peptidases, making it a potential therapeutic agent for acute lung injury.Support or Funding InformationSupported by the Summer Undergraduate Research Fellowship and NIH Grants: 5R25ES020721‐09 (MPI)