Figure 8. Acid aspiration results in release of mitochondrial DAMPs (damage-associated molecular patterns) (MTD) into the pulmonary airspaces. CDDO-Im limits endothelial cell permeability induced by MTDs and by TLR-9-primed PMNs.
A) WT mice were injured by i.t. instillation of acid then sacrificed 5, 30, 60, or 300 min later (0 min represents data from uninjured mice), and BAL performed. Cells and debris recovered in the BAL were removed by centrifugation and the supernatant analyzed for mtDNA, an MTD, by qPCR. N = 3 – 5 for each group from 2 independent experiments. * P < 0.05 compared to uninjured control. B) EA.hy926 cells (an endothelial cell-derived cell line) were cultured to confluence in wells of an ECIS electrode array and freshly isolated human PMNs (2×105/well) + various dilutions of MTDs derived from rat liver mitochondria added. Capacitance, an indicator of endothelial cell permeability, was monitored in real-time. Unstimulated PMNs did not produce an appreciable change in endothelial cell permeability, but the combination of MTDs and PMNs caused an increase that was dependent on the MTD dose. C) Addition of CDDO-Im (100 nM) reduced MTD+PMN-mediated endothelial cell injury. D) Exposure of endothelial cells to MTDs in the absence of PMNs resulted in an increase in endothelial cell permeability that was reduced by CDDO-Im. E) To investigate the effect of CDDO-Im on PMN-mediated injury separate from direct endothelial injury caused by MTDs, TLR-9-primed PMNs were used. CDDO-Im attenuated endothelial cell permeability caused by PMNs activated by CpG sequences. Together, these results show that CDDO-Im reduces endothelial cell injury induced directly by MTDs and by TLR-9 activated PMNs. Data are from continuous averaging of the capacitance measured by each of the 40 electrodes in the well’s electrode array. Error bars represent mean ± SD of 2 wells at the indicated discrete time point from 1 experiment.