Resolution of Post-Lung Transplant Ischemia-Reperfusion Injury is Mediated via Resolvin D1-Alx/FPR2 and Maresin 1-LGR6 Receptor Signaling

Abstract

Purpose Dysregulation of inflammation-resolution pathways can lead to post-lung transplant (LTx) ischemia-reperfusion injury (IRI) and allograft dysfunction. We investigated the role of ω-3-derived specialized pro-resolving lipid mediators, i.e. Resolvin D1 (RvD1) and Maresin-1 (MaR1), and the respective receptor signaling in the resolution of lung IRI. Methods RvD1 and MaR1 expressions were analyzed in bronchoalveolar lavage (BAL) from patients undergoing LTx on post-operative days 0, 1 and 7 using mass spectrometry and ELISA. C57BL/6 mice underwent sham surgery or lung IRI (1hr left lung ischemia followed by 6hr reperfusion) using an in vivo hilar ligation model. RvD1 and/or MaR1 (100ng/kg each; given intratracheally 1hr prior to IR) was administered to mice that were previously treated with/without siRNA for FPR2 (receptor for RvD1) or LGR6 (receptor for MaR1; 10μg given i.p. each). Orthotopic left lung transplants were performed between Balb/C brain dead donors and C57Bl/6 recipients to analyze FPR2 and LGR6 receptor expressions at 6hrs and 48hrs post-transplant. Macrophage (MH-S) and epithelial (MLE12) cells were exposed to hypoxia/reoxygenation (HR; 3hr/3hr or 24hrs) followed by cytokine analysis. Groups (n=3-10) were compared using ANOVA with post-hoc Tukey's test. Results RvD1 and MaR1 levels were significantly increased in BAL of LTx patients on day 7 compared to days 0 and 1. In the hilar ligation IR model, concomitant treatment with recombinant RvD1 and MaR1 significantly attenuated lung dysfunction after 6hrs compared to either treatment or IR alone, that was abolished by pre-treatment with siRNA for FPR2 and LGR6, respectively. Lung inflammation (IL-17, TNF-α, CXCL1, HMGB1), edema (wet/dry ratio) and injury (neutrophil infiltration) were significantly attenuated in mice treated with RvD1+MaR1 compared to either treatment or IR alone. FPR2 and LGR6 expressions were increased in lung tissue after 48hrs compared to 6hrs post-LTx. RvD1 attenuated HMGB1 and TNF-α secretion in HR-exposed MH-S cells that was abolished by siRNA for FPR2, and MaR1 treatment mitigated CXCL1 secretion by HR-exposed MLE12 cells that was abolished by siRNA for LGR6. Conclusion Our results suggest that resolution of lung IRI is mediated by RvD1-FPR2 and MaR1-LGR6 signaling pathways on macrophages and epithelial cells, respectively.