Fig. 4. Physiologic hypoxia enhances macrophage efferocytosis of sRBC and apoptotic neutrophils via endogenous RvE4-SPM production.
Human M2 macrophages (M2) were preincubated in physiologic hypoxia or normoxia for 24 hours, 37°C before addition of carboxyfluorescein succinimidyl ester (CFSE)–labeled human sRBC (1:20 M2:sRBC) or CFSE-labeled human aPMN (1:3 M2:aPMN) for 60 min, 37°C. (A) Increased M2 macrophage efferocytosis of apoptotic neutrophils with physiologic hypoxia versus normoxia assessed by flow cytometry (left) and confocal microscopy (right); arrows indicate apoptotic neutrophils; n = 5 healthy human donors. (B) Increased M2 macrophage efferocytosis of sRBC with physiologic hypoxia versus normoxia; *P < 0.05 for physiologic hypoxia versus normoxia; n = 6 healthy human donors. (C) Inhibition of M2 macrophage efferocytosis of apoptotic neutrophils (left) and apoptotic RBC (right) after incubation in physiologic hypoxia and addition of a lipoxygenase inhibitor (baicalein; 20 μM) and rescue of efferocytosis with addition of SPMs produced by M2 macrophages in physiologic hypoxia as shown in fig. S1B (hypoxia SPMs = RvD2, RvD5, RvD6, MaR1, PD1, and RvE4; each together at 10 nM); *P < 0.05 for +baicalein versus physiologic hypoxia; n = 6 healthy human donors. (D) Enhancement of M2 macrophage efferocytosis of aPMN (left) and sRBC (right) with 15-min preincubation of RvD5 (10 nM), RvD6 (10 nM), RvE4 (10 nM), or the hypoxia SPM panel [as in (C); each at 10 nM] assessed by flow cytometry. Results are the means ± SEM of six healthy human donors; *P < 0.05 and **P < 0.01 for individual SPM and hypoxia SPM panel versus vehicle.