Figure 5: EPO promotes macrophage efferocytosis during inflammation resolution via PPARγ.

(a) ZymA (i.p., 1 mg per mouse) with or without rhEPO or PBS was given to EPOR-MKO or EPOR-C mice and in vivo efferocytosis of ANs were measured (n=5). (b) ZymA (i.p., 1 mg per mouse) with rhEPO or PBS was given to WT mice and in vivo efferocytosis were measured (n=5). (c) CGD mice were given rhEPO (i.p.) or PBS for 1 day before zymA (i.p., 1 mg per mouse) and every 24 h thereafter and in vivo efferocytosis were measured (n=5). (d) Peritoneal macrophages from WT or EPOR-MKO mice were incubated with pHrodor-labelled ANs for 1 h and then assesed for efferocytosis (n=3). (e) Peritoneal macrophages were pre-stimulated with rhEPO for indicated time and then analysed for efferocytosis of ANs (n=3). (f) Peritoneal macrophages from CGD mice were pre-stimulated with rhEPO for 24 h and then analysed for efferocytosis of ANs (n=3). (g) CGD mice received rhEPO (i.p.) treatment for 1 day and their peritoneal macrophages were collected for analysis of efferocytosis of ANs in vitro (n=3). (h) Peritoneal macrophages were incubated with or without rhEPO (40 IU ml⁻¹) or ANs for 24 h and their supernatants were collected for analysis of cytokines (n=3). (i) ZymA (i.p., 1 mg per mouse) was given to WT, EPOR-MKO or CGD mice and levels of macrophage PPARγ (F4/80⁺PPARγ⁺) were measured by flow cytometry (n=3). (j) WT, CGD or EPOR-MKO mice were i.p. given rhEPO or PBS for 1 day before zymA (i.p., 1 mg per mouse) and every 24 h thereafter and levels of macrophage PPARγ were measured (n=3). (k) EPOR-MKO mice or PPARγ-MKO mice were given rosiglitazone (rosiglitazone, 10 mg kg⁻¹ per day via oral gavage) or control (carboxymethyl cellulose), or rhEPO (i.p.) or PBS for 1 day before zymA (i.p., 1 mg per mouse) and every 24 h thereafter. Lavages were collected and efferocytosis of ANs were analysed (n=3). (l) Peritoneal macrophages from EPOR-MKO or PPARγ-MKO mice were pre-stimulated with rhEPO, RSG or control for 24 h and then analysed for efferocytosis of ANs (n=3). Representative data from at least two independent experiments are shown. Error bars represent the s.e.m. *P<0.05, two-tailed unpaired Student’s t-test.