Fig. 6. EGCs stimulate the differentiation of monocytes into anti-inflammatory CD206⁺ Mφs in part via CSF-1 in vitro.

a Experimental outline of in vitro primary bone marrow monocytes stimulated with supernatant of EGCs. b Bone marrow-derived monocytes were stimulated for 24 h with/without supernatant of EGCs. Relative mRNA levels for pro- and anti-inflammatory cytokines normalized to the housekeeping gene rpl32 in bone marrow monocytes cultured with/without EGC supernatant. c Experimental outline of in vitro experiment using sorted Ly6C⁺ MHCII⁻ monocytes stimulated with/without EGC supernatant for 24 h. d Ly6C⁺ MHCII⁻ monocytes were sorted from the muscularis of WT mice 24 h after the induction of muscularis inflammation and were stimulated for 24 h with/without supernatant of EGCs. Relative mRNA levels of pro- and anti-inflammatory mediators normalized to the housekeeping gene rpl32 in sorted Ly6C⁺ MHCII⁻ monocytes stimulated with/without EGC supernatant. e–h Bone marrow monocytes were cultured for 24–48 h with/without EGC supernatant and supplemented with anti-CSF1r antibody. e Contour plots of bone marrow monocytes showing expression of CD206 (top) or CCR2 (bottom) upon culture for 48 h with/without EGC supernatant and supplemented with anti-CSF1r antibody. f Brightfield images of monocytes upon culture for 24 h with/without EGC supernatant. g Quantification of 7-AAD⁺ cells in bone marrow monocytes cultured for 24 h or 48 h with/without EGC supernatant and supplemented with anti-CSF1r antibody. h Percentages of CCR2⁺ and CD206⁺ cells in live CD45⁺ CD11b⁺ Ly6G⁻ CD64⁺ population. i Ly6C⁺ MHCII⁻ monocytes were sorted from the muscularis of WT mice 24 h after the induction of muscularis inflammation and were cultured with/without EGC supernatant and supplemented with anti-CSF1r antibody for 48 h. Percentages of CCR2⁺ and CD206⁺ cells in live CD45⁺ CD11b⁺ Ly6G⁻ CD64⁺ population. a–d T-test. g–i one-way ANOVA. *p < 0.05; **p < 0.01.