Fig. 2. Emergence of Ly6C^hi-proliferative monocytes was CCR2 independent and occurred after bacteria-induced TRM loss.

(A and B) Peritoneal macrophages of mice infected with E. coli or PBS were analyzed for live, apoptotic, and necrotic cells using FAM-FLICA and 4′,6-diamidino-2-phenylindole (DAPI) (A) and their percentage quantified (B) after 18 hours of infection. Numbers in FACS plots represent the percentage of positive cells. Results are expressed as means ± SD (n = 5) and representative of one of two experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001 (Student’s t test). (C to E) Mice were administered clodronate or PBS liposomes intraperitoneally before infection with or without E. coli as indicated. (C) Total Ly6C^hi monocytes (bottom left) and proliferative Ly6C^hi monocytes (bottom right) in the blood were quantified. Results are expressed as means ± SD (n = 6) and representative of one of three experiments. n.s., not significant; ****P < 0.0001 (one-way ANOVA). (D) Quantification of the peritoneal bacterial load was quantified. Results are expressed as means ± SD (n = 6) and representative of one of three experiments. ***P < 0.001 (Student’s t test). (E) Correlation graph of blood proliferative Ly6C^hi monocytes versus bacterial load. (F and G) WT and Tlr4^−/− (F) or WT and Ccr2^−/− mice (G) were administered E. coli or PBS intraperitoneally, and the blood was analyzed for proliferative Ly6C^hi monocytes using BrdU incorporation in vivo after 18 hours. Results are expressed as means ± SD (n = 3 to 6) and representative of one of three experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001 (one-way ANOVA).