Figure 5. CD169⁺ peritoneal macrophages are resistant to H₂O₂-induced apoptosis in Tim4^–/– relative to WT mice.

(A) Histograms of gated WT or Tim4^–/– DAPI^–CD11b⁺F4/80⁺ peritoneal macrophages revealed TIM-4 and CD169 expression, but not Ly6C expression. Shaded histograms represent indicated controls. MFI is noted parenthetically. (B–D) Gated WT or Tim4^–/– CD11b⁺F4/80⁺ peritoneal macrophages incubated in the presence or absence of H₂O₂ (125 μM unless otherwise indicated) were analyzed for DAPI and annexin V binding by flow cytometry. (B) Representative flow cytometry plots at 3 hours after treatment. (C) Summary graphs from 1 of 3 independent experiments, showing lower apoptosis (DAPI^–annexin V⁺) and cell death (DAPI⁺annexin V⁺) in Tim4^–/– versus WT CD11b⁺F4/80⁺ macrophages. *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA (P < 0.0001) with Bonferroni post-test. (D) Dose-response curve showing less apoptosis (DAPI^–annexin V⁺) in Tim4^–/– versus WT CD11b⁺F4/80⁺ macrophages at 3 hours. ***P < 0.001, ANOVA (P < 0.01) with Bonferroni post-test. (E and F) Gated CD11b⁺F4/80⁺ macrophages treated with 125 μM H₂O₂ for 3 hours were permeabilized and stained for intracellular active caspase-3 in triplicate. Shown are representative histograms (E) and summary graphs showing active caspase-3 MFI and frequency of active caspase-3⁺ macrophages from 1 of 2 independent experiments (F). Active caspase-3 levels in gated Tim4^–/– macrophages was reduced compared with WT macrophages. P values were determined by ANOVA (P < 0.01) with Bonferroni post-test. All graphs depict mean ± SEM from a representative experiment performed in triplicate.