Panel A shows representative flow cytometry plots and a scatter graph comparing efferocytosis in untreated macrophages, or macrophages exposed to control apoptotic hepatocytes (treated with DMSO as vehicle control), or apoptotic hepatocytes pre-loaded with differet bile acids (TCA, TCDCA, GCDCA). The scatter graph quantifies percentages of phagocytic macrophages under the different conditions. Panel B presents paired scatter plots quantifying macrophage efferocytosis of apoptotic thymocytes after prior exposure to control or bile acid-loaded apoptotic hepatocytes. Panel C shows paired scatter plots comparing Tnfa and Il1b messenger RNA expression levels in macrophages exposed to control or bile acid-loaded apoptotic hepatocytes. Panel D shows flow cytometry gating strategy plots and scatter graphs analyzing CCR2, ICAM1, and SIRPα expression in live CD11b+F4/80low macrophages after exposure to apoptotic hepatocytes pre-loaded with different bile acids. Panel E shows scratch-wound microscopy images and a scatter plot evaluating fibroblast wound-healing capacity using a culture insert assay, after incubation with conditioned media from macrophages exposed to control or bile acid-loaded apoptotic hepatocytes. Images compare wound closure at 0 and 24 hours, while the scatter graph quantifies wound-healing index values.
Efferocytosis of hepatocytes loaded with distinct bile acids alters Mφ responses in vitro. BMDMs were left untreated (Untr) or exposed to aH that were either treated with a vehicle control (Ctr-aH) or preloaded with different bile acids (TCA, TCDCA, and GCDCA). (A–E) Efferocytosis efficiency is shown through representative FACS plots and a bar graph reporting pooled data of BMDMs phagocytosing aH for 45 min at either 4°C (binding only) or 37°C (binding + uptake). n = 7 biological replicates, mean ± SEM; Friedman test followed by Dunn’s multiple comparison. BMDMs exposed to aH treated as described in A were then rested for 2 h and either (B) subsequently exposed to additional dying cells (aT) for 45 min and their efferocytosis efficiency analyzed; n = 7 biological replicates, mean ± SEM, Wilcoxon test; or (C) Tnfa and Il1b mRNA expression quantified by qPCR; n = 7, mean ± SEM, Wilcoxon test; or (D) analyzed for phenotypic marker expression after 24 h by flow cytometry (on a population of live, CD11b+F4/80low Mφ); n = 7 biological replicates, mean ± SEM, Friedman test followed by Dunn’s multiple comparison test; or (E) analyzed for their capacity to induce wound closure in fibroblasts. A culture insert was added to a fibroblast layer, and then fibroblasts were exposed to Mφ-conditioned media (collected from Mφ treated as in A) for 24 h. n = 4 biological replicates, mean, ±SEM, Friedman test followed by Dunn’s multiple comparison test. *P < 0.05; **P < 0.01.