Panel A shows flow-cytometry plots and a paired scatter plot comparing macrophage efferocytosis of control or TLCA-treated apoptotic hepatocytes. Panel B reports a bar graph quantifying TLCA levels detected in macrophages following uptake or binding of TLCA-treated apoptotic hepatocytes, as measured by high-performance liquid chromatography. Panel C shows t-SNE plots and a bar graph comparing macrophage efferocytosis of apoptotic thymocytes, hepatocytes, and neutrophils. Different colors identify the engulfment of distinct apoptotic-cell types separately, while the bar graph quantifies the percentage of phagocytic macrophages engulfing all three apoptotic cell types concomitantly at a given time point. Panel D presents immunofluorescence images and a bar graph tracking macrophage efferocytosis over time. White labels macrophages, green labels apoptotic thymocytes, blue labels apoptotic hepatocytes, and red labels apoptotic neutrophils at 5-, 15-, 30-, 60-, and 90-minute intervals. Panel E shows flow-cytometry plots and a scatter plot comparing macrophage engulfment of apoptotic thymocytes after treatment with control or TLCA-treated apoptotic hepatocytes. The scatter plot shows fold-change differences relative to the control condition.
Bile acid–laden apoptotic hepatocytes are a carriers of bile acids within the efferocytic Mφ. (A) Apoptotic hepatocytes (aH) were treated with the vehicle DMSO as control (Ctr-aH) or with 50 µM of the secondary bile acid TLCA (TLCA-aH) for 75 min. aH were then added to BMDMs and cocultured with for 45 min. Representative flow cytograms and data reporting the frequency of Mφ phagocytosing Ctr-aH or TLCA-aH are shown. n = 16 biological replicates; mean ± SEM. Wilcoxon test. (B) Amount of TLCA detected in Mφ after uptake of TLCA-aH (37°C) or upon binding to TLCA-aH (4°C), as detected by HPLC. Bars represent pooled data from two independent experiments, each with three biological replicates per condition. (C and D) BMDMs were exposed to aH, apoptotic thymocytes (aT), or apoptotic neutrophils (aN) individually or all the three cell types concomitantly (aT+aH+aN). Apoptotic cells were stained with different cell dyes, and the phagocytic capacity of Mφ was assessed via flow cytometry or immunofluorescence. n = 3 biological replicates/condition. (C) t-SNE plots, concatenated from three mice/condition, report the fraction of Mφ that have been phagocytosing aT (in light blue), aH (in dark green), or aN (in pink), after 45 min of coculture. The black fraction in each t-SNE plot represents the non-phagocytic Mφ. The gate (black circle) shows the Mφ that have been phagocytosed aT, aH, and aN simultaneously. Representative t-SNE plots and quantification are reported. One representative experiment with BMDMs isolated from three WT mice is shown. (D) Immunofluorescence was performed on Mφ (F4/80, white) during the efferocytosis of aT (green), aH (blue), and aN (red). The frequency of phagocytic Mφ was analyzed 5, 15, 30, 60, and 90 min after adding the apoptotic cells to the Mφ culture. IF images representative of three independent experiments are reported. Quantification has been performed by counting a total of 16 different areas/condition. Scale bar, 50 μm. (E) Mφ treated as reported in A are further tested for their capacity to phagocyte apoptotic cells (aT) in vitro. Representative dot plots and pooled data reporting the frequency of Mφ phagocytosing aT in three independent experiments. n = 6 biological replicates; mean ± SEM. Mann–Whitney U test. *P < 0.05; ***P < 0.001.