Panel A reports scatter plots depicting mRNA levels of Tgfb, Il6, and Ifng in macrophages exposed to control apoptotic hepatocytes (Ctr-aH) and TLCA-loaded apoptotic hepatocytes (TLCA-aH). Panel B displays a heatmap of mRNA transcripts analyzed by bulk mRNA sequencing in macrophages exposed to Ctr-aH and TLCA-aH. The heatmap includes rows representing different genes and columns representing different conditions, with color intensity indicating expression levels. Panel C shows flow-cytometry plots and paired scatter plots analyzing CCR2 and inducible nitric oxide synthase (iNOS) expression in CD11blowF4/80low macrophages after engulfment of apoptotic hepatocyte or TLCA-laden apoptotic hepatocytes. Panel D presents flow-cytometry plots and paired scatter plots comparing CCR2, CX3CR1, MHC-II, and CD206 expression in macrophages exposed either to apoptotic hepatocytes in the presence of free TLCA (free TLCA+aH) or apoptotic hepatocytes pre-loaded with TLCA (TLCA-aH). Panel E shows microscopy images and paired scatter plots evaluating fibroblast wound-closure capacity after mechanical injury, following exposure to conditioned media from macrophages that engulfed control or TLCA-treated apoptotic hepatocytes. Panel F shows microscopy images and paired scatter plots measuring the fibroblast wound-healing index using a culture insert assay, after incubation with conditioned media from macrophages that engulfed control or TLCA-treated apoptotic hepatocytes.
Engulfment of bile acid–laden apoptotic hepatocytes shapes Mφ activation status. (A–C) BMDMs were exposed for 45 min to apoptotic hepatocytes (aH), either treated with a vehicle control (Ctr-aH) or preloaded with TLCA (TLCA-aH). After 45 min, the medium was washed away, and the cells were kept in culture for an additional 24 h. On those BMDMs, (A) cytokine mRNA levels, detected by qPCR, n = 3–7 biological replicates, mean ± SEM, Wilcoxon test; (B) mRNA transcripts analyzed via bulk mRNA sequencing, n = 2 biological replicates; (C) CCR2 and iNOS, detected via flow cytometry (on a live, CD11blowF4/80low Mφ population), were analyzed. n = 12 biological replicates, mean ± SEM, Wilcoxon test. (D) BMDMs were exposed for 45 min to aH, either preloaded with TLCA (TLCA-aH) or to untreated apoptotic hepatocytes in the presence of free TLCA (free TLCA+ aH). After 45 min, the medium was washed away, and cells were kept in culture for an additional 24 h. Representative FACS plot and corresponding bar graph reporting frequency of BMDMs (live, CD11blowF4/80low) expressing different polarization markers are shown. n = 8, mean ± SEM, Wilcoxon test. (E and F) Representative images and quantification of scratch closure capacity in fibroblasts (WEHI-164 cell line), either (E) creating a gap by mechanically damaging the cell layer or (F) using a culture insert, followed by treatment for 24 h with conditioned media isolated from WT BMDMs exposed for 45 min to aH, either treated with a vehicle control (Mφ+Ctr-aH) or preloaded with TLCA (Mφ+TLCA-aH). Dotted lines indicate the area covered by migration of the cells into the wound 24 h later. Scale bar, 100 μm. n = 6–8, mean ± SEM, Wilcoxon test. *P < 0.05; **P < 0.01.