Skip to main content
. 2021 May 3;131(9):e132305. doi: 10.1172/JCI132305

Figure 9. Monocyte-derived CCR2+ macrophages promote BEC proliferation through ITGβ6 in vitro.

Figure 9

(A) Sorted primary MoMFs (CD45+CD11b+CCR2hiGR-1int) isolated from 48-hour ILY-treated ihCD59BEC-TG mouse livers were cocultured with BECs for 24 hours. BECs were then fixed in 4% paraformaldehyde, and Ki67 staining was performed (representative images are shown). Scale bar: 40 µm. (B) Ki67+ BECs cultured with primary MoMFs were counted (n = 4–8 per group). (C and D) Bone marrow–derived macrophages (BM-MFs) were isolated and stimulated with TLCA (20 μM). Conditioned media (CM) were then transferred to BECs. BECs were also directly treated with TLCA (20 μM). Itgb6 mRNA expression in BECs was assessed in C; Ki67 staining of BECs was analyzed in D. (E) RAW264.7 murine macrophages (RAW) were similarly treated with TLCA, and conditioned media were transferred to BECs, or BECs were directly treated (Direct trt) with TLCA (20 μM). BEC numbers were assessed by an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] absorbance assay (n = 5 per group). (F) RAW264.7 conditioned media were added to BEC culture with or without an ITGβ6-blocking antibody, followed by measurement of BEC proliferation (n = 10 per group). Data represent the mean ± SEM. *P < 0.05 and **P < 0.01, by unpaired Student’s t test for B–E, compared with the TLCA vehicle condition, and for F, compared with the ITGβ6-blocking antibody control condition. (G) Proposed mechanisms by which BEC injury alone triggers the early signals that induce BEC proliferation via the interaction of bile acids, macrophages, and ITGβ6. BEC injury leads to the release of chemoattractants (e.g., CCL2) and DAMPs, which rapidly recruit and activate circulating CCR2+ monocytes to the injured area. Macrophages induce portal fibrogenesis and further increase bile acid release. Macrophages, myofibroblasts, and bile acids upregulate ITGβ6 expression in BECs, which contributes to BEC proliferation. The illustration in G was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com.