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. 2025 Aug 30;15(1):125.
doi: 10.1186/s13578-025-01467-x.

M1-BMDMs with Wnt5a deletion attenuate liver fibrosis by suppression of Wnt5a/Frizzled 2 axis in hepatic progenitors

Fei-Fei Xing #  1 , Dan-Yang Wang #  1 , Yan-Nan Xu  1 , Xin-Rui Zheng  1 , Shi-Hao Zhang  1 , Meng-Yao Zong  1 , Jun-Yi Zhan  1 , De-Xin Wang  1 , Wei Liu  1 , Jia-Mei Chen  1 , Gao-Feng Chen  1 , Ping Liu  1 , Cheng-Hai Liu  2 , Yong-Ping Mu  3
Affiliations

M1-BMDMs with Wnt5a deletion attenuate liver fibrosis by suppression of Wnt5a/Frizzled 2 axis in hepatic progenitors

Fei-Fei Xing et al. Cell Biosci. .

Abstract

Background: Bone marrow-derived macrophages (BMDMs) regulate hepatic progenitor cells (HPCs) differentiation, potentially via the Wnt signaling pathway. While M1-polarized BMDMs (M1-BMDMs) exert anti-fibrotic effects in the liver, Wnt5a is implicated in fibrosis progression. The specific influence of Wnt5a levels within M1-BMDMs on HPCs fate and cirrhosis development remains unclear. This study aimed to elucidate the relationship between M1-BMDM-derived Wnt5a and HPCs differentiation during cirrhosis progression.

Methods: First, Wnt5a protein expression was assessed in liver biopsy tissues from patients with hepatitis B-associated liver fibrosis. Second, cirrhosis was induced in rats using CCl4/2-AAF. In week 9, rats received intravenous injections of M1-BMDMs with Wnt5a knockdown (M1-BMDM Wnt5a−KD) or overexpression (M1-BMDM Wnt5a−OE); peripheral BMDMs recruitment was blocked using a CCR2 inhibitor. Fibrosis progression, ductular reaction (DR), and HPC differentiation were evaluated. In vitro, WB-F344 cells subjected to frizzled 2 (Fzd2) knockdown (WB-F344 Fzd2−KD) or overexpression (WB-F344 Fzd2−OE) were cultured with conditioned medium from M1-BMDM Wnt5a−KD (CM Wnt5a−KD) or M1-BMDM Wnt5a−OE (CM Wnt5a−OE).

Results: In patients with hepatitis B-related fibrosis, hepatic Wnt5a expression increased progressively with METAVIR fibrosis grade. In the rat cirrhosis model, M1-BMDMs Wnt5a−KD attenuated fibrosis, whereas M1-BMDMs Wnt5a−OE exacerbated it. Mechanistically, in vivo injection of M1-BMDMs Wnt5a−KD significantly inhibited HPCs differentiation into biliary epithelial cells (BECs), while M1-BMDM Wnt5a−OE promoted this differentiation. In vitro, CM Wnt5a−KD inhibited the differentiation of WB-F344 cells into BECs; this inhibition was potentiated by Fzd2 knockdown in WB-F344 cells but abrogated by Fzd2 overexpression. Conversely, under CM Wnt5a−OE conditions, WB-F344 Fzd2−OE cells exhibited increased cholangiocytic differentiation, an effect largely negated by Fzd2 knockdown.

Conclusions: M1-BMDMs Wnt5a−KD demonstrated superior therapeutic efficacy against cirrhosis compared to unmodified M1-BMDMs. Wnt5a/Fzd2 signaling mediated the crosstalk between M1-BMDMs Wnt5a−KD and HPCs, revealing a novel therapeutic target for cirrhosis treatment.

Supplementary Information: The online version contains supplementary material available at 10.1186/s13578-025-01467-x.

Keywords: Bone marrow-derived macrophage; Ductular reaction; Hepatic progenitor cells; Liver cirrhosis; Wnt5a/Frizzled2 signaling axis.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: Liver tissue slices from patients with hepatitis B-associated liver fibrosis were obtained from the biobank of the clinical study titled “Chinese Herbal Medicine Combined with Entecavir in Treating Hepatitis B Cirrhosis”. This clinical trial was approved by the Ethics Committee of Shuguang Hospital Affiliated to Shanghai University of TCM (Ethics approval No. 2014-331-27-01). The ethics of animal experimentation are as follows: (1) Title of the approved project: The effector mechanism of macrophage polarization in the regulation of hepatic progenitor cells differentiation and intervention in liver cirrhosis. (2) Name of the institutional approval committee or unit: Animal Research Committee at Shanghai University of TCM. (3) Ethics approval No. PZSHUTCM210611009. (4) Date of approval: June 11, 2021. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of BMDMs and M1-BMDMs. (a) Flow cytometry analysis of BMDMs purity after 6-day M-CSF induction revealed the following surface marker expression profile: CD45 (+), CD68 (+), and CD11b (+). (b) The mRNA expression levels of M1-BMDMs markers including CD86, iNOS, IL-12β and TNF-α after LPS treatment 24 h. (c) M0-BMDMs and M1-BMDMs morphology (×100). (d) Immunofluorescence staining of CD68 and TNF-α in M1-BMDMs (×200). *P < 0.05, **P < 0.01
Fig. 2
Fig. 2
M1-BMDM Wnt5a −KD injection alleviates the progression of liver cirrhosis. (a) Immunostaining of Wnt5a and Fzd2 in patients with HBV-related liver fibrosis and positive area analysis (n = 5/per group) (×100). The box shows a higher magnification of the square area (×400). (b) Experimental flow chart. (c) EGFP fluorescence images of LV-transfected M1-BMDMs after 48 h. (d) Wnt5a protein and mRNA expression levels. (e) Four weeks after cell transplantation, tracking the distribution of M1-BMDMsKD−EV and M1-BMDMsWnt5a−KD in the liver through EGFP staining (×200). (f) H&E and Sirius Red staining (×100). (g) Serum biochemistry. (h) Sirius Red staining positive area. (i) hydroxyproline content. (j) α-SMA and CD68 immunostaining (×200). (k) protein and mRNA levels of α-SMA, CD68 TNF-α and TGF-β1. *P < 0.05, **P < 0.01
Fig. 3
Fig. 3
M1-BMDM Wnt5a −KD injection inhibits HPCs activation and DR. (a) CK7 and CK19 immunostaining (×200). (b) EpCam and Sox9 immunostaining (×200). (c) Protein and mRNA expression levels of CK7, CK19, EpCAM and SOX9. (d) OV6/CK7 immunofluorescence co-staining (×400), and the costaining cells ratio of OV6/CK7. (e) OV6/CK19 immunofluorescence co-staining (×400), and the costaining cells ratio of OV6/CK19. (f) EpCam/CK19 immunofluorescence co-staining (×400), and the costaining cells ratio of EpCam/CK19. (g) Sox9/CK19 immunofluorescence co-staining (×400), and the costaining cells ratio of Sox9/CK19. *P < 0.05; **P < 0.01
Fig. 4
Fig. 4
M1-BMDMs Wnt5a −KD inhibit DR by suppressing the Wnt5a/Fzd2 signaling axis. (a) Wnt5a and Fzd2 immunostaining (×200). (b) Protein expression levels of Wnt5a and Fzd2. (c) Flow chart of in vitro experiments and the protein and mRNA expression levels of Wnt5a in M1-BMDMs and Fzd2 in WB-F344 cells. (d) CK19 immunofluorescence staining of WBF344 cells (×600) positive area ratio (%). (e) The mRNA expression levels of CK19 in WB-F344 cell. (f) The mRNA expression levels of Fzd-1/-2/-3/-4/-5/-6/-7 in WB-F344 cells. (g) CK19 immunofluorescence staining of Fzd2 gene modified WBF344 cells (×600) and positive area ratio (%). (h) The mRNA expression levels of CK19 in Fzd2 gene modified WB-F344 cells. *P < 0.05; **P < 0.01
Fig. 5
Fig. 5
M1-BMDM Wnt5a −OE injection promotes the liver inflammatory response and the progression of cirrhosis. (a) Experimental flow chart. (b) EGFP fluorescence images of LV-transfected M1-BMDMs after 48 h. (c) Wnt5a protein and mRNA expression levels. (d) Four weeks after cell transplantation, tracking the distribution of M1-BMDMsOE−EV and M1-BMDMsWnt5a−OE in the liver through EGFP staining (×200). (e) H&E and Sirius Red staining (×100). (f) Serum biochemistry. (g) Sirius Red staining positive area (%). (h) Hyp content of liver tissue. (i) α-SMA and CD68 immunostaining (×200). (j) Protein and mRNA expression levels of α-SMA, CD68, TNF-α and TGF-β1. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
M1-BMDM Wnt5a −OE injection promotes HPCs activation and DR. (a) CK7 and CK19 immunostaining (×200). (b) EpCam and Sox9 immunostaining (×200). (c) Protein and mRNA expression levels of CK7, CK19, EpCam and Sox9. (d) Immunofluorescence costaining of OV6/CK7 (×400) and the costaining area ratio (%). (e) Immunofluorescence costaining of OV6/CK19 (×400) and the costaining area ratio (%). (f) Immunofluorescence costaining of EpCam/CK19 (×400) and the costaining area ratio (%). (g) Immunofluorescence costaining of Sox9/CK19 (×400) and the costaining area ratio (%). *P < 0.05; **P < 0.01
Fig. 7
Fig. 7
M1-BMDM Wnt5a −OE injection promotes DR by activating the Wnt5a/Fzd2 signaling axis. (a) Wnt5a and Fzd2 immunostaining (×200). (b) Protein expression levels of Wnt5a and Fzd2. (c) Experimental flow chart of in vitro experiments and the protein and mRNA expression levels of Wnt5a in M1-BMDMs. (d) CK19 immunofluorescence staining of WBF344 cells (×600) positive area ratio (%). (e) The mRNA expression levels of CK19 in WB-F344 cells. (f) The mRNA expression levels of FZD-1/-2/-3/-4/-5/-6/-7 in WB-F344 cells. (g) CK19 immunofluorescence staining of Fzd2 gene modified WBF344 cells (×600) and positive area ratio (%). (h) The mRNA expression levels of CK19 in Fzd2 gene modified WB-F344 cells. *P < 0.05; **P < 0.01
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