Transplantation of Mesenchymal Stem Cells Attenuates Acute Liver Failure in Mice via an Interleukin-4-dependent Switch to the M2 Macrophage Anti-inflammatory Phenotype

Abstract

Background and aims: Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow (BM) is an alternative treatment of acute liver failure (ALF) mainly because of the resulting anti-inflammatory activity. It is not known how MSCs regulate local immune responses and liver regeneration. This study explored the effects of MSCs on hepatic macrophages and the Wnt signaling pathway in ALF.

Methods: MSCs were isolated from BM aspirates of C57BL/6J mice, and transplanted in mice with ALF induced by D-galactosamine (D-Gal). The proliferation of hepatocytes was assayed by immunohistochemical (IHC) staining of Ki-67 and proliferating cell nuclear antigen (PCNA). The levels of key proteins in the Wnt signaling pathway were assayed by western blotting and cytokines were determined enzyme-linked immunosorbent assays (ELISAs). A macrophage polarization assay characterized the M1/M2 ratio. The potential role of interleukin-4 (IL-4) in the biological activity of MSCs was determined by silencing of IL-4.

Results: Transplantation of allogeneic MSCs significantly attenuated D-Gal-induced hepatic inflammation and promoted liver regeneration. MSC transplantation significantly promoted a phenotypic switch from proinflamatory M1 macrophages to anti-inflammatory M2 macrophages, leading to significant Wnt-3a induction and activation of the Wnt signaling pathway in mice with D-Gal-induced ALF. Of the paracrine factors secreted by MSCs (G-CSF, IL-6, IL-1 beta, IL-4, and IL-17A), IL-4 was specifically induced following transplantation in the ALF model mice. The silencing of IL-4 significantly abrogated the phenotypic switch to M2 macrophages and the protective effects of MSCs in both the ALF model mice and a co-culture model in an IL-4 dependent manner.

Conclusions: In vivo and in vitro studies showed that MSCs ameliorated ALF through an IL-4-dependent macrophage switch toward the M2 anti-inflammatory phenotype. The findings may have clinical implications in that overexpression of IL-4 may enhance the therapeutic effects of allogeneic MSC transplantation in the treatment of ALF.

Keywords: Acute liver failure; Interleukin 4; Macrophage; Mesenchymal stem cells; Wnt signaling pathway.

Fig. 1. Mesenchymal stem cell transplantation attenuated D-Gal-induced ALF.

(A) Effects of mesenchymal stem cell (MSC) transplantation on serum levels of alanine transaminase (ALT), aspartate transaminase (AST), prothrombin (PT), and NH₃ at 24, 48, 72, 96, 120, 144, and 168 h after transplantation. Transplantation significantly decreased the levels of liver enzymes (ALT and AST), PT, and NH₃ after infusion; (B) Effects of MSC transplantation on survival included a significant reduction in mortality; (C) Hematoxylin and eosin staining of liver sections; (D) TUNEL staining assay of apoptosis; (E) Immunohistochemical staining of Ki67 and PCNA for assay of cell proliferation; (F) Western blots of β-catenin and active-β-catenin protein expression. *p<0.05 vs. phosphate buffered saline. Normal C57BL/6J mice without transplantation were controls.

Fig. 2. Mesenchymal stem cell (MSC) transplantation increased hepatic Wnt-3a mRNA and protein expression.

(A) mRNA expression of HGF, c-Myc, and Cyclin D1; (B) Western blot assays of TCF1, HGF, c-Myc, and cyclin D1 protein expression; (C) Liver cytokines associated with the Wnt signaling pathway measured by ELISA. (D) Western blots of Wnt-3a protein expression; (E) qRT-PCR assay of Wnt-3a mRNA expression. *p<0.05 vs. phosphate buffered saline. Normal C57BL/6J mice without transplantation were controls.

Fig. 3. Effects of Wnt-3a and the Wnt signaling inhibitor on mesenchymal stem cell (MSC) transplantation-promoted liver regeneration.

(A) Immunohistochemical staining of Ki67 and PCNA (200×). Wnt-3a treatment significantly increased the number of Ki-67- and PCNA-positive hepatocytes. Wnt-C59 treatment significantly abrogated promotion of cell proliferation by MSCs. *p<0.05 vs. Wnt-C59; (B) Western blots of β-catenin, active-β-catenin, and Wnt-3a expression. Wnt-C59 significantly decreased the expression of hepatic β-catenin, active-β-catenin, and Wnt-3a proteins. *p<0.05 vs. MSCs transplantation; (C) Western blots of TCF1, HGF, c-Myc, and Cyclin D1 protein expression. Wnt-C59 significantly decreased the expression of TCF1, HGF, c-Myc, and Cyclin D1. Data are means±standard deviations. *p<0.05 vs. MSC transplantation.

Fig. 4. Role of hepatic macrophages in mesenchymal stem cell (MSC) transplantation-mediated effects on acute liver failure (ALF).

(A) F4/80, CD4, CD8, Foxp3, and CD49b mRNA expression. *p<0.05 vs. phosphate buffered saline (PBS). (B) F4/80 staining (200×). *p < 0.05 vs. PBS transplantation group. (C) IVIS was used to identify distribution of DiR-labeled mesenchymal stem cells (MSCs). (D) F4/80 staining of liver sections after chloroethanol administration (200×); (E) Serum ALT, AST, PT, and NH₃ after MSC transplantation. *p<0.05 vs. MSC transplantation. (F) Hematoxylin and eosin staining of liver tissue (200×). *p<0.05 vs. MSC transplantation. (G) Ki67 and PCNA staining (200×). *p<0.05 vs. CL+MSCs. (H) Survival analysis. *p<0.05 vs. MSCs transplantation group. Normal C57BL/6J mice without transplantation were controls.

Fig. 5. Mesenchymal stem cell (MSC) transplantation induced the phenotypic switching of liver macrophages to the M2 phenotype.

(A) The mRNA expression levels of nitric oxide synthase (iNOS), TNF-α, and MCP-1 markers of M1 phenotype and arginase 1 (Arg1), Mrc-2, and CD163 markers of M2 phenotype, in liver tissue. ^#p<0.05 vs. control. *p<0.05 vs. phosphate buffered saline (PBS). Representative images of dihydroethdium (DHE) staining of (B) Wnt-3a, (C) iNOS, and (D) Arg-1 after mesenchymal stem cell transplantation or PBS. Green and red fluorescence indicate positive staining of target proteins. Normal C57BL/6J mice without transplantation were controls.

Fig. 6. Mesenchymal stem cell (MSC) transplantation increased hepatic IL-4 expression in D-Gal-induced acute liver failure (ALF).

(A) G-CSF, IL-6, IL-1 beta, IL-4, and IL-17A paracrine factors secreted by MSCs determined by enzyme-linked immunosorbent assay. (B) Western blots of hepatic IL-4 protein after MSC transplantation or phosphate buffered saline; (C) qRT-PCR assay of IL-4 mRNA expression in the MSCs transplantation and control groups. *p<0.05 vs. PBS.

Fig. 7. Silencing of IL-4 abrogated mesenchymal stem cell (MSC) transplantation promoted a switch to the macrophage M2 phenotype.

(A) Western blots of hepatic IL-4 protein in the MSCs transplantation and scrambled short hairpin RNA groups. (B) Serum alanine transaminase (ALT), aspartate transaminase (AST), prothrombin (PT), and NH₃ in the MSCs transplantation and scrambled short hairpin RNA groups. (C) Hematoxylin and eosin staining of liver tissue (200×). (D) Ki67 staining and PCNA staining (200×). (E) Survival analysis. (F) Expression of iNOS and Arg-1 mRNA. *p<0.05 vs. Scrambled short hairpin RNA negative control. ^#p<0.05 vs. scramble. Normal C57BL/6J mice without transplantation were controls.

Fig. 8. Hepatic macrophage switch toward the M2 phenotype was dependent on IL-4.

(A) Liver macrophages (400×). (B) Flow cytometry of liver macrophage purity. (C) Flow cytometry of the macrophage phenotype switch. (D) Graphical abstract. *p<0.05 vs. D-Gal(+) short hairpin RNA-IL-4 MSC(−), scrambled short-MSC(−) group.