Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9

Abstract

Background: Mesenchymal stem cells (MSCs) play an anti-inflammatory role by secreting certain bioactive molecules to exert their therapeutic effects for disease treatment. However, the underlying mechanism of MSCs in chronic autoimmune liver diseases-primary biliary cholangitis (PBC), for example-remains to be elucidated.

Methods: Human umbilical cord-derived MSCs (UC-MSCs) were injected intravenously into 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)-induced autoimmune cholangitis mice. Serum levels of biomarkers and autoantibodies, histologic changes in the liver, diverse CD4⁺ T-cell subsets in different tissues, and chemokine activities were analyzed. Moreover, we investigated galectin-9 (Gal-9) expression and its function in UC-MSCs.

Results: In this study, UC-MSC transplantation (UC-MSCT) significantly ameliorated liver inflammation, primarily by diminishing T helper 1 (Th1) and Th17 responses as well as modifying liver chemokine activities in experimental autoimmune cholangitis mice. Mechanistically, UC-MSCs significantly repressed the proliferation of CD4⁺ T cells and suppressed the differentiation of Th1 and Th17 cells, which was likely dependent on Gal-9. Furthermore, the signal transducer and activator of transcription (STAT) and c-Jun N-terminal kinase (JNK) signaling pathways were involved in the production of Gal-9 in UC-MSCs.

Conclusions: These results suggest that Gal-9 contributes significantly to UC-MSC-mediated therapeutic effects and improve our understanding of the immunomodulatory mechanisms of MSCs in the treatment of PBC.

Keywords: Galectin-9; Inflammation; Primary biliary cholangitis; Umbilical cord–derived mesenchymal stem cells.

Fig. 1

Umbilical cord–derived mesenchymal stem cell transplantation alleviates 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)–induced autoimmune cholangitis. a Schematic illustration of the experimental procedure. b Representative images of liver tissue sections by hematoxylin-and-eosin staining from control, primary biliary cholangitis, or umbilical cord–derived mesenchymal stem cell (UC-MSC)–treated mice, showing liver inflammation and the extent of bile duct damage. c Histological scores were assessed on the basis of lymphocytic infiltration and bile duct damage in the liver. d The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and glutamyl transpeptidase (GGT) were measured. e The levels of anti-PDC-E2 autoantibodies were measured. Bars represent the mean ± standard error of the mean (SEM); n = 6 per group. *P <0.05, **P <0.01, ***P <0.001

Fig. 2

Umbilical cord–derived mesenchymal stem cell transplantation inhibits the aberrant T helper 1 (Th1) and Th17 responses in 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)–induced autoimmune cholangitis. a Representative flow cytometric profiles showing the frequencies of Th1 cells in the liver, spleen, and blood. b The frequencies of Th1 cells were quantified. c Representative flow cytometric profiles showing the frequencies of Th17 cells in the liver, spleen, and blood. d The frequencies of Th17 cells were quantified. e Intrahepatic mRNA expressions of interferon-gamma (IFN-γ), interleukin-17A (IL-17A), IL-12, IL-23, T-bet, and RAR-related orphan receptor gamma t (RORγt) were determined by real-time polymerase chain reaction. f Levels of IFN-γ, IL-17A, IL-12, and IL-23 in serum were detected by Luminex. Bars represent the mean ± standard error of the mean (SEM); n = 6 per group. *P <0.05, **P <0.01, ***P <0.001. Abbreviations: PB peripheral blood, SP spleen

Fig. 3

Galectin-9 (Gal-9) is highly expressed in umbilical cord–derived mesenchymal stem cells (UC-MSCs). a Total mRNA of UC-MSCs was extracted after activation with or without interferon-gamma (IFN-γ) and analyzed by real-time polymerase chain reaction for the expression of Gal-1, Gal-3, and Gal-9. Bars represent the mean ± standard error of the mean (SEM); n = 4 per group. b, c UC-MSCs were treated with or without IFN-γ and analyzed by Western blot and enzyme-linked immunosorbent assay for the expression of Gal-9. Bars represent the mean ± SEM; n = 3 per group. d The fluorescence microscope was employed to detect green fluorescent protein (GFP)-positive UC-MSCs in the livers of primary biliary cholangitis mice. Colocalization (white arrow) of the GFP (green) with Gal-9 (red) staining indicated that UC-MSCs expressed Gal-9. Scale bar = 50 μm. Abbreviation: DAPI 4′,6-diamidino-2-phenylindole

Fig. 4

Umbilical cord–derived mesenchymal stem cells (UC-MSCs) suppress CD4⁺ T-cell proliferation as well as T helper 1 (Th1) and Th17 cell differentiation via galectin-9. a Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled CD4⁺ T cells were cultured in the presence of UC-MSCs or UC-MSCs plus α-lactose for 4 days. The proliferation of murine CD4⁺ T cells was assessed on the basis of the fluorescence intensity of CFSE. Bars represent the mean ± standard error of the mean (SEM); n = 3 per group. b Purified naïve murine CD4⁺ T cells were cultured under Th1 polarization conditions in the presence of UC-MSCs or UC-MSCs plus α-lactose. Representative flow cytometric profiles that show the frequencies of Th1 cells are presented. The frequencies of Th1 cells were quantified. c Purified naïve murine CD4⁺ T cells were cultured under Th17 polarization conditions in the presence of UC-MSCs or UC-MSCs plus α-lactose. Representative flow cytometric profiles that show the frequencies of Th17 cells are presented. The frequencies of Th17 cells were quantified. Bars represent the mean ± SEM; n = 4 per group. *P <0.05, **P <0.01, ***P <0.001

Fig. 5

Umbilical cord–derived mesenchymal stem cell (UC-MSC) transplantation downregulates proinflammatory chemokines to repress inflammatory responses. a–d Intrahepatic mRNA expressions of CCL5, CCL20, CXCL10, and CX3CL1 were determined by real-time polymerase chain reaction. Bars represent the mean ± standard error of the mean (SEM); n = 6 per group. e–h CCL5, CCL20, CXCL10, and CX3CL1 mRNA expression in unstimulated, interferon-gamma (IFN-γ)–stimulated, UC-MSCs or UC-MSCs plus α-lactose co-cultured with biliary epithelial cells (BECs) demonstrated by real-time polymerase chain reaction. Bars represent the mean ± SEM; n = 4 per group. *P <0.05, **P <0.01, ***P <0.001. Abbreviation: 2OA-BSA 2-octynoic acid coupled to bovine serum albumin

Fig. 6

Interferon-gamma (IFN-γ)–induced galectin-9 (Gal-9) upregulation of umbilical cord–derived mesenchymal stem cells (UC-MSCs) is mediated through the c-Jun N-terminal kinase (JNK) and signal transducer and activator of transcription (STAT) signaling pathways. a The expression of STAT, p38/mitogen-activated protein kinase (p38/MAPK), extracellular-regulated protein kinase (ERK), JNK, and their phosphorylation forms were detected by Western blot analysis in unstimulated and IFN-γ–stimulated UC-MSCs. b Total mRNA of UC-MSCs was extracted after the activation of IFN-γ with JNK or STAT inhibitors and then analyzed by real-time polymerase chain reaction for the expression of Gal-9. Bars represent the mean ± standard error of the mean (SEM); n = 3 per group. ***P <0.001