Metabolic Reprogramming of CD4+ T Cells by Mesenchymal Stem Cell-Derived Extracellular Vesicles Attenuates Autoimmune Hepatitis Through Mitochondrial Protein Transfer

Abstract

Background: Autoimmune hepatitis (AIH) is a serious liver disease characterized by immune disorders, particularly effector T-cell overactivation. This study aimed to explore the therapeutic effect and underlying mechanism of mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment on CD4⁺ T-cell overactivation and liver injury in AIH.

Methods: The metabolic changes of CD4⁺ T cells were assayed in human AIH and mouse hepatitis models. The liver protective effect of MSC-EVs was evaluated by transaminase levels, liver histopathology and inflammation. The effect of MSC-EVs on the metabolic state of CD4⁺ T cells was also explored.

Results: Enhanced glycolysis (eg, ~1.5-fold increase in hexokinase 2 levels) was detected in the CD4⁺ T cells of AIH patient samples and mouse hepatitis models, whereas the inhibition of glycolysis decreased CD4⁺ T-cell activation (~1.8-fold decrease in CD69 levels) and AIH liver injury (~6-fold decrease in aminotransferase levels). MSC-EV treatment reduced CD4⁺ T-cell activation (~1.5-fold decrease in CD69 levels) and cytokine release (~5-fold decrease in IFN-γ levels) by reducing glycolysis (~3-fold decrease) while enhancing mitochondrial oxidative phosphorylation (~2-fold increase in maximal respiration) in such cells. The degree of liver damage in AIH mice was ameliorated after MSC-EV treatment (~5-fold decrease in aminotransferase levels). MSC-EVs carried abundant mitochondrial proteins and might transfer them to metabolically reprogram CD4⁺ T cells, whereas disrupting mitochondrial transfer impaired the therapeutic potency of MSC-EVs in activated CD4⁺ T cells.

Conclusion: Disordered glucose metabolism promotes CD4⁺ T-cell activation and associated inflammatory liver injury in AIH models, which can be reversed by MSC-EV therapy, and this effect is at least partially dependent on EV-mediated mitochondrial protein transfer between cells. This study highlights that MSC-EV therapy may represent a new avenue for treating autoimmune diseases such as AIH.

Keywords: autoimmune hepatitis; extracellular vesicles; inflammation; metabolic reprogramming; mitochondria.

Figure 1

CD4⁺ T cells are hyperactivated in AIH patients and mice. (A) Schematic illustration of human sample collection. (B) The mRNA levels of CD25 and CD69 were normalized to β-actin mRNA levels in human peripheral blood CD4^` T cells. (C) The mRNA levels of IFN-γ, TNF-α and IL-2 were normalized to β-actin mRNA levels in human peripheral blood CD4⁺ T cells. (D) Schematic illustration of the animal experiments. (E) Serum ALT and AST levels in NC and AIH mice. (F) Representative images of H&E-stained liver sections from NC and AIH mice (scale bars = 500 μm and 100 μm). (G) Representative flow cytometry plots of CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells among MNCs from NC and AIH mouse livers. (H) Statistical analysis of flow cytometry data for CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells among MNCs from NC and AIH mouse livers.

Figure 2

Inhibiting glycolysis downregulates CD4⁺ T-cell activation. (A) Immunofluorescence staining for CD4 and GLUT1 in the livers from AIH patients and HCs (scale bar = 10 μm). (B) Schematic illustration and the mRNA levels of HK2, GLUT1, PFKFB3, and LDHA normalized to β-actin mRNA levels in human peripheral blood CD4⁺ T cells. (C) Immunofluorescence staining for CD4 and GLUT1 in the livers from NC and AIH mice (scale bar = 10 μm). (D) Schematic illustration and Western blot analysis of HK2, GLUT1, and PFKFB3 levels in CD4⁺ T cells. (E) Representative flow cytometry plots of CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells. (F) Statistical analysis of flow cytometry data for CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells.

Figure 3

Inhibiting glycolysis suppresses CD4⁺ T-cell activation and AIH injury. (A) Schematic illustration of the animal experiments. (B) The mRNA levels of HK2, GLUT1, and PFKFB3 in livers from different groups. (C) Representative flow cytometry plots of CD4⁺CD69⁺ T cells among MNCs in the livers of mice from different groups. (D) Statistical analysis of flow cytometry data for CD4⁺CD69⁺ T cells among MNCs in livers of mice from different groups. (E) The mRNA levels of IFN-γ, TNF-α, IL-2, and IL-6 normalized to β-actin mRNA levels in the livers of mice from different groups. (F) Serum ALT and AST levels in different mice. (G) Representative images of H&E-stained liver sections from different mice (scale bars = 500 μm and 100 μm). (H) Suzuki score of liver injury.

Figure 4

MSC-EV treatment inhibits proinflammatory CD4⁺ T-cell activation in vitro. (A) Schematic illustration of MSC-EV isolation. (B) Representative TEM images of MSC-EVs (scale bar = 200 nm). (C) Size distributions of MSC-EVs measured by NTA. (D) Western blot analysis of positive and negative markers of MSC-EVs. (E) Representative fluorescence images of MSC-EV (red) uptake by CD4⁺ T cells (green). The yellow box indicates the colocalization of red and green signals (scale bar = 10 μm). (F) Representative flow cytometry plots of CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells. (G) Statistical analysis of flow cytometry data for CD4⁺CD69⁺ T cells and CD4⁺CD25⁺ T cells. (H) The mRNA levels of IFN-γ, TNF-α, IL-2 and IL-6 normalized to β-actin mRNA levels in different groups of CD4⁺ T cells. (I) The protein levels of IFN-γ, TNF-α, IL-2 and IL-6 in the supernatants of CD4⁺ T-cell cultures were measured via ELISA.

Figure 5

MSC-EV treatment reduces liver damage in AIH mice. (A) Representative IVIS images of different mouse organs. (B) Statistical analysis of the fluorescence intensity in different mouse organs. (C) Schematic illustration of the animal experiments. (D) Serum ALT and AST levels in the mice. (E) Representative images of H&E-stained liver sections from different mice (scale bars = 500 μm and 100 μm). (F) Suzuki’s score of liver injury.

Figure 6

MSC-EV treatment reduces CD4⁺ T-cell activation in vivo. (A) Schematic illustration of the animal experiments. (B) Representative fluorescence images of dye-labeled EVs (red) in liver sections. Liver tissues were stained with a CD4 antibody (green) and DAPI (blue). The yellow box indicates the colocalization of red and green signals (scale bar = 20 μm). (C) Representative flow cytometry plots of CD4⁺CD69⁺ T cells among MNCs in livers from mice in the different groups. (D) Statistical analysis of flow cytometry data for CD4⁺CD69⁺ T cells among MNCs in the livers of mice from different groups. (E) The mRNA levels of HK2, GLUT1, and PFKFB3 were normalized to β-actin mRNA levels in livers of mice from different groups. (F) Immunofluorescence staining for CD4 and GLUT1 in the liver (scale bar = 10 μm).

Figure 7

MSC-EV treatment induces metabolic reprogramming in CD4⁺ T cells in vitro. (A) PCA score plot. (B) Heatmap analysis of the differentially expressed genes. (C) Volcano plot of differentially expressed genes (Padj≤0.05 and |log2(fold change)| ≥ 1). (D) KEGG pathway enrichment analysis. (E) Assay of 2-NBDG uptake in CD4⁺ T cells. (F) The mRNA levels of HK2, GLUT1, and LDHA were normalized to β-actin mRNA levels in different groups of mouse CD4⁺ T cells. (G) Western blot analysis of HK2, GLUT1, and PFKFB3 levels in CD4⁺ T cells. (H) Lactate levels in the supernatant of CD4⁺ T cells.

Figure 8

MSC-EVs induce the metabolic switch of CD4⁺ T cells via mitochondrial transfer. (A) Western blot analysis of the levels of mitochondrial electron transport chain (ETC) proteins in MSCs and MSC-EVs. (B) Schematic illustration and fluorescence images. The yellow box indicates the colocalization of red and green signals (scale bar = 50 μm). (C) Schematic illustration and confocal images. The yellow box indicates the colocalization of red and green signals (scale bar = 5 μm). (D) Western blot analysis of the levels of mitochondrial ETC proteins in different groups of CD4⁺ T cells. (E) Measurement of the OCR in CD4⁺ T cells. (F) Measurement of the ECAR of CD4⁺ T cells. (G) Basal and maximal ECAR:OCR ratios.

Figure 9

Disruption of mitochondrial transfer impairs the therapeutic effects of MSC-EVs on CD4⁺ T-cell activation. (A) Schematic illustration of Rho-EV isolation. (B) Representative TEM images of Ctrl-EVs and Rho-EVs (scale bar = 200 nm). (C) Size distributions of Ctrl-EVs and Rho-EVs measured by NTA. (D) Western blot analysis of Ctrl-EVs and Rho-EVs. (E) Representative flow cytometry plots and statistical analysis of CD4⁺CD69⁺ T cells. (F) The protein levels of IFN-γ, TNF-α, IL-2 and IL-6 in the supernatants of CD4⁺ T-cell cultures. (G) Measurement of the OCR in CD4⁺ T cells. (H) Measurement of the ECAR in CD4⁺ T cells. (I) Basal and maximal ECAR:OCR ratios.