GDF15 deficiency exacerbates chronic alcohol- and carbon tetrachloride-induced liver injury

Abstract

Growth differentiation factor 15 (GDF15) has recently been shown to have an important role in the regulation of mitochondrial function and in the pathogenesis of complex human diseases. Nevertheless, the role of GDF15 in alcohol-induced or fibrotic liver diseases has yet to be determined. In this study, we demonstrate that alcohol- or carbon tetrachloride (CCl₄)-mediated hepatic GDF15 production ameliorates liver inflammation and fibrosis. Alcohol directly enhanced GDF15 expression in primary hepatocytes, which led to increased oxygen consumption. Moreover, GDF15 reduced the expression of pro-inflammatory cytokines in liver-resident macrophages, leading to an improvement in inflammation and fibrosis in the liver. GDF15 knockout (KO) mice had more TNF-α-producing T cells and more activated CD4⁺ and CD8⁺ T cells in the liver than wild-type mice. Liver-infiltrating monocytes and neutrophils were also increased in the GDF15 KO mice during liver fibrogenesis. These changes in hepatic immune cells were associated with increased tissue inflammation and fibrosis. Finally, recombinant GDF15 decreased the expression of pro-inflammatory cytokines and fibrotic mediators and prevented the activation of T cells in the livers of mice with CCl₄-induced liver fibrosis. These results suggest that GDF15 could be a potential therapeutic target for the treatment of alcohol-induced and fibrotic liver diseases.

Figure 1

Treatment with alcohol induces GDF15 production in primary murine hepatocytes. (a) Gdf15 expression in liver tissue, hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs). (b) Real-time PCR analysis of primary hepatocytes treated with vehicle or alcohol. (c) Levels of GDF15 in culture supernatants of primary hepatocytes treated with vehicle or alcohol. (d) Gdf15 expression in D1 HSCs and D7 HSCs (cultured for 1 day and 7 days, respectively). (e) Gdf15 expression in D1 HSCs treated with alcohol. (f) Real-time PCR analysis of primary hepatocytes treated with vehicle, oligomycin, or rotenone. (g) Levels of GDF15 in culture supernatants of primary hepatocytes treated with vehicle, oligomycin, or rotenone. (h,i) Body weight gain and dietary intake of mice fed a control or alcohol liquid diet for 6 weeks (n = 5/group). (j,k) Gdf15 mRNA and GDF15 protein expression in the livers of WT and GDF15 KO (GKO) mice fed a control or alcohol liquid diet for 6 weeks. Relative quantification of each protein was done by densitometry. (l) Serum levels of GDF15 in WT or GKO mice fed vehicle or an alcohol liquid diet for 6 weeks. All data are representative of three independent experiments and are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, versus the corresponding controls.

Figure 2

Recombinant GDF15 enhances oxygen consumption in hepatocytes and inhibits the expression of pro-inflammatory cytokines in Kupffer cells. (a,b) Oxygen consumption rates were measured in primary murine hepatocytes treated with vehicle or recombinant GDF15 (rGDF15, 100 or 200 ng/mL). Oligomycin (2 µg/mL) and rotenone (1 µM) were used as mitochondrial OXPHOS inhibitors. CCCP (5 µM), a mitochondrial uncoupler, was used to measure maximal mitochondrial respiration. (c) Real-time PCR analysis of Kupffer cells treated with lipopolysaccharide (LPS; 10 ng/mL) and/or rGDF15 (50 or 100 ng/mL). (d) Schematic description of co-culturing Kupffer cells with hepatocytes. (e) Real-time PCR analysis of Kupffer cells co-cultured with WT or GDF15 KO hepatocytes in medium containing lipopolysaccharide (10 ng/mL). (f) Real-time PCR analysis of HSCs co-cultured with WT or GDF15 KO hepatocytes in medium containing lipopolysaccharide (10 ng/mL) for 3 hours. All data are representative of three independent experiments and are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, versus the corresponding controls.

Figure 3

GDF15 protects chronic alcohol-fed mice from liver injury and fat deposition. After 6 weeks of feeding with a control or alcohol liquid diet, WT and GDF15 KO (GKO) mice (n = 6/group) were sacrificed. (a) Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured. (b) Triglyceride content in the liver of the mice was measured. (c) Hematoxylin-eosin (H&E) and Oil Red O staining of liver sections (original magnification, × 200; bars, 100 μm). (d) Serum levels of TNF-α and IL-6. (e) Real-time PCR analysis of hepatic Tnf-α, Il1b, Il-6, and Ccl2. All data are representative of three independent experiments and are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, versus the corresponding controls.

Figure 4

GDF15 deficiency exacerbates CCl₄-induced liver fibrosis. (a) Serum levels of AST and ALT. (b) Serum levels of TNF-α. (c) H&E and Sirius Red staining of liver sections (original magnification, ×200; bars, 100 μm). (d) Real-time PCR analysis of hepatic Tnf-α, Acta2, and Col1a1 of the liver from the mice. All data are representative of three independent experiments and are expressed as the mean ± SEM. *P < 0.05, versus the corresponding controls.

Figure 5

GDF15 suppresses activation of hepatic immune cells during CCl₄-induced liver fibrogenesis. (a,b) Percentages of hepatic CD4⁺ and CD8⁺ T cells in WT and GDF15 KO (GKO) mice with liver fibrosis. (c–e) Percentages of hepatic CD4⁺CD44⁺ and CD8⁺CD44⁺ T cells in WT and GDF15 KO mice with liver fibrosis. (f–h) Percentages of hepatic monocytes (CD11b⁺Ly6C^high) and neutrophils (CD11b⁺Ly6G⁺) in WT and GDF15 KO mice with liver fibrosis. (i–l) Intracellular staining for TNF-α and IL-17A in hepatic CD4⁺ and CD8⁺ T cells. All data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, versus the corresponding controls.

Figure 6

Recombinant GDF15 ameliorates CCl₄-induced liver inflammation and fibrosis. WT and GDF15 KO (GKO) mice (n = 5/group) were treated with CCl₄ (2 mL/kg in olive oil, 20% v/v) and recombinant GDF15 (rGDF15, 0.5 mg/kg). (a) H&E and Sirius Red staining of liver sections (original magnification, × 200; bars, 100 μm). (b) Serum levels of AST and ALT. (c) Serum levels of TNF-α and IL-6. (d) Real-time PCR analysis of hepatic Gdf15, Tnf-α, Acta2, and Col1a1. (e) Western blots of liver tissue. (f) Percentages of hepatic CD4⁺CD44⁺ and CD8⁺CD44⁺ T cells in GDF15 KO mice treated with vehicle or rGDF15. (g) Percentages of hepatic monocytes (CD11b⁺Ly6C^high) and neutrophils (CD11b⁺Ly6G⁺) in GDF15 KO mice treated with vehicle or rGDF15. (h) Intracellular staining for TNF-α in CD8⁺ T cells in the livers of GDF15 KO mice treated with vehicle or rGDF15. All data are expressed as the mean ± SEM. **P < 0.01, versus the corresponding controls.

Figure 7

Graphical summary. Alcohol and CCl₄ increase GDF15 production in hepatocytes. During alcohol-induced and fibrotic liver injury, GDF15 decreases hepatic inflammation and fibrosis via regulation of liver immune cells.