M2-like Kupffer cells in fibrotic liver may protect against acute insult

Abstract

Aim: To investigate the mechanism of hepatoprotection conferred by liver fibrosis through evaluating the activation phenotype of kupffer cells.

Methods: Control and fibrotic mice were challenged with a lethal dose of D-GalN/lipopolysaccharide (LPS), and hepatic damage was assessed by histology, serum alanine transferase (ALT) levels, and hepatic expression of HMGB1, a potent pro-inflammatory mediator. The localization of F4/80 (a surrogate marker of KCs), HMGB1, and type I collagen (Col-1) was determined by immunofluorescence staining. The phenotype of KCs was characterized by real-time PCR. KCs isolated from control or fibrotic mice were challenged with LPS or HMGB1 peptide, and HMGB1 translocation was analyzed.

Results: Liver fibrosis protected mice against D-GalN/LPS challenge, as shown by improved hepatic histology and reduced elevation of ALT compared with the normal mice treated in the same way. This hepatoprotection was also accompanied by inhibition of HMGB1 expression in the liver. Co-localization of F4/80, HMGB1, and Col-1 was found in fibrotic livers, indicating the close relationship between KCs, HMGB1 and liver fibrosis. KCs isolated from fibrotic mice predominantly exhibited an M2-like phenotype. In vitro experiments showed that HMGB1 was localized in the nucleus of the majority of M2-like KCs and that the translocation of HMGB1 was inhibited following stimulation with LPS or HMGB1 peptide, while both LPS and HMGB1 peptide elicited translocation of intranuclear HMGB1 in KCs isolated from the control mice.

Conclusion: M2-like Kupffer cells in fibrotic liver may exert a protective effect against acute insult by inhibiting the translocation of HMGB1.

Keywords: Injury resistance; Kupffer cell activation; Liver fibrosis; Translocation; high-mobility group box 1.

Figure 1

Inhibition of High mobility group box 1 expression is closely associated with the injury resistance in the setting of liver fibrosis. Control and fibrotic mice (treated with CCl₄ for 6 wk) were challenged with a lethal dose of D-GalN (1 mg/g)/LPS (50 ng/g), and hepatic damage was assessed by histology (A: HE staining; original magnification, × 200) and serum ALT levels (B). ^aP < 0.05 vs the control group, ^bP < 0.05 vs the fibrosis group, ^cP < 0.05 vs the fibrosis + D-GalN/LPS group. The expression of HMGB1 was determined by immunohistochemical staining (C: original magnification, × 200). Data are expressed as mean ± SEM. CCl₄: Carbon tetrachloride; D-GalN: D-galactosamine; HMGB1: High mobility group box 1; LPS: Lipopolysaccharide.

Figure 2

Kupffer cells may be involved in High mobility group box 1-mediated injury resistance. The expression and localization of F4/80 (a surrogate marker of KCs), HMGB1, and Col-1 were determined by immunofluorescence staining (original magnification, × 100). Col-1: Type I collagen; HMGB1: High mobility group box 1; KCs: Kupffer cells.

Figure 3

Kupffer cells in the fibrotic liver exhibit predominantly a M2-like activation. KCs were isolated from the livers of normal, acutely injured (D-GalN/LPS) and fibrotic mice, and M1 and M2 gene signatures were then determined by quantitative real-time PCR. Data are expressed as mean ± SEM. ^aP < 0.05 vs the control group, ^bP < 0.05 vs the D-GalN/LPS group, ^cP < 0.05 vs the control group, ^dP < 0.05 vs the fibrosis group. D-GalN: D-galactosamine; KCs: Kupffer cells; LPS: Lipopolysaccharide.

Figure 4

Translocation of High mobility group box 1 triggered by lipopolysaccharide or High mobility group box 1 peptide is inhibited in M2-like Kupffer cells. KCs were isolated from the livers of control and fibrotic mice, and then treated with LPS (10 ng/mL) or HMGB1 peptide (30 μg/mL). The translocation of HMGB1 in KCs was analyzed by immunofluorescence staining (original magnification, × 200). HMGB1: High mobility group box 1; KCs: Kupffer cells; LPS: Lipopolysaccharide.