M2-like macrophages in the fibrotic liver protect mice against lethal insults through conferring apoptosis resistance to hepatocytes

Abstract

Acute injury in the setting of liver fibrosis is an interesting and still unsettled issue. Most recently, several prominent studies have indicated the favourable effects of liver fibrosis against acute insults. Nevertheless, the underlying mechanisms governing this hepatoprotection remain obscure. In the present study, we hypothesized that macrophages and their M1/M2 activation critically involve in the hepatoprotection conferred by liver fibrosis. Our findings demonstrated that liver fibrosis manifested a beneficial role for host survival and apoptosis resistance. Hepatoprotection in the fibrotic liver was tightly related to innate immune tolerance. Macrophages undertook crucial but divergent roles in homeostasis and fibrosis: depleting macrophages in control mice protected from acute insult; conversely, depleting macrophages in fibrotic liver weakened the hepatoprotection and gave rise to exacerbated liver injury upon insult. The contradictory effects of macrophages can be ascribed, to a great extent, to the heterogeneity in macrophage activation. Macrophages in fibrotic mice exhibited M2-preponderant activation, which was not the case in acutely injured liver. Adoptive transfer of M2-like macrophages conferred control mice conspicuous protection against insult. In vitro, M2-polarized macrophages protected hepatocytes against apoptosis. Together, M2-like macrophages in fibrotic liver exert the protective effects against lethal insults through conferring apoptosis resistance to hepatocytes.

Figure 1

Fibrosis protects mice against various lethal hepatic insults. Control and fibrotic (CCl₄, 6 weeks) Balb/c mice were challenged with CCl₄ (3 μl/g), D-GalN (1 mg/g)/LPS (50 ng/g), or APAP (1 mg/g) through intraperitoneal injection. (a) The survival rate of control and fibrotic mice upon CCl₄ challenge (n = 12). (b–d) The hepatic damage in control and fibrotic mice with or without acute insult was assessed by serum ALT and AST levels, H&E staining and apoptosis detection by TUNEL staining (n = 8). Data were expressed as mean ± SEM. (e) The survival rate of control and fibrotic mice subjected to APAP (n = 8) or D-GalN/LPS (n = 10) treatment.

Figure 2

Injury resistance is accompanied by the suppression of pro-inflammatory immune responses. Control and fibrotic (CCl₄, 6 weeks) mice were challenged with lethal doses of CCl₄ (a,c,d) or D-GalN/LPS (b) (details in Methods and Materials). (a and b) mRNA levels of injury mediators, including IL-1β, IL-12, TNF-α, IL-6, MMP-9, and MMP-13, were detected by qRT-PCR analysis. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (c) The MMP-9 activities in the liver tissues and plasma were detected by zymography. (d) The expression and translocation of HMGB1 was analysed by immunohistochemistry.

Figure 3

Macrophages exert dichotomous roles in cases of homeostasis and hepatic fibrosis. Macrophages in the control and fibrotic mice were depleted with liposome-clodronate (Clo) for 48 hours, and the resultant mice were then given a lethal dose of CCl₄ by intraperitoneal injection. Sera and liver tissues were harvested 24 hours after toxin challenge. (a) Hepatic injury was assessed by serum ALT levels. (b and c) The mRNA and protein expressions of injury markers were detected by qRT-PCR and Luminex Assay, respectively. (d) Histological severity of liver injury was assessed and scored blindly by experienced pathologists. (e) Apoptotic cells were identified using TUNEL staining. Data were expressed as mean ± SEM.

Figure 4

Macrophages in the fibrotic liver exhibit an M2-preponderant signature. Control and fibrotic mice were subjected to a lethal dose of CCl₄. Twenty-four hours after acute challenge, macrophages were isolated from the livers by pronase/collagenase digestion and differential centrifugation using Percoll. F4/80 + macrophages were obtained by positive selection using magnetic MicroBeads. (a) qRT-PCR analysis was performed to characterize the phenotypes of macrophages. (b) The ratio of M2/M1 gene expression was calculated. Data were expressed as mean ± SEM. *p < 0.05. (c) The protein expression of iNOS (M1 marker) and CD206 (M2 marker) was analysed by immunofluorescent staining in control and fibrotic livers with or without acute insult.

Figure 5

Adoptive transfer of M2-preponderant macrophages confers resistance to lethal insult. Macrophages isolated from the livers of fibrotic mice were adoptively transferred into control mice by tail vein. Twenty hours later, a lethal dose of CCl₄ was given for an additional 24 hours. (a and b) The gene and protein levels of injury mediators were assessed by qRT-PCR and Luminex Assay, respectively. Data were expressed as mean ± SEM. Liver injury was visualized by H&E staining (c) and TUNEL assay (d).

Figure 6

M2-polarized macrophages confer apoptosis resistance to hepatocytes (in vitro). M0 macrophages isolated from control mice were polarized in vitro by IFN-γ for M1 activation or IL-4/IL-13 for M2 activation. Then, conditioned media (CM) from M0, M1, and M2 macrophages were applied to primary hepatocytes for 6 hours, followed by apoptosis induction for another 12 hours. (a) The mRNA levels of M1 markers (iNOS, TNF-α, IL-1β), M2 markers (ARG-1, FIZZ-1, YM-1, CCL17), and a macrophage marker (CD68) were measured by qRT-PCR analysis. *p < 0.05, **p < 0.01, ***p < 0.001. (b and c) Hepatocyte apoptosis induced by TNF-α/D-GalN was quantitatively analysed by Annexin V-FITC/PI double-labelled flow cytometry. *p < 0.05. Data were expressed as mean ± SEM.