Fibrinogen-like protein 2 aggravates nonalcoholic steatohepatitis via interaction with TLR4, eliciting inflammation in macrophages and inducing hepatic lipid metabolism disorder

Abstract

Rationale: The functions of fibrinogen-like protein 2 (fgl2) have been studied in many inflammatory and neoplastic diseases, but the role of fgl2 in nonalcoholic fatty liver disease has not yet been elucidated. In this study, we sought to investigate the role of fgl2 in the pathogenesis of nonalcoholic steatohepatitis (NASH). Methods: Hepatic fgl2 expression was tested in patients with nonalcoholic fatty liver (NAFL) or NASH and controls. Wild-type and fgl2-/- C57BL/6 mice were subjected to a methionine/choline-deficient (MCD) diet or a high-fat diet (HFD) to establish NASH models. Bone marrow-derived macrophages (BMDMs) stimulated with LPS or free fatty acids were used for the in vitro study. Results: In both humans and mice with NASH, macrophage accumulation was concomitant with significantly increased fgl2 expression in the liver. Fgl2 deficiency attenuated liver steatosis and inflammation in diet-induced murine models of NASH. In both liver tissues and BMDMs from NASH mice, fgl2 deficiency resulted in reduced levels of proinflammatory cytokines and reactive oxygen species (ROS) compared with levels in wild-type controls. Activation of NF-κB, p38-MAPK and NLRP3 inflammasomes was also suppressed upon fgl2 disruption. Moreover, lipogenic genes (Fasn and SREBP-2) were downregulated while lipolytic genes (PPAR and CPT1A) were upregulated in the livers of fgl2-/- NASH mice. Primary hepatocytes incubated with the medium collected from fgl2-/- BMDMs showed less fat deposition than those incubated with WT BMDMs. Furthermore, we discovered that fgl2 combined with TLR4 mediates the activation of the Myd88-dependent signaling pathway, which may contribute to inflammation and lipid metabolism disorders. Conclusions: These data suggest that fgl2 aggravates the progression of NASH through activation of NF-κB, p38-MAPK and NLRP3 inflammasomes in macrophages, which consequently induces overproduction of proinflammatory cytokines and lipid metabolism disorders. An interaction of fgl2 and TLR4 may in part contribute to the activation of inflammatory signaling pathways in macrophages.

Keywords: Fibrinogen-like protein 2; Lipid metabolism; Macrophage; Nonalcoholic steatohepatitis; Toll-like receptor 4.

Figure 1

Fgl2 expression was enhanced concomitantly with hepatic macrophage accumulation in patients with NASH. Liver sections of patients with NAFL and NASH and controls (human subjects without NAFLD) were stained with HE (A). CD68+ macrophages (red) and fgl2 (green) were observed by immunofluorescent staining (C). The NAFLD active score was evaluated in controls (n=6), NAFL (n=6) and NASH (n=8) human subjects (B). The fluorescence intensity of fgl2-, CD68- and fgl2-positive macrophages (fgl2 in CD68+) was evaluated by ImageJ software (C). Correlations between fgl2 expression in liver/hepatic macrophages and NAFLD activity score were also analyzed (D). Five microscopic fields per liver section from 3 patients in each group were counted. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by one-way ANOVA with Bonferroni correction and Spearman's rank correlation coefficient analysis was applied to analyze the correlation between fgl2 expression levels and NAFLD activity score. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 2

The accumulated hepatic macrophages showed increased expression of fgl2 in the livers of NASH mice. Mice were subjected to an MCD diet (A) for 6 weeks and HFD (B) for 24 weeks to establish NASH models. The MCS diet (A) and chow diet (B) were used as controls. Mice were sacrificed at the indicated time points. Hepatic fgl2 expression was analyzed by western blotting (A, B). For bar graphs, n=6-10 in each group. F4/80+ macrophages (red) and fgl2 (green) were observed by immunofluorescent staining (C). Five microscopic fields per liver section from 3 animals in each group were counted. The data represent the mean ± SD from at least three independent experiments. For multiple group comparisons, significant differences were determined by one-way ANOVA with Bonferroni correction. Differences between two experimental groups were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 3

Fgl2 deficiency attenuated liver inflammatory injury in NASH mice. In MCD-fed or HFD-fed WT and fgl2-/- mice, HE staining was performed to detect histological changes in the liver (A). The NAFLD activity score was evaluated (B). BMDMs were isolated from WT or fgl2-/- mice and injected into macrophage-depleted WT NASH mice (C). Histological changes were detected by HE staining (D, arrows indicate inflammatory infiltration). Serum ALT, AST, LDH (E) and fasting glucose (F) were tested by an automatic biochemical analyzer (n=10 in each group). The levels of serum insulin were tested by an ELISA kit (F). The levels of the proinflammatory cytokines TNF-α, MCP-1, IL-6, IL-1β and IL-18 in the liver were tested by ELISAs (G). For bar graphs, n=6-10 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 4

Fgl2 deficiency ameliorated liver steatosis in HFD-induced NASH by regulating lipid metabolism. In MCD-fed (A) or HFD-fed (B) WT and fgl2-/-mice, oil red O staining was performed to detect liver steatosis. The levels of cholesterol and triglycerides in the liver were examined (C, D). Hepatic mRNA levels of genes involved in lipogenesis (Fasn, SREBP-2) (E, F) or lipolysis (PPARα, CPT1A) (G, H) were tested by real-time PCR. For bar graphs, n=8 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 5

Fgl2 deficiency reduced lipid accumulation in hepatocytes by inhibiting the secretion of proinflammatory cytokines in macrophages. BMDMs from WT and fgl2-/- mice were stimulated with LPS (100 ng/ml) or FFA (800 μmol/L). The levels of proinflammatory cytokines, including TNF-α, MCP-1, IL-6, IL-1β and IL-18, in the supernatant of cell cultures were tested by ELISAs (A). Primary hepatocytes were isolated from C57BL/6J mouse livers and incubated with LPS- or FFA-BMDM-CM for 24 hours. The brief experimental procedure is shown in a diagram. Oil red O staining was used to detect fat deposition in primary hepatocytes after treatment with BMDM-CM (B). Then, the mRNA levels of genes involved in lipogenesis (Fasn, SREBP-2) (C) or lipolysis (PPARα, CPT1A) (D) in primary hepatocytes were tested by real-time PCR. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 6

Fgl2 disruption suppressed activation of the NF-κB and p38-MAPK signaling pathways in NASH. For in vivo examination, the total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NF-κB-p65, p38-MAPK and their phosphorylated forms were analyzed by western blotting (n=6) (A). In vitro, BMDMs from WT or fgl2-/- mice were stimulated with LPS or FFA and tested for the normal and phosphorylated levels of NF-κB and p38-MAPK by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 7

Fgl2 disruption inhibited activation of the NLRP3 inflammasome in NASH. Total protein was obtained from liver tissues of MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls. NLRP3, pro-caspase-1, cleaved caspase-1 (caspase-1 p10), pro-IL-1β, mature IL-1β and IL-18 were analyzed by western blotting (A). BMDMs from WT and fgl2-/- mice were stimulated with LPS or FFA and tested for inflammasomes by western blotting (B). Image density was quantified using ImageLab software. For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 8

Fgl2 interacted with TLR4 on macrophages and activated the MyD88-dependent signaling pathway in NASH. The expression of TLR4, MyD88 and TRAF6 in liver tissues was tested by western blotting in MCD-fed or HFD-fed WT and fgl2-/- mice. MCS-fed and chow-fed mice were used as controls (A). The expression of TLR4 on F4/80+ hepatic macrophages was detected by flow cytometry (B). Fgl2 was overexpressed in THP-1 cells by infection with HBLV-h-Fgl2-GFP-PURO. HBLV-GFP-PURO was used as a control. Then, the expression of fgl2, TLR4, MyD88 and TRAF6 was detected (C). Coimmunoprecipitation (CoIP) of endogenous TLR4 and fgl2 was performed in differentiated THP-1 cells stimulated by LPS or FFA. Protein extracts were immunoprecipitated with an antibody against fgl2 or TLR4, followed by immunoblotting with the indicated antibodies (bottom panel, D). Fgl2 and TLR4 were also detected in total cell lysates (top panel, D). For bar graphs, n=6 in each group. The data represent the mean ± SD from at least three independent experiments. Statistical differences were determined by one-way ANOVA with Bonferroni correction or two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Figure 9

Fgl2 contributes to the progression of NASH. In macrophages stressed by LPS, FFA or other stimulators in NAFLD, fgl2 induces ROS production and activates NF-κB and p38-MAPK signaling pathways and the NLRP3 inflammasome. An interaction of fgl2 and TLR4 and subsequent activation of the TLR4-MyD88-TRAF6 axis may be involved in this process. Proinflammatory cytokines such as TNF-α, IL-6, MCP-1, IL-1β and IL-18 are secreted to induce liver injury and lipid metabolism disorders. TNF-α and IL-6 promote the expression of SREBP-2 or Fasn, which are involved in the synthesis of cholesterol, triglycerides and fatty acids. TNF-α and IL-1β downregulate the expression of PPARα and CPT1A, which contribute to lipolysis. These changes result in more significant liver steatosis and further aggravate hepatic inflammatory injury, leading to the progression of NASH.