Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis

Abstract

Background/aims: Studies in animal models and humans suggest a link between endotoxemia and non-alcoholic steatohepatitis. Since Kupffer cells are responsible for clearing endotoxin and are activated via endotoxin interaction with Toll-like receptor 4 (TLR-4), we examined the relationship between hepatic TLR-4 expression and Kupffer cell content during the genesis of steatohepatitis.

Methods: Male C57BL/6, C3H/HouJ and TLR-4 mutant C3H/HeJ mice were fed control or methionine/choline-deficient diet (MCDD). In one group of C57BL/6 mice, Kupffer cells were depleted by weekly intraperitoneal injections of clodronate liposomes. After 3 weeks, serum ALT activity and portal endotoxin levels were measured. Real-time PCR was used to examine mRNA expression of TLR-4, TLR-2, CD14, MD-2, TGFbeta, TNFalpha, CD36, PPAR-alpha, liver fatty acid binding protein (L-FABP) and collagen alpha1.

Results: We observed histological evidence typical of steatohepatitis, portal endotoxemia and enhanced TLR-4 expression in wild type mice fed MCDD. In contrast, injury and lipid accumulation markers were significantly lower in TLR-4 mutant mice. Destruction of Kupffer cells with clodronate liposomes blunted histological evidence of steatohepatitis and prevented increases in TLR-4 expression.

Conclusions: These findings demonstrate the importance of TLR-4 signaling and underscore a direct link between TLR-4 and Kupffer cells in the pathogenesis of steatohepatitis.

Figure 1. Effect of feeding MCDD on TLR-4 signaling molecules

(A) For assessment of TLR-4, MD2 and CD14 expression, livers were collected from male C57BL/6 mice fed CD or MCDD for 3 weeks. Pre-developed assays for real-time PCR were used according to the manufacturer’s instructions (Applied Biosystems). Expression of each target mRNA was calculated relative to average values in the control group using a comparative CT method and presented as mean ± SEM of at least 4 observations/group. (B) Western blot analysis of hepatic TLR-4 expression relative to β-actin was used to confirm enhanced protein levels. Values are mean ± SEM of at least 4 observations per group. *p< 0.05, **p< 0.005 compared to control using Student’s t test.

Figure 2. Hepatic Histopathology

Representative H&E-stained photomicrographs of (A) C3H/HouJ + CD; (B)) C3H/HeJ + CD (C)) C3H/HouJ + MCDD (D) C3H/HeJ + MCDD; endotoxin sensitive (left panels) mice fed MCDD display obvious NASH. Injury was clearly diminished in endotoxin resistant mice (right panels). Additional sections of liver from mice fed MCDD were subjected to immunohistochemical staining for neutrophils (E&F) and Kupffer cells (F4/80; G&H). The appearance of brown staining indicates the presence of these cells in response to MCDD.

Figure 3. Hepatic triglyceride content and serum ALT activity

Serum levels of the liver-specific injury marker ALT (A) and triglyceride levels (B) in 20% liver homogenates were measured using standard spectrophotometric assays. Statistical comparisons were made using two-way ANOVA. *p< 0.05 compared to PBS controls or ^#diet-matched C3H/HouJ mice.

Figure 4. Hepatic PPAR-αand L-FABP expression

Expression of PPAR-α and L-FABP mRNA was analyzed by real-time PCR and calculated relative to average values in the control group using a comparative CT method. Data are presented as mean ± SEM of 5 observations/group. Statistical comparisons were made using two-way ANOVA. *p< 0.05 compared to PBS controls or ^#diet-matched C3H/HouJ mice.

Figure 5. TNF-α and Collagen mRNA in C3H/HeJ mice

Expression of TNF-α and Collagen α1 mRNA was analyzed by real-time PCR and calculated relative to average values in the control group using a comparative CT method. Data are presented as mean ± SEM of 5 observations/group. Statistical comparisons were made using two-way ANOVA. *p< 0.05 compared to PBS controls or ^#diet-matched C3H/HouJ mice.

Figure 6. Effect of clodronate liposomes on Kupffer cells

To confirm destruction of Kupffer cells, livers of mice fed control diet or MCDD were preserved in zinc fixative and subjected to immunohistochemical staining with primary antibodies directed against macrophages (F4/80). Hematoxylin was used as a counter stain. Positive cells are stained brown. Photomicrographs are representative of 6 mice/dietary group.

Figure 7. Hepatic expression of (A) TNF-α, (B) TGF-β and (C) Collagen

Livers were collected from male C57BL/6 mice fed control (CD) or methionine/choline deficient (MCDD) for 3 weeks. In addition, mice were administered clodronate liposomes (200 µg/mouse) to destroy Kupffer cells or PBS (phosphate-buffered saline) as a vehicle control as described in Methods. Pre-developed assays for real-time PCR were used to assess target mRNA levels. Statistical comparisons were made using two-way ANOVA (n= 5/group). *p< 0.05 compared to mice fed control diet; ^#diet-matched PBS (phosphate-buffered saline) controls.

Figure 8. Effect of Kupffer cell destruction on toll-like receptor expression

After 3 weeks of feeding MCDD the effect of Kupffer cell destruction on expression of TLR-4, CD36 and TLR-2 was examined using real-time PCR. Statistical comparisons were made using two-way ANOVA (n= 5/group). *p< 0.05 compared to mice fed control diet or ^#p< 0.05 compared to diet-matched PBS (phosphate-buffered saline) controls.