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

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, TGFβ, TNFα, CD36, PPAR-α, liver fatty acid binding protein (L-FABP) and collagen α1.

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.

Introduction

Non-alcoholic steatohepatitis (NASH) in the setting of obesity is believed to occur via a “two-hit” mechanism involving hepatic fat accumulation and oxidative stress. Studies in animal models suggest that gut-derived endotoxin mediates oxidative stress and the development of NASH. In support of the idea that gut-derived bacterial products play a causal role in the pathogenesis of NASH in genetically overweight mice, treatment with probiotic dietary supplements that sterilized the gut of endotoxin-bearing Gram-negative organisms prevented histological alterations and insulin resistance associated with steatohepatitis [1], [2], [3]. In humans, severe steatohepatitis often occurs following jejuno-ileal bypass and in patients placed on total parenteral nutrition, both of which are situations believed to cause bacterial overgrowth and endotoxemia. In fact, Wigg et al. evaluated 22 patients with NASH for small bowel bacterial content and found that bacterial overgrowth was prevalent among NASH patients [4]. In a study by Vanderhoof et al. liver injury and steatosis in a rodent model of jejuno-ileal bypass were prevented by therapies that minimized bacterial load [5]. Thus, leakage of endotoxin from the gut is a likely stimulus of NASH.

Since blood leaving the gut empties directly into the portal vein, the liver is exposed to gut-derived endotoxin. As a result of endotoxemia, Kupffer cells, the resident hepatic macrophages, are activated via the Toll-like receptor 4 complex (TLR-4) on the cell surface. This receptor is a member of the Toll-like family of pattern recognition receptors that are of central importance during host defense against invading pathogens. TLR-4 interaction with endotoxin results in the release of a myriad of pro-inflammatory mediators that induce hepatic injury and fibrosis [6]. In addition, cytokines have profound effects on lipid metabolism [7].

The present study investigated the potential importance of Kupffer cells and TLR-4 in the pathogenic mechanisms underlying NASH. Accordingly, mice were fed a methionine and choline deficient diet (MCDD), which results in hepatic microvascular dysfunction and pronounced pathological changes within 3–4 weeks [8], [9], [10]. Administration of this diet in mice depleted of Kupffer cells via treatment with liposome encapsulated clodronate as well as in mice deficient in TLR-4 signaling due to a spontaneous mutation (i.e. C3H/HeJ mice) provides evidence that TLR-4 expression on Kupffer cells is a critical component of diet-induced NASH.