Seki E, De Minicis S, Österreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF. TLR4 enhances TGF-β signaling and hepatic fibrosis. Nature Med 2007;13:1324-1332. Available at: www.nature.com (Reprinted with permission.)
Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-β pseudoreceptor Bambi to sensitize HSCs to TGF-β–induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-β is mediated by a MyD88–NF-κB–dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-β signaling by a TLR4-MyD88–NF-κB axis provides a novel link between proinflammatory and profibrogenic signals.
The liver is an exceptional organ in terms of its multiple functions. It has the unique potential to regenerate after tissue loss and, for example, plays an important role in the regulation process that keeps blood glucose stable. These and many other functions represent the organ's ability to react to molecular signals and to execute the proper reaction toward the body's demands. Therefore, the liver appears to be a preferred source for and target of cytokine signaling; at present, one can only imagine the incredible complexity of the network connecting cytokines, receptors, and signaling pathways in the liver. Dysregulation of certain pathways plays a key role underlying the pathogenesis of liver disease. In terms of chronic liver injury, several studies have highlighted the role of transforming growth factor beta (TGF-β) in activating hepatic stellate cells (HSCs), the main producers of extracellular matrix in the fibrotic liver, and the promotion of a fibrogenic phenotype.1, 2 On the other hand, chronic liver inflammation is a key prerequisite for triggering liver fibrosis. Inflammatory signaling pathways and innate immune responses modulate the development of hepatitis and the progression toward hepatocellular carcinoma.3, 4 However, until now the cell-type–specific molecular mechanisms linking signaling pathways driving inflammation and innate immune responses on one hand and liver fibrogenesis on the other hand have not been defined yet.
The liver, due to its anatomic location, is primarily confronted with gut-derived bacterial products, including lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria. LPS and other microbiological molecules are sensed by the so-called Toll-like receptors (TLRs), currently consisting of 10 members in humans divided into 5 subfamilies, which coordinate defense mechanisms against these pathogens.5 TLR4 acts as a receptor for LPS and mediates its intracellular actions via the adapter molecule MyD88. Despite the constant confrontation with gut-derived bacterial products, the normal liver does not show signs of inflammation, which on one hand can be explained by the relatively low expression of TLRs and their adaptor molecules in the liver in comparison with other organs.6 On the other hand, hepatocyte-specific deletion of the nuclear factor κB (NF-κB) activating kinase subunit NEMO results in spontaneous liver inflammation, triggered by activation of hepatic Kupffer cells—the liver resident macrophages—by gut-derived bacterial products,4 which shows that intact NF-κB in hepatocytes prevents chronic liver injury from gut derived bacterial endotoxins. Previous studies in animals have supported an important role of TLR4-dependent signaling in liver pathology. Experiments in MyD88 knockout mice demonstrated that MyD88 is required for induction of liver injury in response to hypoxia and LPS.7, 8 Moreover, a recent publication showed that MyD88 is involved in mediating liver cancer upon administration of the chemical carcinogen diethylnitrosamine.9 This effect was attributed to the MyD88-dependent regulation of interleukin-6 production in hepatic Kupffer cells, highlighting again that hepatic Kupffer cells might be primary mediators of TLR-dependent signaling in the liver.
The groups of Robert Schwabe and David Brenner, who already in the past contributed major findings to the understanding of molecular mechanisms underlying the development of liver fibrosis, have published a study in which they demonstrated that TRL4-dependent signaling is an important mediator of liver fibrosis.10 Using a genetic approach, they demonstrated that TLR4 knockout mice displayed significantly less liver fibrosis upon bile duct ligation compared to wild-type controls. Although these mice showed the same level of liver injury, recruitment of macrophages to the liver was significantly reduced in these mice, coinciding with reduced expression of Ccl2 and Ccl4, two potent mediators of macrophage chemotaxis. Moreover, treatment of mice with nonabsorbable broad-spectrum antibiotics also resulted in a clear reduction in the fibrotic response of mice, supporting the hypothesis that gut-derived bacterial products are the natural ligands activating hepatic TLR4 and aggravating the hepatic fibrogenic response.
To analyze the cell-specific molecular mechanism underlying this phenotype, the authors performed elegant experiments using a combination of clodronate-mediated Kupffer cell depletion, irradiation, and bone-marrow transplantation to generate TLR4-chimeric mice. Surprisingly, these experiments revealed that bone-marrow–derived cells, including Kupffer cells, are not the primary cells mediating hepatic fibrosis in response to TLR4 ligands. Instead, they could demonstrate that HSCs express high levels of TLR4 in the nonactivated and activated state, and that LPS directly activates TLR4-dependent pathways and leads to expression of TLR4-dependent genes in HSCs but not in hepatocytes, among them genes encoding chemokines and adhesion molecules. The authors provide evidence that reduced expression of chemokines by HSCs is a causative event for decreased Kupffer cell chemotaxis in livers of TLR4 knockout mice, demonstrating that LPS mediates its fibrogenic effects primarily through HSCs (Fig. 1).
In further experiments, the authors describe a surprising molecular link between TRL4 and the TGF-β pathway. They demonstrate that LPS stimulation alone is not sufficient to activate HSCs to a myofibroblast-like phenotype, but that pretreatment with LPS strongly enhanced their response to the profibrogenic cytokine TGF-β. Because hepatic Kupffer cells are a major source of TGF-β in the fibrotic liver, increased TLR4-dependent expression of chemokines, increased chemotaxis of macrophages, and a subsequent increase in Kupffer-cell-dependent TGF-β excretion is one likely mechanism underlying increased fibrosis in wild-type mice compared to TLR4-mutant animals. On the other hand, the authors performed microarray analysis, quantitative polymerase chain reaction analysis, and immunoblotting and identified strong down-regulation of the TGF-β pseudoreceptor Bambi in HSCs in a TLR4-dependent manner. Overexpression of Bambi by adenoviral vectors prevented activation of HSCs, whereas a dominant negative form of Bambi led to strong susceptibility of HSCs to TGF-β signaling, suggesting that next to increased chemotaxis of Kupffer cells, down-regulation of Bambi in HSCs is an independent but complementary mechanism by which TLR4 enhances HSC activation and hepatic fibrosis. Finally, by using adenoviral vectors expressing an inhibitor of NFκB kinase (IκB)-superrepressor and knockout mice for MyD88 and the adapter molecule Trif, the authors demonstrate that TLR4-dependent down-regulation of Bambi is mediated via a pathway involving MyD88 and NF-κB, but not Trif.
The results presented by Seki and co-workers have far-ranging implications for the current concept of molecular mechanisms underlying fibrogenesis in the liver. Independently of the causative agent, chronic hepatitis results in hepatic fibrosis and finally cirrhosis if not successfully treated, but up to now the molecular link between proinflammatory and fibrogenic pathways in the liver was missing. The authors demonstrate that hepatic stellate cells represent the primary liver cell compartment integrating these signals. They demonstrate that LPS acts in a profibrogenic manner via two independent mechanisms: It induces the secretion of chemokines from HSCs and chemotaxis of Kupffer cells which secrete the profibrogenic cytokine TGF-β. Additionally, TLR4-dependent signals augment TGF-β signaling on an intracellular level via down-regulation of the TGF-β pseudoreceptor Bambi (Fig. 1).
The fact that overexpression of dominant negative Bambi in livers of mice resulted in increased fibrogenesis even without any other fibrogenic stimulus10 underlines the crucial function and the potential of this molecule as a target for future molecular therapies against hepatic fibrosis. This strategy might be more specific and linked with lower risks than, for example, molecular inhibition of TLR4 signaling, which in Kupffer cells might play an essential role in the clearance of systemic endotoxins and bacterial infection.6, 11, 12 Finally, given the rising importance of probiotics, for example, in the treatment of inflammatory bowel disease,13 the findings presented by Seki et al. provide a molecular explanation for the hypothesis that modification of the intestinal microflora by antibiotics or probiotics might be a useful strategy in the treatment of liver cirrhosis14 and the prevention of fibrogenesis in the chronically inflamed liver.