Functional Linkage of Cirrhosis-Predictive Single Nucleotide Polymorphisms of Toll-like Receptor 4 to Hepatic Stellate Cell Responses

In a recent study, a single nucleotide polymorphism (SNP) of the Toll-like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild-type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4؊/؊ mice and a human HSC line (LX-2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hy- poresponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS-induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein-1, interleukin-6), down- regulation of bone morphogenic protein and the activin membrane-bound inhibitor expression (an inhibitory transforming growth factor ␤ pseudoreceptor), and activation of a nuclear factor ␬B (NF-␬B)–responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF-␬B, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase were greatly increased in HSCs from either TLR4؊/؊ or myeloid differentiation factor 88؊/؊ (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; in- creased apoptosis in these lines was accompanied by decreased phospho-ERK and Bcl-2. Conclusion: TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4-mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression.