3.1.6. TLR9

In humans, hepatic TLR9 expression is upregulated in NASH patients [100,133], while high serum levels of bacterial DNA (TLR9 ligand) have also been linked with liver cirrhosis and liver fibrosis [134,135]. Indeed, an increase in circulating bacterial DNA has been measured in patients with alcoholic and fatty liver disease prior to fibrotic development [136]. In addition, acute binge drinking has been shown to significantly increase serum bacterial DNA and pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β to indirectly contribute to liver fibrosis [135,139,157].

The role of TLR9 in chronic liver inflammation has been well established in animal models. In mouse models of CDAA-induced NASH, activation of TLR9 can stimulate IL-1β expression in KCs, mediating steatohepatitis and hepatocyte apoptosis driven by lipid accumulation [137,138]. In addition, the pro-inflammatory role of TLR9 in NASH has been further confirmed in atherogenic diet-fed NASH and *foz* mouse models, demonstrating reduced pro-inflammatory cytokine production and attenuated hepatic neutrophil infiltration in TLR9−/<sup>−</sup> mice [133]. Similarly, evidence for TLR9-mediated liver injury and inflammation in chronic-binge-ethanol-fed mice was shown to be driven by IL-1β expression as well as TLR9/TLR2-dependent hepatic neutrophil infiltration mediated by CXCL1/2/5 expressed from hepatocytes and HSC [102,139].

Despite the inflammatory role of TLR9 in human NASH, TLR9 KO failed to improve hepatic fibrosis in murine NASH models [133]. TLR9 does, however, seem to influence fibrosis development in other models of fibrosis including murine BDL, where TLR9−/<sup>−</sup> mice demonstrated significantly alleviated fibrogenesis and HSC activation compared to WT mice [140]. Similar results were found using the spontaneous fibrosis Tak1ΔHep mouse model, also demonstrating a reduction in liver inflammation and fibrosis in TLR9 KO mice [124]. Moreover, stimulation of TLR9 using CpG DNA directly activated the fibrogenic phenotype in primary mouse HSCs and the immortalized human HSC LX-2 cell line [158].
