Meccanismi di fibrogenesi epatica associata ad epatite HCV-correlata ed a steatosi non alcolica

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection and non-alcoholic fatty liver disease (NAFLD), the localization and level of TLR4 expression in the liver of patients with both diseases have never been investigated. Moreover, during NAFLD, portal and interface chronic inflammation (PCI and ICI) are strongly associated with fibrosis, but the determinants subtending this association still remain to be elucidated. Therefore, in the present study, we aimed: 1) to evaluate, by means of immunohistochemistry and real time polymerase chain reaction (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection and NAFLD; 2) to investigate if PCI and ICI observed in NAFLD are associated with hepatic progenitor cell compartment activation. Fifty patients who had undergone liver biopsy and 11 patients transplanted due to chronic HCV infection, 55 patients who had undergone liver biopsy and received histological diagnosis of NAFLD, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, –smooth muscle actin and cytokeratin-7, and a subgroup of patients with chronic hepatitis C and all controls by rt-PCR for TLR4. Immunohistochemistry for –smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for cytokeratin-7 to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients with chronic HCV-related hepatitis, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti- cytokeratin-7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (p<0.001), the degree of inflammation (p<0.001), and the activity of portal/septal myofibroblasts (p<0.001). Moreover, rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (p<0.001). In these 26 patients, TLR4 gene expression positively correlated with fibrosis (p<0.05) and inflammation (p<0.05). Also in patients with NAFLD, hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts showed the highest TLR4 level of expression by immunohistochemistry. However, a marked anti-TLR4 positivity was frequently observed also in hepatocytes and in inflammatory cells. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of fibrosis (p<0.001), the activity of portal/septal myofibroblasts (p<0.001), the activity of hepatic stellate cells (p<0.001), and the degree of portal (p<0.001) and interface inflammation (p<0.01). While the score of hepatocyte TLR4-positivity did not show any significant correlation, the score of TLR4 positivity of portal inflammatory infiltrate showed significant correlations with ballooning (p<0.01), fibrosis (p<0.001), activity of portal/septal myofibroblast (p<0.001) and activity of hepatic stellate cells (p<0.01). PCI and ICI strongly correlated with HPC compartment activation and with the activity of portal/septal myofibroblasts (p0.001). Lobular inflammation, ballooning and HPC compartment activation were all associated with both PCI (p<0.01) and ICI (p<0.05) by univariate analysis. In the multivariate models, HPC compartment activation was independently associated with PCI and ICI (OR 4.4, 1.7-11.5; OR 3.4, 1.5-7.9, respectively). The present results suggest that TLR4 expression by hepatic progenitor cells, biliary epithelial cells and inflammatory cells contribute to the progression of liver damage in the course of chronic HCV-related infection and NAFLD. Moreover, our data propose that, during NAFLD, PCI and ICI are strongly associated with activation of the HPC compartment and this association is likely one determinant subtending the strong association between PCI/ICI and fibrosis.