Kuklin A. Expression of innate immunity related genes in intact hepatocytes and regenerating rat liver

Liver regeneration after partial hepatecomy is a very complex and highly regulated process. Sequential action of different signaling molecules directs quiescent liver cells to proliferate and restore liver mass. The earliest phase of liver regeneration - priming phase, is known to be regulated by a number of innate immunity factors such as IL-6 and TNF-alfa that are crucial for triggering of liver regeneration. In this study, we addressed the question whether interferon alpha (IFN-alfa), the active player in innate immunity, is involved in the triggering of liver regeneration. We investigated expression of Ifn-alfa gene in total liver, hepatocytes and non-parenchymal cells after partial hepatectomy and during acute phase response after laparotomy. We revealed early (at 1 h) up-regulation of Ifn-alfa mRNA abundance after partial hepatectomy and its down-regulation during 12 h after laparotomy. In order to determine which genes might be regulated by IFN-alfa in regenerating liver we investigated the transcriptome of primary hepatocytes cultivated during 3 h and 6 h with quasi-physiological concentration of IFN-alfa (250 U/ml) resembling the previously estimated concentration in regenerating rat liver. IFN-alfa induced up-regulation of differentially expressed genes encoding both activators and inhibitors of apoptosis, inflammation, T-cells activity etc. pointed to the balanced hepatocytes response to quasi-physiological dose of IFN-alfa unlike toxic effect of higher dose of IFN-alfa during longer incubation. Functional annotation and time course analysis of differentially expressed genes showed that different sets of genes respond in a distinct manner to IFN-alfa. The earliest responding genes were "antiviral genes", whose products directly inactivate pathogens within host cell, followed by ISGylation-related genes, with a subsequent activation of genes encoding transcription factors and others. The differentially expressed genes were enriched with the genes possessing specific transcription factors binding sites in their promoters. It is pointed that Jak/STAT/ISGF3, Jak/STAT, PI3K/AKT and p38/MAPK are involved in intracellular transition of IFN-alfa signal in a descending extent. ISGylation-related genes (Ube1l, Ube2l6, Usp18, Isg15 and Trim25) were the earliest responders to IFN-alfa among the differentially-expressed genes in intact hepatocytes. They were selected for investigation of their expression in the liver after partial hepatectomy and laparotomy. The most prominent elevation of their mRNAs abundances was observed during acute phase response with maximum up-regulation at 1 h for Trim25 and at 3 h for Usp18 and Isg15 and down-regulation after partial hepatectomy. To determine if other IFN-alfa-stimulated genes have the same expression profile we investigated Pkr and Irf7 mRNA abundances. The changes in Irf7 mRNA abundance were similar to ISGylation-related genes peaking at 3 h during acute phase response and were down-regulated to 12 h after PHE. The Pkr abundance increased dramatically to 12 h after PHE. Comparing the time scale of genes expression with that of Ifn? abundance we conclude that expression of Ube1l, Ube2l6, Usp18, Isg15 and Irf7 genes after partial hepatectomy and laparotomy was not associated with expression of Ifn-alfa gene unlike Pkr gene that was associated. It means that indicated genes that are known as classical IFN-stimulated genes might be responsible for other factors. The detailed in silico analysis of these genes promoters revealed the potential regulatory role of transcription factors from IRF family particularly IRF3 which is known to be activated independently from IFN-alfa. The expression of two house-keeping genes, Tbp and 18S rDNA which reflects integral processes of transcription and translation, respectively, revealed the principal difference in the earliest response to both operations. We presume that the earliest response is characterized by an enhanced transcription and translation after partial hepatectomy with concomitant down regulation of post-translational modification by ISG15 and enhanced protein modification by ISG15 at the background of down-regulated total transcription and translation after laparotomy.