4). To test whether continuous NF-κB activation is needed for the observed changes, we compared gene expression in 4-week-old mice with sustained NF-κB activation with animals MI-503 where CAIKK2 expression was inhibited for 3 days by DOX readministration. DOX readministration for 3 days inhibited CAIKK2 expression

(Fig. 5A). Histological analyses revealed that the livers from DOX-readministered mice exhibited less inflammation (Desmet score: control 0, CAIKK2LAP 0.8 ± 0.5) compared to those from mice without DOX readministration (Desmet score: control 0, CAIKK2LAP 2.5 ± 0.8, P = 6 × 10−3) (Fig. 5B,C). Expression of stress response genes and chemokines was reversed in DOX-treated animals, suggesting that these processes require

continuous NF-κB activation (Fig. 5D). Furthermore, 12-week-old animals were readministered DOX for 3 weeks. Histological analyses, as well as hydroxyproline assay, revealed that despite the NF-κB system being switched off, hepatic fibrosis did not regress within this time frame (Supporting Fig. S5). Although 3 weeks might have been too short to significantly reduce the extent of liver fibrosis, HSC activation markers were all down-regulated after readministration of DOX (Fig. 5E), suggesting that activation of HSCs is dependent on signaling pathway sustained activation of the hepatic NF-κB system. Given the increased presence of macrophage surface markers as well as genes involved in macrophage activation in our microarray analysis (Fig. 4D), we hypothesized that activation of hepatocellular NF-κB signaling leads to liver fibrosis development by way of macrophage recruitment. To test whether hepatocyte-mediated recruitment of macrophages contributes to liver fibrogenesis, we injected clodronate liposomes, an established macrophage-depleting agent. Injection of clodronate liposomes from age 3 to 12 weeks resulted in a marked decrease in macrophage surface marker F4/80, as well as attenuated lysozyme

M and Cxcl10 expression, whereas CAIKK2 expression was intact (Fig. 6A,B; Supporting Fig. S6B). Unaltered levels of F4/80 expression were observed in animals injected with control liposomes (Fig. MCE 6B). Clodronate administration did not reduce the extent of hepatic damage (Supporting Fig. S6A), or the extent of overall inflammation as suggested by unaltered levels of several inflammation-related genes (Supporting Fig. S6B). On the other hand, we observed attenuated fibrogenesis as evidenced by Sirius-red staining (Fig. 6C) and by Desmet scoring (Fig. 6D). A reduction of collagen deposition was also confirmed by hydroxyproline assay and morphometrical analysis of Sirius-red-stained sections (Fig. 6C,E). Thus, our data indicate that sustained hepatocellular NF-κB activation leads to liver fibrosis development by way of recruitment and activation of macrophages.