As the up-regulation of CCR9 mRNA in HSCs and the existence of CCR9+ HSCs were confirmed in fibrotic livers (Fig. 3), this suggested that HSCs might be affected PD0325901 order by the CCL25/CCR9 axis. By flow cytometry, significantly increased CCR9 expression was noticed in HSCs from fibrotic livers (Fig. 6A). Importantly, CCR9-expressing HSCs isolated from CCl4-treated WT mice had activated and profibrogenic phenotypes, as shown by the increase of α-SMA, TGF-β1, collagen 1α1, and TIMP-1 mRNA expression (Fig. 6B). A transwell migration assay demonstrated that WT HSCs had significant potential to migrate along CCL25 gradients compared with HSCs from CCR9−/− mice (Fig. 6C). After

48 hours of culture with 300 ng/mL CCL25, fibrosis marker mRNAs increased in HSCs from WT mice compared with HSCs from CCR9−/− mice (Fig. 6D), but to a lesser extent compared with activated HSCs in vivo (Fig. 6B). Accordingly, accumulation of CD11b+CCR9+ macrophages might be influential upon chronic liver injury and subsequent hepatic fibrosis. To examine the interactions of HSCs that produce the majority of collagen leading to liver fibrosis,3 hepatic CD11b+ Decitabine concentration macrophages were isolated from CCl4-injected WT or CCR9−/− mice and cocultured with quiescent HSCs isolated from

WT mice. The mRNA levels of fibrosis markers, including α-SMA, TGF-β1, collagen 1α1, and TIMP-1, were significantly higher in HSCs cocultured with CD11b+ macrophages from WT mice compared with those with CD11b+ macrophages from CCR9−/− mice (Fig. 7A). TNF-α is a key factor of HSC activation.3, 25 Addition of anti-TNF-α antibodies significantly decreased the levels of fibrosis marker mRNAs in HSCs from each group. Furthermore, the neutralization of TGF-β1 caused decreased levels of fibrosis marker mRNAs in HSCs as well. This suggested both TNF-α and TGF-β released from CCR9+ macrophages are important for HSCs activation. We also confirmed that fibrosis marker mRNAs

in HSCs were not affected by CD8+ T lymphocytes or other non-CD11b immune cells (Fig. 7A). To confirm the significance of TNF-α-mediated HSC activation click here by CD11b+CCR9+ macrophages, quiescent HSCs isolated from mice deficient for TNF receptor super family 1a (TNFRsf1a−/−) were cocultured with WT CD11b+ macrophages from fibrotic livers, or cultured with TNF-α at 500 pg/mL, as TNF-α is known to activate HSCs through TNF receptor 1.30 The addition of TNF-α did not activate TNFRsf1a−/− HSCs, and the degree of HSC activation evaluated by α-SMA and collagen 1α1 mRNA expression when cocultured with WT CD11b+ macrophages was significantly diminished, while that evaluated by TGF-β1 and TIMP-1 mRNA expression was only slightly elevated (Fig. 7B). Immune cells, including macrophages, play a critical role in the initiation of liver injury and subsequent liver fibrosis.