54 ACTIVATED HEPATIC NKT CELLS RECRUIT CXCR3 EXPRESSING REGULATORY T CELLS (T REGS) INTO THE LIVER VIA A CYTOKINE-TO-CHEMOKINE CASCADE

Parallel Session 6: HEPATIC FIBROSIS AND INFLAMMATION performed, the former by expert pathologists. In addition, measurement of the gene expression of 95 genes using Taqman Low Density Arrays that were designed to study polymorphonuclear (PMN) cell infiltration and hepatocyte proliferation was undertaken. Results: Acute carbon tetrachloride injury demonstrated no differences in hepatocyte damage between genotypes as assessed by ALT/AST levels and necroinflammatory scores. After chronic injury, large numbers of infiltrating cells (neutrophils, macrophages, T-cells) were present in the fibrotic bands of wild type mice. Mice deficient for c-Rel had significantly lower numbers of infiltrating cells. These correlated with lower transcript levels of Monocyte Chemotactic Proteins (MCP-1/-2 and -3), RANTES and Chemokine (C-X-C motif) Ligands (CXCL2 and 5). Consequently, c-Rel knockout mice had on average stage 2.5 fibrosis at peak injury compared with stage 3.5 in wild type mice. On the other hand, at days 3, 7, and 10 (recovery phase), c-Rel deficient mice showed reduced levels of hepatocyte proliferation and a consequent retardation of recovery from fibrosis. In addition, c-Rel knockout HSC showed reduced levels of pro-collagen1 and a-smooth muscle actin mRNA and ChIP analysis demonstrated direct binding of c-Rel to the RANTES promoter in WT HSC. Conclusion: This data suggests that c-Rel plays biphasic roles in liver fibrosis – acting during injury to enhance infiltration of neutrophils to the liver and increasing fibrotic tissue deposition and during recovery to stimulate hepatocyte proliferation and resolution of fibrosis. 54 ACTIVATED HEPATIC NKT CELLS RECRUIT CXCR3 EXPRESSING REGULATORY T CELLS (T REGS) INTO THE LIVER VIA A CYTOKINE-TO-CHEMOKINE CASCADE T. Santodomingo-Garzon, J.-L. Han, T. Le, Y. Yang, M.G. Swain. Medicine, University of Calgary, Calgary, Canada E-mail: [email protected] Background and Aims: Possible interactions between NKT cells and Tregs in the regulation of liver inflammation remains unexplored. In this study we determined the potential role played by activated NKT cells in the hepatic recruitment of CD4+FOXP3+ T cells (ie. Tregs). Methods: For these experiments we administered the NKT cell-activating ligand a-Gal-C18-Cer into C57BL/6 mice (10 mg/mouse ip; a derivative of a galactosylceramide which does not typically induce hepatitis). After a-Gal-C18-Cer administration livers were collected and cells isolated for FACS. Results: a-Gal-C18-Cer activated hepatic NKT cells to produce IFN˜a (by FACS) and resulted in a ~60-fold increase in hepatic levels of the chemokine CXCL10/IP-10 (by ELISA); an effect completely prevented in a-Gal-C18-Cer-treated CD1d knockout mice which lack NKT cells. In addition, a-Gal-C18-Cer administration resulted in the significant recruitment of Tregs into the liver. ~50% of Tregs recruited into the liver expressed CXCR3 (cognate receptor for CXCL10), and ~36% expressed the surface integrin CD103. Moreover, there was a significant 5-fold increase in the percentage of hepatic Tregs producing IL-10 after a-GalC18-Cer treatment, compared to vehicle-treated controls (by FACS). In contrast, surface TGFb1 expression was low and similar on Tregs in both treated and untreated groups. Recruitment of CXCR3+ Tregs into the liver after a-Gal-C18-Cer administration was completely prevented in CD1d knockout mice, as well as in mice pre-treated with a specific CXCL10 neutralizing antibody. Moreover, a-Gal-C18-Cer-treated CXCL10-neutralized mice demonstrated obvious biochemical (ALT levels) and histological evidence of hepatitis, which were absent in a-Gal-C18-Cer-treated control mice. Hepatitis in a-Gal-C18-Cer-treated CXCL10-neutralized mice was associated with the increased hepatic recruitment and activation of NK cells. Conclusions: We demonstrate that hepatic NKT cells can orchestrate the hepatic inflammatory response, not only through their well characterized pro-inflammatory properties, but also by regulating the recruitment of Tregs into the liver via a novel pathway driven by NKT cell mediated

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production of CXCL10 interacting with CXCR3 expressed on Tregs. These findings have important implications for the development of clinical therapeutics aimed at preventing the recruitment of CXCR3-expressing Th1 effector cells during hepatitis, as well as for the use of NKT cell activating ligands for the treatment of liver cancers.