789 PRECLINICAL CHARACTERIZATION OF NOVEL CYCLOPHILIN INHIBITORS BASED ON THE POLYKETIDE, SANGLIFEHRIN

Abstract

Introduction: Progressive hepatic fibrosis is a common feature of chronic hepatitis C. Natural Killer (NK) cells represent a major component of intra-hepatic lymphocytes and have been shown to be important for the early control and natural course of HCV infection. Moreover, in mouse models it has been shown that natural killer cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSC) in a NKG2D and TRAIL dependent manner. Here, we analyzed the interactions of human NK cells from HCVpositive patients with activated primary HSC and the potential effects of interferon-a (IFN-a) treatment. Material and methods: NK cells from untreated HCV-RNA(+) patients (n = 11), interferon-a (IFN-a) treated patients (n = 10) and healthy controls (n = 12) were co-incubated with activated primary HSC (ScienCell). NK cells from healthy persons were incubated in the presence or absence of IFN-a (25 IU/ml). Cytotoxic activity of NK cells was studied using the CD107a assay. In addition, INF-g and TNFa production of NK cells was measured by FACS analysis. Induction of HSC apoptosis (active Caspase-3) was analyzed flowcytometrically. Results: Analyzing cytolytic activity of NK cells following coincubation with HSC only discrete CD107a expression could be observed on both NK cells from HCV(+) patients and healthy controls ([mean±SEM: 5.7±0.7% vs. 4.7±0.8%; p =n.s.). Furthermore, only negligible secretion of IFN-g and TNF-a could be observed. However, NK cells from untreated HCV-infected patients were significantly more effective in induction of HSC apoptosis (20.8±2.1%) than NK cells from healthy controls (6.2±0.6%). Of note, NK cells from IFN-a/RBV treated HCV(+) patients displayed an even stronger capability to kill HSC (26.9±4.3%). In vitro stimulation of NK cells with IFN-a resulted in a significant up-regulation of TRAIL and was associated with increased killing of HSC as compared to un-stimulated NK cells. This effect could be blocked with both NKG2Dand TRAIL-specific antibodies. Conclusion: NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated hepatic stellate cells. This function of NK cell is increased following IFN-a treatment. Thus, NK cells may play an important anti-fibrotic role in chronic hepatitis C.