The Role Of IFN-α-induced CD100Expression On Natural Killer Cells In Control Of Hepatitis C Virus

Hepatitis C virus (HCV) infection is a major health problem. An estimated130-170million people worldwide have HCV infection, and approximately70%-80%of thepatients undergo viral persistence. Partial chronic HCV patients progress into severe liverdisease, such as hepatic cirrhosis and/or hepatocellular carcinoma. The current therapy forchronic hepatitis C mainly involves Pegylated interferon-(Peg-IFN-) in combinationwith ribavirin, which provides sustained control of the HCV replication in around55%ofpatients. Therefore, it is very important to fully understand the mechanisms underlyingIFN-mediated antiviral effect. In addition to a direct antiviral action, IFN-likely exertsimmunomodulatory activities on the elimination of HCV-infected hepatocytes. However,the potential molecular mechanisms of IFN--mediated anti-HCV immune responseremain elusive.Immune responses are thought to play vital roles in virus control and diseaseprogression. Natural killer (NK) cells, an important innate immune cell population,provide early defense against viral infections through killing virus-infected cells andproducing antiviral cytokines, such as IFN-γ, that inhibit viral replication. NK cells are known to be functionally impaired during HCV infection, whereas the underlyingmechanism has not been well elucidated. Sema4D, also called CD100, was the firstimmune semaphorin discovered and is constitutively expressed by resting T cells and NKcells. As an adhesion molecule, CD100has important immunoregulatory effects on NKfunctions by enhancing the adhesion between NK effectors and target cells. However,related studies about CD100involved in chronic HCV infection have not been reported sofar.To identify the underlying molecular mechanisms whereby NK cells exert killingfunctions on HCV infection, we selectly examined CD100, CD69and TRAIL expressionon NK cells in75chronic HCV patients (30treatment-na ve patients,25HCV patientswith early virological response (EVR) and20patients achieved sustained virologicalresponse (SVR)) with or without Peg-IFN-treatment by flow cytometry. We alsoexamined the effects of HCV and IFN-on CD100and its receptors Plexin-B1/B2expression, and the role of CD100on NK cell killing activity via blockade experiments invitro.The main materials and methods:1. The role of CD100expression on NK cells in chronic HCV infected patientsCD100was reported involved in immunoregulation during human immunodeficiencyvirus (HIV) and hantaan virus infection. However, the studies on association betweenCD100and HCV infection have not been reported. The distribution of NK cell and subsetsas well as the expression of CD100, CD69and TRAIL on NK subpopulations in chronicHCV patients was examined by flow cytometry and the level of sCD100was detected byELISA. Spearman rank test was employed to analyze the correlation between the threemolecules and clinical parameters, such as alanine aminotransferase (ALT) and HCVRNA.2. The effects of HCV and IFN-on CD100and its receptors, Plexin-B1/B2expressionTo identify the role of HCV infection and IFN-on CD100and Plexin-B1/B2expression, the experiments below was carried out. QRT-PCR and flow cytometry wereused to detect the expression of Plexin-B1/B2on K562and Huh7.5cells. We investigated the changes of CD100and Plexin-B1/B2expression upon stimulation of peripheral bloodmononuclear cells (PBMC) or Huh7.5cells with HCV or IFN-in vitro.3. The molecular mechanisms involved in the regulation of NK cell functions by IFN-To investigated the underlying mechanisms of enhanced NK cell functions by IFN-,we examined the percentages of NK cell and subsets as well as CD100, CD69and TRAILexpression in chronic HCV patients with EVR after the initiation of Peg-IFN-andribavirin treatment and in patients with SVR. The level of sCD100in serum was detectedby ELISA in HCV patients following IFN-therapy. The relationship betweenCD100/TRAIL expression and virological response was observed. We also investigatedthe alteration of NK killing activity upon co-culturing isolated NK cells with K562,Huh7.5or HCV-infected Huh7.5cells after stimulation with IFN-or blockingCD100/Plexin-B1/B2interation.The main results are as below:1. The frequency of CD56dimNK cells was significantly decreased and CD56negNKsubsets increased in chronic HCV patients compared to healthy donors.2. The level of sCD100was significantly declined in HCV patients and the expressionlevels of CD100, CD69and TRAIL on total NK and subsets were slightly different,but no significance.3. The expression of CD100and TRAIL were positively correlated with ALT levels, andwas negatively with HCV-RNA titers in HCV patients.4. In in vitro studies, both HCV and IFN-could affect CD100and its receptors,Plexin-B1/B2expression.5. The percentages of CD100, CD69and TRAIL expression on NK cells as well as thelevel of sCD100in serum were markedly up-regulated in chronic HCV patients withEVR after the initiation of Peg-IFN-and ribavirin therapy, and the abnormaldistribution of NK subsets and the expression of the three molecules were returned tonormal in patients with SVR.6. HCV infection markedly enhanced NK cytotoxicity, but not IFN-γ production; IFN-treatment significantly up-regulated NK cell killing activity, including increased cytotoxicity and IFN-γ secretion in response to different target cells (K562, Huh7.5and HCV FL-J6/JFH-1infected Huh7.5cells).7. NK cell functions were markedly inhibited, showing decreased CD107a expressionand IFN-γ production via blockade of CD100-Plexin-B1/B2ligations between NKsand target cells.In conclusion, we found that CD100expression on circulating NK cells wasdown-regulated during HCV infection, and consequently mediating weaker adhesionbetween NKs and target cells, which apparently affects NK cell killing activity and HCVclearance. Correlation analysis between CD100expression and ALT or HCV-RNA levelsdemonstrated that CD100might be involved in immune-mediated hepatic inflammationand elimination of virus. IFN-based therapy efficiently activated NK cells and increasedNK cell killing functions through up-regulation of immune molecules’ expression, likeCD100, TRAIL, CD107a and IFN-γ expression, which facilitated HCV clearance. Ourstudy indicated the underlying mechanisms whereby CD100-Plexin-B1/B2interactionplays an important role in regulation of NK functions, which provide experimental andacademic evidence in the development of immunotherapeutic approaches for treatment ofHCV infection.