Expression Of TIPE2, FOXP3, CTLA-4mRNA In PBMCs Of Patients With Chronic Hepatitis C Virus

Objective:Hepatitis C virus (HCV) is a single-stranded positive strandRNA virus, with a full length of9.6kb. Currently, nearly3%of the world’spopulation is infected with HCV, of whom more than50-80%of patients fromacute to chronic persistent infection, even the development of liver cirrhosis,hepatocellular carcinoma. It is known that innate and adptive immutity areinvolved in the progression of HCV infection, it’s chronic process andclearance. But, the exact mechanim underling the pathogenesis of hepaticinflammation induced by HCV is not yet clear. TIPE2(Tumor necrosis factor-α-induced protein8like2, TNFAIP8L2), is a newly discovered regulator ofimmune singal transduction pathways. TIPE2is a negative regulator of innateand adapvie immunity, plays an important role in inflammatory diseases, andit provides a molecular bridge from inflammation to cancer by targing Rassignaling pathway. Previous study have shown that not only TIPE2highlyexpressed in immune cells and tissues such as lymph nodes, lymphocytes andmonocytes, but also expressed in other non-immune cells, such as epithelialcells, preudo stratified epithelium cells, liver cells, neuronal cells andchondrocytes. And some research about the characteristics of human TIPE2showed TIPE2may be involved in the occurrence and development of manydiseases, such as systemic lupus erythematosus, diabetic nephropathy, chronichepatitis B, hepatocellular carcinoma, renal cell carcinoma, but the specificmechanism was large unkown. Recently Luan et al has reported that naturallyoccurring CD4+CD25+Tregs isolated from murine spleens expressed TIPE2,and the TIPE2could enhance the immune suppressive activity of Tregs. Theresults suggested that TIPE2gene might be related to the immune regulationmediated by Tregs, but the specific mechanism was unclear. Treg cells are agroup of immunosuppressive T cell subsets, palyed an important role in infection, tumor immunity and transplantation. According to reports, Tregcells secrete immunosuppressive factors which directly inhibit the function ofeffector T cells. In many tumor diseases, extensive infiltration of Treg cellsprompts the poor prognosis and low survival rate. Human forkhead/wingedhelix transcription factor p3(FOXP3), a member of the forkhead/winged-helixfamily of transcriptional regulators, which has been identified as a specificmarker of Tregs, could regulated development, differentiation and maturationof Treg. Cytotoxic T lymphocyte-associated antigen4(CTLA-4) is a negativeregulator of T cell activation, is the cell surface molecules, has inhibitoryeffects on T cell activation. Considering normal expression of TIPE2gene inthe immune system could prevent hyper-responsiveness and maintain immunehomeostasis, we hypothesized that the TIPE2gene and FOXP3/CTLA-4mightbe related to the pathogenesis of chronic hepatitis C (CHC).Therefore, the main aim of this project is to detect the expression ofTIPE2, FOXP3and CTLA-4gene in peripheral blood mononuclear cells(PBMCs) of patients with CHC. On one hand understanding the expression ofTIPE2gene in PBMCs of patients with chronic hepatitis C, on the other handto explore the role of TIPE2, FOXP3and CTLA-4gene in the pathogenesis ofchronic hepatitis C, and further explore whether TIPE2regulated developmentof CHC.Methods:1Human subjectsThis study consisted of60CHC patients (Male/Famale,31/29) with anaverage age of49.40±14.31years, who were admitted to Department ofTraditional and Western Medical Hepatology of The Third Hospital of He BeiMedical University, from May2012to October2013. There was no serologicevidence of co-infection with other hepatotropic viruses. The study wasconducted according to the criteria from the Prevention and Therapy Regimenof Viral Hepatitis revised at Xian Conference in2000and the Prevention andTherapy Regimen of Viral Hepatitis revised diagnostic criteria in2004.42healthy controls (Male/Female,12/30) were also recruited with an average of 50.21±8.51years.7ml of anticoagulation blood from all procedures was usedto separated peripheral blood mononuclear cells, and5ml of vein blood wasobtained to separate serum.2Treatment methodsInterferon plus ribavirin were used to antiviral therapy. According ofleukocyte and neutropenia case, drugs of promoting leukocyte wereadministerded. Patients with abnormal liver function were given protect liverand jaundice treatment.3Detection of the mRNA expression levels of TIPE2, FOXP3and CTLA-4inPBMCs from CHC patientsBlood was added to acid citrate dextrose anticoagulant and separated byFicoll density gradient centrifugation and then stored in Trizol reagent tostabilize the RNA. Total RNA was subjected to RNase free DNase to removegenomic DNA concentration. We used ultraviolet spectrophotometer to detectof the purity and concentration of RNA, and elected the absorbance(A) on260/280nm is between1.8-2.0. cDNA was synthesized using5units ofReverse Transcription System. Primer5.0software was used to design specificprimers for the TIPE2, FOXP3and CTLA-4fragments. The mRNAexpression levels of TIPE2, FOXP3and CTLA-4were evaluated by RT-PCR.4Serum levels of clinical parameters of CHCWe collected serums from CHC patients and used ELISA to testanti-HCV and automatic biochemistry analyzer to determine serum alanineaminotransferase(ALT), aspartate aminotransferase(AST) and total bilirubin(TBil) levels. Detection of HCV-RNA was performed with qualitativeRT-PCR.Results:1The mRNA expression levels of TIPE2in PBMCs from CHC patients andthe correlation with antiviral therapyWe examined the mRNA expression levels of TIPE2in PBMCs of theHCV infected patients and healthy controls. The TIPE2mRNA expressionwas dramatically reduced in HCV patients compared with healthy controls (0.50±0.19vs0.71±0.17, P＜0.01). In21patients with interferon plusribavirin antiviral therapy, the TIPE2mRNA expression in PBMCs wasevidently increased than pre-treatments (0.48±0.12vs0.59±0.12, P＜0.01).2The mRNA expression levels of FOXP3in PBMCs from CHC patients andthe correlation with antiviral therapyThe mRNA levels of FOXP3was significantly higher in peripheral bloodmononuclear cells from chronic hepatitis C patients than in healthy controls(0.77±0.38vs0.40±0.09, P＜0.01),21patients after interferon plusribavirin antiviral therapy, the FOXP3mRNA in PBMCs evidently decreasedthan pre-treatments (0.85±0.35vs0.50±0.06, P＜0.01).3The mRNA expression levels of CTLA-4in PBMCs from CHC patients andconnection with antiviral therapyRT-PCR analysis showed that the mRNA levels of CTLA-4wassignificantly higher in peripheral blood mononuclear cells from chronichepatitis C patients than in healthy controls (0.67±0.19vs0.44±0.13, P＜0.01);21patients after interferon plus ribavirin antiviral therapy, the CTLA-4mRNA in PBMCs evidently decreased than pre-treatments(0.70±0.19vs0.55±0.14, P＜0.01).4Correlation between mRNA expression levels of TIPE2and clinicalparameters of CHCWe analyzed clinical parameters of these HCV patients, according to theserum levels of HCV-RNA, patients were subdivided into HCV-RNA lowgroup (HCV-RNA≤1×105IU/ml) and HCV-RNA high group (HCV-RNA＞1×105IU/ml). Similarly, based on the serum levels of ALT, AST and TBil,HCV patients were also subdivided into normal and abnormal groups.Statistical analysis results showed that the mRNA expression levels of TIPE2in PBMCs in the low/normal groups were much higher than that in thehigh/abnormal groups（HCV-RNA,0.63±0.17vs0.37±0.10, P＜0.01; ALT,0.61±0.17vs0.37±0.10, P＜0.01; AST,0.61±0.17vs0.36±0.10, P＜0.01;TBil,0.59±0.18vs0.36±0.10, P＜0.01), and the mRNA expression levelsof TIPE2were negatively corralated with clinical parameters of CHC (ALT, r=-0.529, P＜0.01; AST, r=-0.567, P＜0.01), however, there was no directcorrelation between TIPE2mRNA levels with HCV-RNA or TBil activities.5Correlation between mRNA expression levels of TIPE2, FOXP3andCTLA-4Furthermore, SPSS13.0analysis showed there was a negative correlationbetween the mRNA expression levels of TIPE2and FOXP3, CTLA-4(FOXP3,r=-0.563, P＜0.01; CTLA-4, r=-0.613, P＜0.01).Conclusion:1Patients with chronic hepatitis C had evidently reduced levels of TIPE2expression in peripheral blood mononuclear cells compared to healthyindividuals, and after antiviral and anti-flammation therapy this levels wassignificantly increased.2The mRNA levels of FOXP3, CTLA-4was much higher on peripheral bloodmononuclear cell in patients with chronic hepatitis C than those in healthyindividuals, and after antiviral and anti-flammation therapy there levels wassignificantly reduced.3Down-regulation of TIPE2mRNA expression may result in elevated hepaticinflammation and fibrosis.4TIPE2、FOXP3、CTLA-4may invoved in the pathogenisis of chronicHepatitis C from regulating CD4+CD25+Treg.5TIPE2may involve in the pathogenesis of HCV-induced hepatitis andregulate the cellular immunity, determine the progression of chronic hepatitisC.