献血感染丙型肝炎病毒人群的T细胞免疫及与肝损伤的相关性研究
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摘要
丙型肝炎是由感染丙型肝炎病毒(Hepatitis C virus,HCV)引起的,至今仍是世界公共卫生的严重威胁。为研究HCV感染者的T细胞免疫状态及其对疾病进展的影响,我们招募了62位研究对象,包括20位健康对照、42位HCV感染者。我们通过HCV主要的T细胞免疫原非结构蛋白3(NS3蛋白)多肽库体外刺激外周血淋巴细胞,利用流式细胞方法检测CD8~+T细胞和CD4~+T细胞的γ-干扰素(IFN-γ)、白介素-2(IL-2)和肿瘤坏死因子-α(TNF-α)的分泌水平。同时,我们检测了研究对象血清中白介素-6(IL-6)和白介素-10(IL-10)的水平。结果表明,HCV感染者外周血中仍持续存在针对NS3的特异性CD8~+T细胞和CD4~+T细胞。感染者血清中IL-6、IL-10显著高于对照人群。相关性分析显示,HCV感染者外周血中分泌IFN-γ和TNF-α的HCV特异性CD8~+T细胞水平及血清中IL-10水平与血清中的谷丙转氨酶(ALT)和谷草转氨酶(AST)呈现正相关。本研究表明,HCV感染者体内针对病毒的特异性细胞免疫持续保持一定的水平,并且可能与病人的肝损伤有关,该研究对于了解HCV感染的细胞免疫特征及研发相应的免疫干预策略具有一定的意义。
Hepatitis C which is caused by the infection of hepatitis C virus(HCV),remains a serious threat to public health all over the world. The antigen-specific T cells play a pivotal role in the anti-virus immune responses and the disease progress of the infected patients. To study the T cell status and its correlation with liver injury in patients with HCV infection,we recruited 62 subjects,including 20 healthy controls and 42 patients with HCV infection.The over-lapping peptide library covering the whole length of non-structure protein3(NS3),a dominant cellular immunogen of HCV,was used to stimulate the peripheral blood mononuclear cells. We analyzed the secreting levels of interferon-γ(IFN-γ),interleukin-2(IL-2)and tumor necrosis factor-α(TNF-α) of CD8~+T cells and CD4~+T cells by flow cytometry. The levels of interleukin-6(IL-6) and interleukin-10(IL-10)in the serum were also measured. Combined with clinical information,we investigated the correlation between cellular immunity and alanine aminotransferase(ALT)and aspartate aminotransferase(AST)which are the important indicators of liver injury. The results showed that HCV-specific CD8~+T cells and CD4~+T cells responses in patients with HCV infection were higher than that in healthy controls. The levels of IL-6 and IL-10 were also significantly higher in the HCV infection group than that in the healthy control group. Further correlation analyses showed that IFN-γ,TNF-α secreted by CD8~+T cells and IL-10 were positively correlated with ALT and AST. Our results shed light on the characteristics of the antigen-specific cellular immunity in the HCV patients and may provide helpful recommendations for the immune intervention in the HCV infection therapy.
Hepatitis C which is caused by the infection of hepatitis C virus(HCV),remains a serious threat to public health all over the world. The antigen-specific T cells play a pivotal role in the anti-virus immune responses and the disease progress of the infected patients. To study the T cell status and its correlation with liver injury in patients with HCV infection,we recruited 62 subjects,including 20 healthy controls and 42 patients with HCV infection.The over-lapping peptide library covering the whole length of non-structure protein3(NS3),a dominant cellular immunogen of HCV,was used to stimulate the peripheral blood mononuclear cells. We analyzed the secreting levels of interferon-γ(IFN-γ),interleukin-2(IL-2)and tumor necrosis factor-α(TNF-α) of CD8~+T cells and CD4~+T cells by flow cytometry. The levels of interleukin-6(IL-6) and interleukin-10(IL-10)in the serum were also measured. Combined with clinical information,we investigated the correlation between cellular immunity and alanine aminotransferase(ALT)and aspartate aminotransferase(AST)which are the important indicators of liver injury. The results showed that HCV-specific CD8~+T cells and CD4~+T cells responses in patients with HCV infection were higher than that in healthy controls. The levels of IL-6 and IL-10 were also significantly higher in the HCV infection group than that in the healthy control group. Further correlation analyses showed that IFN-γ,TNF-α secreted by CD8~+T cells and IL-10 were positively correlated with ALT and AST. Our results shed light on the characteristics of the antigen-specific cellular immunity in the HCV patients and may provide helpful recommendations for the immune intervention in the HCV infection therapy.
引文
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[13]Rosen H R,Miner C,Sasaki A W,Lewinsohn D M,Conrad A J,Bakke A,Bouwer H G,Hinrichs D J.Frequencies of HCV-specific effector CD4+T cells by flow cytometry:correlation with clinical disease stages[J].Hepatology,2002,35(1):190-198.
[14]Rosen H R.Hepatitis C pathogenesis:mechanisms of viral clearance and liver injury[J].Liver Transpl,2003,9(11):S35-43.
[15]Chen N,Liu Y,Guo Y,Chen Y,Liu X,Liu M.Lymphocyte activation gene 3 negatively regulates the function of intrahepatic hepatitis C virus-specific CD8+T cells[J].J Gastroenterol Hepatol,2015,30(12):1788-1795.
[16]Irshad M,Gupta P,Irshad K.Immunopathogenesis of liver injury during hepatitis C virus infection[J].Viral Immunol,2019,32(3):112-120.
[17]Liu B S,Groothuismink Z M,Janssen H L,Boonstra A.Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections[J].J Leukoc Biol,2011,89(6):981-988.
[18]Shaker O G,Sadik N A.Polymorphisms in interleukin-10 and interleukin-28B genes in Egyptian patients with chronic hepatitis C virus genotype 4 and their effect on the response to pegylated interferon/ribavirin-therapy[J].J Gastroenterol Hepatol,2012,27(12):1842-1849.
[19]Zhang L,Hao C Q,Miao L,Dou X G.Role of Th1/Th2 cytokines in serum on the pathogenesis of chronic hepatitis C and the outcome of interferon therapy[J].Genet Mol Res,2014,13(4):9747-9755.
[2]Mohd Hanafiah K,Groeger J,Flaxman A D,Wiersma S T.Global epidemiology of hepatitis C virus infection:new estimates of age-specific antibody to HCVseroprevalence[J].Hepatology,2013,57(4):1333-1342.
[3]Nguyen L H,Nguyen M H.Systematic review:Asian patients with chronic hepatitis C infection[J].Aliment Pharmacol Ther,2013,37(10):921-936.
[4]Shoukry N H,Grakoui A,Houghton M,Chien D Y,Ghrayeb J,Reimann K A,Walker C M.Memory CD8+T cells are required for protection from persistent hepatitis C virus infection[J].J Exp Med,2003,197(12):1645-1655.
[5]Lechner F,Wong D K,Dunbar P R,Chapman R,Chung R T,Dohrenwend P,Robbins G,Phillips R,Klenerman P,Walker B D.Analysis of successful immune responses in persons infected with hepatitis Cvirus[J].J Exp Med,2000,191(9):1499-1512.
[6]Klenerman P,Thimme R.T cell responses in hepatitis C:the good,the bad and the unconventional[J].Gut,2012,61(8):1226-1234.
[7]Napoli J,Bishop G A,McGuinness P H,Painter D M,McCaughan G W.Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Th1-associated cytokines[J].Hepatology,1996,24(4):759-765.
[8]Priimiagi L S,Tefanova V T,Tallo T G,Shmidt E V,Solomonova O V,Tuisk T P.Immune-regulating Th1-and Th2-cytokines in chronic infections caused by hepatitis B and C viruses[J].Vopr Virusol,2003,48(4):37-40.
[9]Falasca K,Ucciferri C,Dalessandro M,Zingariello P,Mancino P,Petrarca C,Pizzigallo E,Conti P,Vecchiet J.Cytokine patterns correlate with liver damage in patients with chronic hepatitis B and C[J].Ann Clin Lab Sci,2006,36(2):144-150.
[10]Bozkaya H,Bozdayi A M,Aslan N,Turkay C,Sarioglu M,Cetinkaya H,Akdogan M,Cinar K,Erden E,Kose K,Senturk H,Akkiz H,Karayalcin S,Yurdaydin C,Uzunalimoglu O.Circulating IL-2 and IL-10 in chronic active hepatitis C with respect to the response to IFN treatment[J].Infection,2000,28(5):309-313.
[11]Schulze Zur Wiesch J,Ciuffreda D,Lewis-Ximenez L,Kasprowicz V,Nolan B E,Streeck H,Aneja J,Reyor L L,Allen T M,Lohse A W,McGovern B,Chung RT,Kwok W W,Kim A Y,Lauer G M.Broadly directed virus-specific CD4+T cell responses are primed during acute hepatitis C infection,but rapidly disappear from human blood with viral persistence[J].JExp Med,2012,209(1):61-75.
[12]Semmo N,Day C L,Ward S M,Lucas M,Harcourt G,Loughry A,Klenerman P.Preferential loss of IL-2-secreting CD4+T helper cells in chronic HCV infection[J].Hepatology,2005,41(5):1019-1028.
[13]Rosen H R,Miner C,Sasaki A W,Lewinsohn D M,Conrad A J,Bakke A,Bouwer H G,Hinrichs D J.Frequencies of HCV-specific effector CD4+T cells by flow cytometry:correlation with clinical disease stages[J].Hepatology,2002,35(1):190-198.
[14]Rosen H R.Hepatitis C pathogenesis:mechanisms of viral clearance and liver injury[J].Liver Transpl,2003,9(11):S35-43.
[15]Chen N,Liu Y,Guo Y,Chen Y,Liu X,Liu M.Lymphocyte activation gene 3 negatively regulates the function of intrahepatic hepatitis C virus-specific CD8+T cells[J].J Gastroenterol Hepatol,2015,30(12):1788-1795.
[16]Irshad M,Gupta P,Irshad K.Immunopathogenesis of liver injury during hepatitis C virus infection[J].Viral Immunol,2019,32(3):112-120.
[17]Liu B S,Groothuismink Z M,Janssen H L,Boonstra A.Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections[J].J Leukoc Biol,2011,89(6):981-988.
[18]Shaker O G,Sadik N A.Polymorphisms in interleukin-10 and interleukin-28B genes in Egyptian patients with chronic hepatitis C virus genotype 4 and their effect on the response to pegylated interferon/ribavirin-therapy[J].J Gastroenterol Hepatol,2012,27(12):1842-1849.
[19]Zhang L,Hao C Q,Miao L,Dou X G.Role of Th1/Th2 cytokines in serum on the pathogenesis of chronic hepatitis C and the outcome of interferon therapy[J].Genet Mol Res,2014,13(4):9747-9755.