白细胞介素-21在乙型肝炎相关慢加急性肝衰竭发病机理中的作用
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摘要
研究背景与目的
慢加急性肝衰竭(ACLF)是指在既往明确诊断或未诊断的慢性肝病基础上,出现急性肝功能损害,表现为黄疸和凝血功能障碍,并且在4周内出现腹水或/和肝性脑病。在我国,90%以上ACLF病例存在HBV感染,被称为乙型肝炎相关慢加急性肝衰竭(HB-ACLF)。 HB-ACLF患者病情重,治疗费用高,预后不良,3个月生存率只有62%,目前唯一有效的治疗手段是肝移植。
目前认为HB-ACLF的发病取决于病毒和宿主两方面因素。一方面研究认为,HBV基因组前C区(PC)和C基因启动子(BCP)核苷酸变异可能引起病毒复制和抗原表达水平的改变,可能与乙型肝炎相关肝衰竭的发生有关。另一方面,更多的研究集中在HB-ACLF发病时机体免疫状况的改变,结果发现单核-巨噬细胞、树突状细胞、自然杀伤细胞、CD4+及CD8+T淋巴细胞、调节性T细胞、Th17细胞以及多种细胞因子都可能参与HB-ACLF发生和发展过程。
白细胞介素-21(IL-21)是由激活的CD4+T细胞和NKT细胞分泌的一种Ⅰ类细胞因子,广泛参与机体免疫活动的调控作用,能够影响多种免疫细胞的发育、活化、扩增和功能表达。研究发现IL-21与炎性肠病、系统性红斑狼疮、Ⅰ型糖尿病、银屑病和风湿性关节炎等多种炎症性、自身免疫性或风湿性疾病发病有关。而且越来越多的证据表明,IL-21参与机体的抗感染免疫,如人类免疫缺陷病毒感染时,IL-21能够促进NK和CD8+T细胞存活、增强其细胞毒活性。最近,来自HBV转基因鼠的研究发现,IL-21的分泌具有年龄依赖性；然而,IL-21在HBV感染导致的肝脏炎症活动,肝细胞损伤和病毒控制中的作用至今未明。
本研究通过检测HB-ACLF、重度和轻度慢性乙型肝炎(S-CHB和M-CHB)患者以及健康对照(HC)血清IL-21及相关细胞因子水平,IL-21分泌细胞及相关CD4+T细胞亚群的频数,以及IL-21对其它炎症因子分泌的影响,探索IL-21在HB-ACLF发病中的作用,并分析及其与疾病严重程度和预后的关系。
研究方法
第一部分：血清IL-21及相关细胞因子的检测
本部分纳入39例HB-ACLF、20例S-CHB、20例M-CHB患者和10例HC。HB-ACLF和S-CHB为2008年1月至2009年7月在南方医院感染内科和广州市第八人民医院住院患者。随访至2010年6月,HB-ACLF患者22例存活,16例死亡,1例接受原位肝移植；S-CHB患者全部存活。对其中7例入院后病情逐渐缓解的HB-ACLF患者进行动态观察,这些患者入院只接受常规内科对症治疗和核苷(酸)类似物(拉米夫定或恩替卡韦)抗HBV治疗,均未接受糖皮质激素和血浆置换术。
HB-ACLF的纳入标准为：(1)血清HBsAg阳性时间大于12个月；和(2)血清总胆红素(TBIL)>17、或每天上升>17.1μmol/L;和(3)凝血酶原活动度(PTA)<40%；和(4)起病(出现黄疸和凝血功能障碍)后4周内出现腹水或／和肝性脑病。
S-CHB的纳入标准为：(1)血清HBsAg阳性时间大于12个月；和(2)血清谷丙转氨酶(ALT)>600U/L;和(3)血清TBIL>51.3μmol/L;和(4)PTA<60%;但不足以诊断为HB-ACLF的患者。
M-CHB患者均为上述时间段南方医院门诊患者,纳入标准：(1)血清HBsAg阳性时间大于12个月；和(2)血清ALT<400U/L且>80U/L:和(3)血清TBIL <51-3μmol/L。
健康对照10例,均为南方医院感染内科实验室学生或工作人员,纳入标准：(1)血清HBsAg阴性；和(2)血清ALT<40U/L;和(3)血清TBIL<17.1μmol/L。
所有研究对象均排除以下情况：(1)年龄>60岁或<18岁；或(2)合并感染人类免疫缺陷病毒(HIV)、甲型肝炎病毒(HAV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)或戊型肝炎病毒(HEV)；或(3)合并甲状腺功能亢进、糖尿病等自身免疫性疾病；或(4)合并恶性肿瘤；或(5)半年内接受过干扰素、糖皮质激素、或其它免疫调节制剂；或(6)影像学或组织学发现明显肝硬化。
分离所有研究对象的血清-80℃贮存,采用流式微球试验(CBA)批量检测血清中包括IL-21在内的12种细胞因子的水平。
第二部分：外周IL-21分泌细胞的检测
本部分纳入21例HB-ACLF(存活14例,未存活7例)、13例S-CHB.19例M-CHB患者和10例HC,并对6例HB-ACLF恢复患者进行动态观察。有8例S-CHB为新增病例,为同期南方医院或广州市第八人民医院的住院患者；其余病例均来自第一部分试验。纳入和排除标准如上述。密度梯度离心法分离患者外周血单个核细胞(PBMC),采用细胞内因子染色(ICS)的方法经流式细胞仪检测外周IL-21分泌细胞及相关CD4+T细胞亚群的频数。
第三部分：IL-21对PBMC分泌其它细胞因子的影响
选择9例CHB患者和6例HC进行IL-21的体外诱导实验。CHB的诊断标准参考2010年新版《慢性乙型肝炎防治指南》。分离研究对象的PBMC,以不同浓度(0、10和100ng/mL)的重组人IL-21(rhIL-21)蛋白刺激培养24小时后,收集培养上清,采用Bender FlowCytomix系统检测11种细胞因子的浓度。
研究结果
第一部分：HB-ACLF患者血清II-21及相关细胞因子水平的变化。
不同人群血清IL-21及相关细胞因子浓度的比较。血清IL-21水平在HC. M-CHB. S-CHB和HB-ACLF依次升高,HB-ACLF患者显著高于其它三组人群(P<0.001,P<0.001和P=0.001),且S-CHB患者显著高于HC(P=0.042)。血清IL-6、IL-8、IL-10、IL-17A、IFN-γ和TNF-a水平在HB-ACLF亦有所升高,而IL-12/IL-23p40低于M-CHB,IL-1p、IL-2、IL-4和IL-12p70在四组人群间无显著差异。
HB-ACLF存活与未存活患者血清IL-21及相关细胞因子的比较。未存活组血清IL-21(P=0.024)与IL-2(P=0.021)、IL-6(P=0.028)和IL-10(P=0.008)浓度显著高于存活组患者。IL-12p70和IL-17A略高于存活组患者,但无统计学差异(P=0.106和P=0.130)。其它细胞因子两组间比较均无显著差异。
HB-ACLF恢复患者血清IL-21及相关细胞因子的动态变化。随着病情缓解,血清IL-21(P=0.003)与IL-1β (P=0.050)、IL-8(P=0.004)、IL-12p70(P=0.018).和IFN-γ(P=0.015)水平均逐渐下降,前后比较有统计学差异；而IL-12/IL-23p40则逐渐升高,尽管统计学差异不显著(P=0.104)。
血清IL-21及相关细胞因子水平与疾病严重程度的相关性。血清IL-21水平与MELD积分(r=0.573, P<0.001)、TBIL (r=0.566, P<0.001)、 PTA (r=-0.520, P<0.001)、白蛋白(ALB, r=-0.369, P=0.001)、肌酐值(CR, r=0.327, P=0.012)显著相关,而与HBV DNA和谷丙转氨酶(ALT)无显著相关性。其它细胞因子,与TBIL显著相关的有IL-6、IL-8、IL-12p70、IL-17A、IL-12/12-23p40和IFN-γ;与MELD积分、PTA和ALB显著相关的有IL-6、IL-8和IL-17A,与CR显著相关的有IL-4、IL-10和IFN-γ。
血清细胞因子对HB-ACLF临床结局的预测价值。以HB-ACLF患者两种不同临床结局(存活或未存活)为因变量,以12种细胞因子浓度为协变量进行Logistic回归分析,构建诊断模型。有6种细胞因子进入回归方程,其中IL-21. IL-2、IL-10和TNF-α为HB-ACLF死亡患者的危险因素,而IL-17A和IL-12/IL-23p40为保护因素。该模型对HB-ACLF患者临床结局的正确判断率为84.6%(33/39),低于MELD积分的正确判断率97.4%(38/39)。进一步对每种细胞因子构建受试者工作特征曲线(ROC),并计算曲线下面积(A),只有IL-21(A=0.695,P=0.039)与IL-2(A=0.718,P=0.021)、IL-6(A=0.707,P=0.028)和IL-10(A=0.749, P=0.008)的诊断意义具有显著性(P<0.05),但其诊断效能仍低于MELD积分(A=0.953,P<0.001)。
第二部分：HB-ACLF患者外周IL-21分泌细胞及相关CD4+T细胞亚群的频数变化。
不同人群IL-21分泌细胞及CD4+T细胞亚群的频数比较。四组人群分泌IL-21、IFN-γ、IL-10或IL-17A的总CD4+T细胞频数整体比较均有显著差异,IL-21+CD4+T细胞在HB-ACLF组最高,IFN-y+CD4+T细胞频数在M-CHB组最高,而IL-10+CD4+T细胞和IL-17A+CD4+T细胞颇数存S-CHB织最高。名重比较发现：HB-ACLF和S-CHB患者的IL-21+CD4+T细胞频数均显著高于M-CHB患者(P<0.001和P=0.002)和HC(P<0.001和P=0.001),而HB-ACLF与S-CHB患者之间无显著差异。M-CHB患者IFN-γ+CD4+T细胞频数显著高于S-CHB(P<0.001)和HB-ACLF (P=0.004)患者,而与HC比较差异无显著性。S-CHB和HB-ACLF患者IL-10+CD4+T细胞频数显著高于M-CHB患者(均P<0.001)和HC(P=0.001和P=0.002),且S-CHB患者显著高于HB-ACLF患者(P=0.028)。三组HBV感染患者之间的IL-17A+CD4+T细胞频数相互比较无显著差异,但均显著高于HC (HB-ACLF, P=0.003; S-CHB, P=0.004; M-CHB, P=0.017)。
进一步对IL-21+CD4+T细胞共表达IFN-γ、IL-10和IL-17A的情况进行分析发现：EL-21+IFN-γ+、IL-21+IL-17A+、IL-21+IL-10+和IL-21+IFN-γ-IL-10IL-17A-的CD4+T细胞亚群在四组人群间整体比较均有显著差异。除IL-21+IL-10+CD4+T细胞亚群的频数在S-CHB最高外,另外三个IL21+CD4+T细胞亚群的频数均以HB-ACLF患者最高。四组人群之间多重比较：HB-ACLF和S-CHB患者的IL-21+IFN-γ-IL-10IL-17A-CD4+T细胞亚群频数显著高于M-CHB患者(均P<0.001)和HC(P<0.001和P=0.001),而S-CHB与HB-ACLF无显著差异。IL-21+IFN-γ+CD4+T细胞亚群频数在HC、M-CHB、S-CHB和HB-ACLF患者递增,HC显著低于三组HBV感染患者(P=0.018,P=0.006和P<0.001),且M-CHB患者显著低于HB-ACLF患者(P=0.021),而S-CHB与HB-ACLF患者比较无显著差异。S-CHB和HB-ACLF患者的IL-21+IL-10+CD4+T细胞亚群频数显著高于M-CHB患者(P<0.001和P=0.001)和HC(P<0.001和P=0.002),且S-CHB高于HB-ACLF患者(P=0.030)。HB-ACLF患者的IL-21+IL-17A+CD4+T细胞亚群频数显著高于M-CHB患者(P=0.004)和HC (P<0.001), S-CHB患者亦高于HC(P=0.012),而其余各组间比较无差异。
HB-ACLF存活与未存活患者IL-21分泌细胞及CD4+T细胞亚群的比较。入院时存活与死亡患者外周分泌IL-21. IFN-γ. IL-10或IL-17A的CD4+T细胞频数均无显著差异。进一步分析IL-21与IFN-γ、IL-10和IL-17A共表达的CD4+T细胞频数在两组间亦无显著差异。
HB-ACLF恢复患者外周IL-21分泌细胞及CD4+T细胞亚群的动态变化。6例恢复期HB-ACLF患者外周IL-21+CD4+T细胞频数随病情缓解而逐渐下降,前后变化具有显著意义(P<0.001)；而分泌IFN-γ、IL-10或IL-17A的CD4+T细胞亚群频数在病程中的变化无统计学差异。进一步对IL-21与IFN-γ、IL-10或IL-17A共表达CD4+T细胞亚群频数的动态变化进行分析,发现IL-21+IL-10+(P=0.007)和IL-21+IFN-γ-IL-10-IL-17A-(P<0.001) CD4+T细胞频数前后比较具有统计学差异,均随疾病恢复而下降；余无显著差异。
外周IL-21分泌细胞及CD4+T细胞亚群与疾病严重程度的相关性。对进行ICS检测的53例处于不同疾病状态(HB-ACLF. S-CHB或M-CHB)的HBV感染者的外周IL-21分泌细胞及CD4+T细胞亚群的频数与反映疾病严重程度的临床参数进行相关性分析。IL-21CD4+T细胞频数与MELD积分(r=0.605,P<0.001)、TBIL (r=0.656, P<0.001)、PTA (r=-0.322, P=0.019)和ALB (r=-0.587, P<0.001)均显著相关,而与HBVDNA、ALT和CR的无显著相关性。IFN-y+CD4+T细胞频数与ALT、TBIL和MELD积分呈负相关,而与ALB、CR和PTA呈正相关。IL-10+CD4+T细胞频数与TBIL和MELD积分呈正相关,而与ALB呈负相关。IL-17A+CD4+T细胞频数与各临床指标均无显著相关性。
对IL-21与IFN-γ、IL-10和IL-17A的共表达CD4+T细胞与疾病严重程度的相关性进行分析发现：在排除上述三种共表达细胞的干扰后,IL-21+IFN-γ-IL-10-IL-17A-CD4+T细胞频数与TBIL (r=0.733, P<0.001)、MELD积分(1-0.649, P<0.001)、ALB (r=-0.674, P<0.001)和PTA (r=-0.377, P=0.005)的相关性更为显著,相关系数更高。三种共表达细胞,IL-21+IFN-γ+CD4+T细胞频数与MELD积分呈正相关；IL-21+IL-10+CD4+和IL-21+IL-17A+CD4+T细胞频数与TBIL和MELD积分呈正相关,而与ALB呈负相关；其余各指标间无显著相关性。
血清IL-21. IFN-γ、IL-10、IL-17A浓度与外周相应因子分泌细胞频数的相关性。共有55例研究对象同时接受了CBA检测血清中IL-21、IFN-γ、IL-10、IL-17A蛋白浓度和ICS检测外周分泌四种细胞因子的CD4+T细胞频数。对两种检测的结果进行Spearman相关性分析,发现只有血清IL-21浓度与IL21+CD4+T细胞频数呈正相关(r=0.482, P<0.001);IFN-y与IFN-y+CD4+T细胞(r=-0.067, P=0.627)、IL-10与IL-10+CD4+T细胞(r=0.114,P=0.406). IL-17A与IL-17A+CD4+T细胞(r=0.073,P=0.596)均无显著相关性。说明外周血中的IL-21主要由CD4+T细胞分泌,而IFN-γ、IL-10和IL-17A的来源则相对广泛。
第三部分：IL-21对PBMC分泌其它细胞因子的影响。
血清IL-21水平与其它细胞因子浓度的相关性分析。对第一部分CBA检测103份血清中各细胞因子的浓度进行相关性分析,发现IL-21与IL-1p(r=0.252,P=0.010)、IL-2(r=0.426, P<0.001)、IL-4(r=0.278, P=0.004)、IL-6(r=0.532, P<0.001)、IL-8(r=0.2424, P<0.001)、IL-10(r=0.207, P=0.036)、IL-12p70(r=0.451, P<0.001)、IL-17A (r=0.330, P=0.001)、IFN-γ (r-0.362, P<0.001)和TNF-α (r=0.303,P=0.002)均呈正相关,但与IL-12/IL-23p40(r=-0.207, P=0.785)无显著相关性。
IL-21体外对PBMC分泌其它细胞因子的影响。无论是对HC还是CHB患者的PBMC, rhIL-21均能显著上调其IL-1p(P=0.009和P<0.001)、IL-6(P=0.006和P<0.001)、IL-10(P=0.002和P<0.001)、IFN-γ(P=0.006和P=0.001)和TNF-α(均P=0.042)的表达,并呈现剂量依赖效应。rhIL-21对IL-2和IL-12p70的分泌也有轻微促进作用,而对IL-8则有轻微抑制作用,尽管两者均无统计学差异。
结论与讨论
相比于M-CHB和S-CHB患者,HB-ACLF患者血清IL-21水平显著升高,且未存活患者高于存活患者,而随着病情恢复,血清IL-21水平又逐渐下降,说明IL-21参与HB-ACLF的病理生理过程。血清IL-21水平与反映肝脏疾病严重程度的临床指标(如TBIL. PTA和MELD积分等)显著相关,并且对HB-ACLF的预后具有一定的判断价值,提示血清IL-21有可能作为反映HB-ACLF肝脏疾病严重性和判断预后的新的生物学指标。
HB-ACLF和S-CHB患者外周IL-21分泌细胞的频数显著高于M-CHB和HC,随病情恢复而逐渐减少,并且与反映肝脏疾病严重程度的临床指标相关,进一步说明IL-21分泌细胞可能与肝脏严重的炎症活动有关。
体外rhIL-21能够促进IL-1β、IL-6、IL-10、IFN-γ和TNF-α的分泌,提示IL-21可能通过调节其它免疫细胞功能、上调前炎症因子的表达的方式参与肝脏炎症活动。
上述证据表明,IL-21参与HB-ACLF的病理生理过程,血清IL-21水平有可能作为评估疾病严重性和判断预后的良好指标,阻断IL-21通路或许是早期干预HB-ACLF发生发展的一个新的治疗靶点。
Bacground and Aims
Acute-on-chronic liver failure (ACLF) is an acute hepatic insult, manifesting as jaundice and coagulopathy, and complicated within four weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. In china, more than90%of ACLF cases were infected with HBV, known as hepatitis B-related ACLF (HB-ACLF). HB-ACLF patients are usually severe, with high cost of treatment and poor prognosis. The three-month survival rate of them is no more than62%. Currently, the only effective treatment is orthotopic liver transplantation (OLT).
Until now, the pathogenesis of HB-ACLF is considered to dependent on two aspects, namely, viral factors and host factors. Studies suggested that, nucleotide mutations in BCP and PC regions of HBV genome may cause changes of viral replication and antigen expression, resulting in the occurrence of hepatitis and liver failure. More researchs focused on the inflammation and immune status changes in HB-ACLF, and they found that the monocytes-macrophages, dendritic cells, natural killer cells, CD4+and CD8+T lymphocytes, regulatory T cells, Th17cells and many kinds of cytokines may participate in the pathophysiological process of HB-ACLF.
Interleukin-21(IL-21) is produced by activated CD4+T cells and NKT cells, belonging to the type I cytokines family. IL-21participates in the regulation of immune activity broadly and it can influence the development, activation, expansion and function of a variety of immune cell. Studies found that IL-21was associated with inflammatory bowel disease, systemic lupus erythematosus, type I diabetes, psoriasis, rheumatoid arthritis, and other inflammatory, autoimmune or rheumatic diseases. More and more evidence suggested that IL-21was involved in the anti-infection immunity, such as the human immunodeficiency virus (HIV) infection, by promoting the survival of NK and CD8+T cells, and enhance their cytotoxic activity. Recently, a study on HBV transgenic mouse model showed that the production of IL-21was age-dependent. However, the role of IL-21in the pathogenesis of liver inflammatory injury caused by HBV infection remains largely unknown.
By assaying the serum levels of IL-21and other cytokines, the frequencies of peripheral IL-21-secreting cells and CD4+Th subsets in HB-ACLF, severe and moderate chronic hepatitis B (S-CHB and M-CHB) patients and health controls (HC), and the effects of IL-21on the production of other inflammatory factor, this study was designed to explore the potential role of IL-21in the pathogenesis of HB-ACLF and its association with disease severity and prognosis.
Materials and Methods
Measurement of serum IL-21and other cytokines
Thirty-nine cases of HB-ACLF,20cases of S-CHB,20cases of M-CHB and10HC subjects were included in this part of study. The HB-ACLF and S-CHB cases are hospitalized patients admitted to Nanfang Hospital or The Eighth People's Hospital of Guangzhou Branch from January2008to July2009. Followed up to June2010,22HB-ACLF patients survived,16patients died, and one underwent OLT, while all the S-CHB patients survived during that period. Seven HB-ACLF patients who got clinical improvement between week1and week5-6after admission were observed continuously. These seven patients were all treated with nucleos(t)ide analogues (Lamivudine or Entecavir), and none received glucocorticiod or plasma-exchange therapy.
The including criteria for HB-ACLF were:(1) positive for serum HBsAg more than12months; and (2) serum total bilirubin (TBIL) of>171μmol/L or rapidly rising levels of TB (TB>17.1μmol/L/day; and (3) plasma prothrombin activity (PTA) of<40%; and (4) the complications of ascites and/or hepatic encephalopathy within4weeks of the first evidence of jaundice or coagulopathy.
The including criteria for S-CHB were:(1) positive for serum HBsAg more than12months; and (2) serum alanine aminotransferase (ALT) of>600IU/L; and (3) serum TBIL of>51.3μmol/L; and (4) plasma prothrombin activity (PTA) of<60%; while not enough to be diagnosed as HB-ACLF.
The M-CHB group was comprised of20out-patients who presented to Nanfang Hospital between2008and2009. The including criteria for them were:(1) positive for serum HBsAg more than12months; and (2) elevated serum ALT levels (80-40IU/L); and (3) serum TBIL of<51.3μmol/L.
HC group was comprised of students or staff in the Laboratory of Infectious Diseases, Nanfang Hospital. The including criteria for them were:(1) negative for serum HBsAg; and (2) normal serum ALT levels (<40IU/L); and (3) normal serum TBIL levels (<17.1μmol/L).
The excluding criteria for all subjects were:(1) more than60or less than18 years old; or (2) co-infection with HIV, hepatitis A virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus; or (3) co-occurrence of hyperthyroidism, diabetes or other autoimmune diseases; or (4) co-occurrence of malignant neoplasm; or (5) received interferon, glucocorticiod, or other immune modulators; or (6) obvious cirrhosis by imaging or histology.
Serum from all subjects were isolated and stored at-80℃until use. Twelve cytokines including IL-21were detected by Cytometric Beads Array.
Assay of the peripheral IL-21-secreting cells
Twenty-one cases of HB-ACLF (14survived and7died),13cases of S-CHB,19cases of M-CHB and10HC subjects were included into the second part of study. And6recovering HB-ACLF patients were observed continuously. All the subjects were from the Part I except8S-CHB patients, who were newly recruited into this study. The including and excluding criteria were the same as Part I. Peripheral blood mononuclear cells (PBMC) from all subjects were isolated with density gradient centrifugation. The frequencies of IL-21-secreting cells and CD4+Th subsets were assayed by the method of intracellular cytokine staining.
The effects of IL-21on the production of other cytokines from PBMC
Nine CHB patients and6HC subjects were included into the third part of study for IL-21inducding experiment. The diagnosis of CHB was made according to the Chinese Guideline for Chronic Hepatitis B (2010version).PBMC were isolated and stimulated with recombinant human IL-21(rhIL-21) at different concentrations (0,10or100ng/mL) for24hours. Then11cytokines in the supernatants were measured by Bender FlowCytomix
Results
Serum levels of IL-21and other cytokines in HB-ACLF patients
Comparison of serum levels of IL-21and other cytokines in the study groups. The serum levels of IL-21increased gradually from HC to M-CHB, S-CHB and HB-ACLF, and they were significantly higher in HB-ACLF than in other three groups (P<0.001, P<0.001and P=0.001), and also higher in S-CHB than in HC (P=0.042). Serum levels of IL-6, IL-8, IL-10, IL-17A, IFN-γ and TNF-a increased in HB-ACLF as well, while the levels of IL-12/IL-23p40reached topmost in M-CHB patients. However, there were no significant differences in the serum levels of IL-1β, IL-2, IL-4or IL-12p70among the four study groups.
Comparison of serum levels of IL-21and other cytokines in HB-ACLF survivors and non-survivors. The serum levels of IL-21(P=0.024), IL-2(P=0.021), IL-6(P=0.028) and IL-10(P=0.008) were significantly higher in HB-ACLF non-survivors than in survivors. Though without significance, the IL-12p70and IL-17A levels were also elevated in the non-survivors, while the other cytokines were of no difference between the two groups.
The dynamics of serum levels of IL-21and other cytokines in seven recovering HB-ACLF patients. With clinical improvement, the serum levels of IL-21(P=0.003), IL-1β (P=0.050), IL-8(P=0.004), IL-12p70(P=0.018) and IFN-y (P=0.015) decreased significantly, while the levels of IL-12/IL-23p40increased though without significance (P=0.104).
The correlations of serum levels of IL-21and other cytokines with the severity of diseases. The serum levels of IL-21correlated with MELD score (r=0.573, P<0.001), TBIL (r=0.566, P<0.001) and creatinine (CR, r=0.327, P=0.012) positively, while correlated with PTA (r=-0.520, P<0.001) and albumin (ALB, r=-0.369, P=0.001) negatively. There were no significant correlations between IL-21and HBV DNA load or ALT levels. For the other cytokines, IL-6, IL-8, IL-12p70, IL-17A, IL-12/12-23p40and IFN-y levels all correlated with TBIL significantly; IL-6, IL-8and IL-17A levels all correlated with MELD score, PTA and ALB; and the IL-4, IL-10and IFN-y levels correlated with CR values significantly.
The predictive value of serum cytokines on the outcome of HB-ACLF. The Logistic regression was carried out in HB-ALCF patients to build a diagnostic model with the outcome (survival or not) as dependent variables and the cytokines concentration as co-variables. There were six cytokines entered the regression equation with IL-21, IL-2, IL-10and TNF-a as risk factors and IL-17A and IL-12/IL-23p4protective factors for the poor outcome of HB-ACLF. With this new model, the outcome of84.6%(33/39) of HB-ALCF patients can be correctly discriminated, which was lower than that of MELD score (97.4%,38/39). The receptor's operating characteristics (ROC) curves was constructed for each cytokine, and the under-line area (A) was calculated. However, only IL-21(A=0.695, P=0.039), IL-2(A=0.695, P=0.039), IL-6(A=0.707, P=0.028) and IL-10(A-0.749, P=0.008) had diagnostic significances (P<0.005), though their power was still not comparable with MELD score (A=0.953, P<0.001).
Frequency changes of peripheral IL-21-secreting cells and CD4+Th subsets in HB-ACLF patients.
Comparison of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets among the four study groups. Globally, there were significant differences in the total frequencies of CD4+T cells secreting IL-21, IFN-y, IL-10and IL-17A. The frequencies of IL-21+CD4+T cell in HB-ACLF, IFN-γ+CD4+T cells in M-CHB, IL-10+CD4+T cells and IL-17A+CD4+T cells in S-CHB group reached topmost, respectively. And the multiple comparisons found that:the frequencies of IL-21+CD4+T cells were significantly higher in HB-ACLF and S-CHB when compared to M-CHB (P<0.001and P=0.002) and HC (P<0.001and P=0.001), but no significance achieved between HB-ACLF and S-CHB. M-CHB patients had significantly increased frequencies of IFN-y+CD4+T cells than S-CHB (P<0.001) and HB-ACLF (P=0.004) patients, though there was no significance between M-CHB and HC. The frequencies of IL-10+CD4+T cells elevated significantly in HB-ACLF and S-CHB when compared to M-CHB (both P<0.001) and HC (P=0.001and P=0.002), and they were even higher in S-CHB than in HB-ACLF (P<0.028). There were no significant differences in IL-17A+CD4+T cell frequencies in the HB-ACLF, S-CHB and M-CHB patients, but they were all higher than in the HC subjects (P=0.003, P=0.004, and P=0.017).
We analyzed the co-expression of IL-21with IFN-y, IL-10or IL-17A in CD4+T cells. Generally, the frequencies of IL-21+IFN-y+, IL-21+IL-17A+, IL-21+IL-10+and IL-21+IFN-γ-IL-10-IL-17A-CD4+T cells were significantly different among the four subject groups. In particular, the frequencies of IL-21+IL-10+CD4+T cells reached topmost in S-CHB group, while the other three IL21+CD4+T cell populations were all highest in HB-ACLF. The interclass comparisons showed that:the frequencies of IL-21+IFN-γ TL-10-IL-17A-CD4+T cells were significantly higher in HB-ACLF and S-CHB when compared to M-CHB (both P<0.001) and HC (P<0.001and P=0.001), however, no significance achieved between S-CHB and HC groups. The frequencies of IL-21+IFN-y+CD4+T increased gradually from HC to M-CHB, S-CHB and HB-ACLF, they were significantly lower in HC than in other three HBV infected groups (P=0.018, P=0.006and P<0.001), and they were also lower in M-CHB than in HB-ACLF (P=0.021), but no significance appeared between S-CHB and HB-ACLF groups. The frequencies of IL-21+IL-10+CD4+T cells increased significantly in S-CHB and HB-ACLF patients when compared to M-CHB patients (P<0.001and P=0.001) and HC subjects (P<0.001and P=0.002), and they were even higher in S-CHB than in HB-ACLF (P=0.030). The frequencies of IL-21+IL-17A+CD4+T cells elevated significantly in HB-ACLF when compared to M-CHB (P=0.004) and HC (P<0.001), and they were also higher in S-CHB than in HC (P=0.012).
Comparison of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets between HB-ACLF survivors and non-survivors. There were no significant differences in the frequencies of peripheral CD4+T cells secreting IL-21, IFN-γ, IL-10and IL-17A in HB-ACLF patients with different outcomes, neither the frequencies of IFN-γ, IL-10or IL-17A co-expressed IL-21+CD4+T cells.
The dynamics of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets in recovering HB-ACLF patients. With clinic improvement, the frequencies of IL-21+CD4+T cells decreased significantly in six recovering HB-ACLF patients (P<0.001), while the frequencies of IFN-γ+, IL-10+or IL-17A+CD4+T cells showed no significant changes during the first5-6weeks after admission. For the IFN-γ, IL-10or IL-17A co-expressed IL-21+CD4+T cells, we found the frequencies of IL-21+IL-10+(P=0.007) and IL-21+IFN-γ IL-10-IL-17A-(P<0.001) CD4+T cells went down significantly with recovering of HB-ACLF.
The correlations of peripheral IL-21-secreting cells and CD4+Th subsets with disease severity. The linear correlations of the frequencies of peripheral IL-21-secreting cells and CD4+Th subsets with clinic parameters reflecting the severity of disease were analyzed in53HBV infected (HB-ACLF, S-CHB or M-CHB) patients. The frequencies of IL-21+CD4T cells correlated with MELD score (r=0.605, P<0.001) and TBIL (r=-0.322, P=0.019) positively, while correlated with PTA (r=-0.322, P=0.019) and ALB (r=-0.587, P<0.00) negatively. For other Th subsets, the frequencies of IFN-γ+CD4+T cells correlated with ALT, CR and PTA positively, while correlated with MELD score and TBIL negatively; IL-10+CD4+T cells correlated with MELD score and TBIL positively, while correlated with ALB negatively; and IL-17A+CD4+T cells correlated with none of the parameters significantly.
The correlations of IFN-y, IL-10or IL-17A co-expressed IL-21+CD4+T cells with disease severity were further analyzed. Without the interference of co-expression, the frequencies of IL-21+IFN-γ-IL-10-IL-17A-CD4+T cells correlated with TBIL (r=0.733, P<0.001), MELD score (r=0.649, P<0.001), ALB (r=-0.674, P<0.001) and PTA (r=-0.377, P=0.005) more significantly. For the three co-expressed IL-21+CD4+T cell populations, the frequencies of IL-21+IFN-y+CD4+T cells correlated with TBIL and MELD score positively; IL-21+IL-10+CD4+and IL-21+IL-17A+CD4+T cells correlated with TBIL and MELD score positively, while correlated with ALB negatively; and no significant correlations existed between any other parameters.
The correlations of serum concentrations of IL-21, IFN-γ, IL-10and IL-17A with frequencies of related cytokine-producing cells. There were55subjects underwent both CBA measurement for the serum cytokines and ICS assay for the cytokine-producing cells. Spearman correlation was performed to find the association between these two assays. And we found only the serum concentrations of IL-21correlated with the frequencies of peripheral IL21+CD4+T cells positively (r=0.482, P<0.001). There were no significant correlations between serum IFN-y and IFN-y+CD4+cells (r=-0.067, P=0.627), serum IL-10and IL-10+CD4+T cells (r=0.114, P=0.406), serum IL-17A and IL-17A+CD4+T cells (r=0.073, P=0.596). It hints that IL-21in serum mainly produced by CD4+T cells, while the sources of IFN-γ, IL-10and IL-17A were relatively broad.
Effects of IL-21on the cytokines production from PBMC
The correlations of serum levels of IL-21with other cytokines. Cytokines in103sera samples were analyzed and we found IL-21significantly correlated with all the cytokines except IL-12/IL-23p40(r=-0.207, P=0.785). IL-21correlated with IL-1β (r=0.252, P=0.010), IL-2(r=0.426, P<0.001), IL-4(r=0.278, P=0.004), IL-6 (r=0.532, P<0.001), IL-8(r=0.2424, P<0.001), IL-10(r=0.207, P=0.036), IL-12p70(r=0.451, P<0.001), IL-17A (r=0.330, P=0.001), IFN-y (r=0.362, P<0.001) and TNF-a (r=0.303, P=0.002) positively.
In vitro effects of IL-21on the secretion of other cytokines from PBMC. With the stimuli of rhIL-21, both in HC and CHB subjects, the secretion of IL-1β (P=0.009and P<0.001), IL-6(P=0.006and P<0.001), IL-10(P=0.002and P<0.001), IFN-y (P=0.006and P=0.001) and TNF-a (both P=0.042) from PBMC increased significantly and showed a dose-dependent phenomenon. Although no significance achieved, rhIL-21could promote the production of IL-2and IL-12p70as well, while inhibit the expression of IL-8slightly.
Conclusion and Discussion
As compared to M-CHB and S-CHB patients, serum IL-21levels elevated significantly in HB-ACLF patients, and they were significantly higher in HB-ACLF non-survivors than in survivors. Further more, IL-21concentrations decreased with the recovery of HB-ACLF. It indicates that IL-21is possibly involved in the pathogenesis of HB-ACLF. Serum IL-21levels correlated significantly with clinic parameters (such as TBIL, PTA and MELD score) reflecting the severity of disease and could predict the outcome of HB-ACLF. This hints that serum IL-21may become a prospective biomarker which could reflect the severity of HB-ACLF and predict the outcome of patients.
The frequencies of peripheral IL-21-secreting cells were significantly increased in HB-ACLF and S-CHB patients when compared to M-CHB and HC subjects, they decreased with the recovery of HB-ACLF, and correlated with the severity of disease significantly. All these evidences demonstrate that IL-21-secreting cells have a causal role in sever liver inflammation.
In vitro rhIL-21could up-regulate the production of IL-1β, IL-6, IL-10, IFN-y and TNF-a significantly, which will sequentially result in the liver inflammation. It indicates that IL-21may participate in the liver inflammation by means of regulating other immune cells or up-regulating the expression of certain pro-inflammatory cytokines.
After all, we found IL-21was possibly involved in the pathogenesis of HB-ACLF, serum IL-21level could be a good index to assess the severity of disease and predict the outcome, and blocking IL-21pathway may be a potential target for the treatment of HB-ACLF.
慢加急性肝衰竭(ACLF)是指在既往明确诊断或未诊断的慢性肝病基础上,出现急性肝功能损害,表现为黄疸和凝血功能障碍,并且在4周内出现腹水或/和肝性脑病。在我国,90%以上ACLF病例存在HBV感染,被称为乙型肝炎相关慢加急性肝衰竭(HB-ACLF)。 HB-ACLF患者病情重,治疗费用高,预后不良,3个月生存率只有62%,目前唯一有效的治疗手段是肝移植。
目前认为HB-ACLF的发病取决于病毒和宿主两方面因素。一方面研究认为,HBV基因组前C区(PC)和C基因启动子(BCP)核苷酸变异可能引起病毒复制和抗原表达水平的改变,可能与乙型肝炎相关肝衰竭的发生有关。另一方面,更多的研究集中在HB-ACLF发病时机体免疫状况的改变,结果发现单核-巨噬细胞、树突状细胞、自然杀伤细胞、CD4+及CD8+T淋巴细胞、调节性T细胞、Th17细胞以及多种细胞因子都可能参与HB-ACLF发生和发展过程。
白细胞介素-21(IL-21)是由激活的CD4+T细胞和NKT细胞分泌的一种Ⅰ类细胞因子,广泛参与机体免疫活动的调控作用,能够影响多种免疫细胞的发育、活化、扩增和功能表达。研究发现IL-21与炎性肠病、系统性红斑狼疮、Ⅰ型糖尿病、银屑病和风湿性关节炎等多种炎症性、自身免疫性或风湿性疾病发病有关。而且越来越多的证据表明,IL-21参与机体的抗感染免疫,如人类免疫缺陷病毒感染时,IL-21能够促进NK和CD8+T细胞存活、增强其细胞毒活性。最近,来自HBV转基因鼠的研究发现,IL-21的分泌具有年龄依赖性；然而,IL-21在HBV感染导致的肝脏炎症活动,肝细胞损伤和病毒控制中的作用至今未明。
本研究通过检测HB-ACLF、重度和轻度慢性乙型肝炎(S-CHB和M-CHB)患者以及健康对照(HC)血清IL-21及相关细胞因子水平,IL-21分泌细胞及相关CD4+T细胞亚群的频数,以及IL-21对其它炎症因子分泌的影响,探索IL-21在HB-ACLF发病中的作用,并分析及其与疾病严重程度和预后的关系。
研究方法
第一部分：血清IL-21及相关细胞因子的检测
本部分纳入39例HB-ACLF、20例S-CHB、20例M-CHB患者和10例HC。HB-ACLF和S-CHB为2008年1月至2009年7月在南方医院感染内科和广州市第八人民医院住院患者。随访至2010年6月,HB-ACLF患者22例存活,16例死亡,1例接受原位肝移植；S-CHB患者全部存活。对其中7例入院后病情逐渐缓解的HB-ACLF患者进行动态观察,这些患者入院只接受常规内科对症治疗和核苷(酸)类似物(拉米夫定或恩替卡韦)抗HBV治疗,均未接受糖皮质激素和血浆置换术。
HB-ACLF的纳入标准为：(1)血清HBsAg阳性时间大于12个月；和(2)血清总胆红素(TBIL)>17、或每天上升>17.1μmol/L;和(3)凝血酶原活动度(PTA)<40%；和(4)起病(出现黄疸和凝血功能障碍)后4周内出现腹水或／和肝性脑病。
S-CHB的纳入标准为：(1)血清HBsAg阳性时间大于12个月；和(2)血清谷丙转氨酶(ALT)>600U/L;和(3)血清TBIL>51.3μmol/L;和(4)PTA<60%;但不足以诊断为HB-ACLF的患者。
M-CHB患者均为上述时间段南方医院门诊患者,纳入标准：(1)血清HBsAg阳性时间大于12个月；和(2)血清ALT<400U/L且>80U/L:和(3)血清TBIL <51-3μmol/L。
健康对照10例,均为南方医院感染内科实验室学生或工作人员,纳入标准：(1)血清HBsAg阴性；和(2)血清ALT<40U/L;和(3)血清TBIL<17.1μmol/L。
所有研究对象均排除以下情况：(1)年龄>60岁或<18岁；或(2)合并感染人类免疫缺陷病毒(HIV)、甲型肝炎病毒(HAV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)或戊型肝炎病毒(HEV)；或(3)合并甲状腺功能亢进、糖尿病等自身免疫性疾病；或(4)合并恶性肿瘤；或(5)半年内接受过干扰素、糖皮质激素、或其它免疫调节制剂；或(6)影像学或组织学发现明显肝硬化。
分离所有研究对象的血清-80℃贮存,采用流式微球试验(CBA)批量检测血清中包括IL-21在内的12种细胞因子的水平。
第二部分：外周IL-21分泌细胞的检测
本部分纳入21例HB-ACLF(存活14例,未存活7例)、13例S-CHB.19例M-CHB患者和10例HC,并对6例HB-ACLF恢复患者进行动态观察。有8例S-CHB为新增病例,为同期南方医院或广州市第八人民医院的住院患者；其余病例均来自第一部分试验。纳入和排除标准如上述。密度梯度离心法分离患者外周血单个核细胞(PBMC),采用细胞内因子染色(ICS)的方法经流式细胞仪检测外周IL-21分泌细胞及相关CD4+T细胞亚群的频数。
第三部分：IL-21对PBMC分泌其它细胞因子的影响
选择9例CHB患者和6例HC进行IL-21的体外诱导实验。CHB的诊断标准参考2010年新版《慢性乙型肝炎防治指南》。分离研究对象的PBMC,以不同浓度(0、10和100ng/mL)的重组人IL-21(rhIL-21)蛋白刺激培养24小时后,收集培养上清,采用Bender FlowCytomix系统检测11种细胞因子的浓度。
研究结果
第一部分：HB-ACLF患者血清II-21及相关细胞因子水平的变化。
不同人群血清IL-21及相关细胞因子浓度的比较。血清IL-21水平在HC. M-CHB. S-CHB和HB-ACLF依次升高,HB-ACLF患者显著高于其它三组人群(P<0.001,P<0.001和P=0.001),且S-CHB患者显著高于HC(P=0.042)。血清IL-6、IL-8、IL-10、IL-17A、IFN-γ和TNF-a水平在HB-ACLF亦有所升高,而IL-12/IL-23p40低于M-CHB,IL-1p、IL-2、IL-4和IL-12p70在四组人群间无显著差异。
HB-ACLF存活与未存活患者血清IL-21及相关细胞因子的比较。未存活组血清IL-21(P=0.024)与IL-2(P=0.021)、IL-6(P=0.028)和IL-10(P=0.008)浓度显著高于存活组患者。IL-12p70和IL-17A略高于存活组患者,但无统计学差异(P=0.106和P=0.130)。其它细胞因子两组间比较均无显著差异。
HB-ACLF恢复患者血清IL-21及相关细胞因子的动态变化。随着病情缓解,血清IL-21(P=0.003)与IL-1β (P=0.050)、IL-8(P=0.004)、IL-12p70(P=0.018).和IFN-γ(P=0.015)水平均逐渐下降,前后比较有统计学差异；而IL-12/IL-23p40则逐渐升高,尽管统计学差异不显著(P=0.104)。
血清IL-21及相关细胞因子水平与疾病严重程度的相关性。血清IL-21水平与MELD积分(r=0.573, P<0.001)、TBIL (r=0.566, P<0.001)、 PTA (r=-0.520, P<0.001)、白蛋白(ALB, r=-0.369, P=0.001)、肌酐值(CR, r=0.327, P=0.012)显著相关,而与HBV DNA和谷丙转氨酶(ALT)无显著相关性。其它细胞因子,与TBIL显著相关的有IL-6、IL-8、IL-12p70、IL-17A、IL-12/12-23p40和IFN-γ;与MELD积分、PTA和ALB显著相关的有IL-6、IL-8和IL-17A,与CR显著相关的有IL-4、IL-10和IFN-γ。
血清细胞因子对HB-ACLF临床结局的预测价值。以HB-ACLF患者两种不同临床结局(存活或未存活)为因变量,以12种细胞因子浓度为协变量进行Logistic回归分析,构建诊断模型。有6种细胞因子进入回归方程,其中IL-21. IL-2、IL-10和TNF-α为HB-ACLF死亡患者的危险因素,而IL-17A和IL-12/IL-23p40为保护因素。该模型对HB-ACLF患者临床结局的正确判断率为84.6%(33/39),低于MELD积分的正确判断率97.4%(38/39)。进一步对每种细胞因子构建受试者工作特征曲线(ROC),并计算曲线下面积(A),只有IL-21(A=0.695,P=0.039)与IL-2(A=0.718,P=0.021)、IL-6(A=0.707,P=0.028)和IL-10(A=0.749, P=0.008)的诊断意义具有显著性(P<0.05),但其诊断效能仍低于MELD积分(A=0.953,P<0.001)。
第二部分：HB-ACLF患者外周IL-21分泌细胞及相关CD4+T细胞亚群的频数变化。
不同人群IL-21分泌细胞及CD4+T细胞亚群的频数比较。四组人群分泌IL-21、IFN-γ、IL-10或IL-17A的总CD4+T细胞频数整体比较均有显著差异,IL-21+CD4+T细胞在HB-ACLF组最高,IFN-y+CD4+T细胞频数在M-CHB组最高,而IL-10+CD4+T细胞和IL-17A+CD4+T细胞颇数存S-CHB织最高。名重比较发现：HB-ACLF和S-CHB患者的IL-21+CD4+T细胞频数均显著高于M-CHB患者(P<0.001和P=0.002)和HC(P<0.001和P=0.001),而HB-ACLF与S-CHB患者之间无显著差异。M-CHB患者IFN-γ+CD4+T细胞频数显著高于S-CHB(P<0.001)和HB-ACLF (P=0.004)患者,而与HC比较差异无显著性。S-CHB和HB-ACLF患者IL-10+CD4+T细胞频数显著高于M-CHB患者(均P<0.001)和HC(P=0.001和P=0.002),且S-CHB患者显著高于HB-ACLF患者(P=0.028)。三组HBV感染患者之间的IL-17A+CD4+T细胞频数相互比较无显著差异,但均显著高于HC (HB-ACLF, P=0.003; S-CHB, P=0.004; M-CHB, P=0.017)。
进一步对IL-21+CD4+T细胞共表达IFN-γ、IL-10和IL-17A的情况进行分析发现：EL-21+IFN-γ+、IL-21+IL-17A+、IL-21+IL-10+和IL-21+IFN-γ-IL-10IL-17A-的CD4+T细胞亚群在四组人群间整体比较均有显著差异。除IL-21+IL-10+CD4+T细胞亚群的频数在S-CHB最高外,另外三个IL21+CD4+T细胞亚群的频数均以HB-ACLF患者最高。四组人群之间多重比较：HB-ACLF和S-CHB患者的IL-21+IFN-γ-IL-10IL-17A-CD4+T细胞亚群频数显著高于M-CHB患者(均P<0.001)和HC(P<0.001和P=0.001),而S-CHB与HB-ACLF无显著差异。IL-21+IFN-γ+CD4+T细胞亚群频数在HC、M-CHB、S-CHB和HB-ACLF患者递增,HC显著低于三组HBV感染患者(P=0.018,P=0.006和P<0.001),且M-CHB患者显著低于HB-ACLF患者(P=0.021),而S-CHB与HB-ACLF患者比较无显著差异。S-CHB和HB-ACLF患者的IL-21+IL-10+CD4+T细胞亚群频数显著高于M-CHB患者(P<0.001和P=0.001)和HC(P<0.001和P=0.002),且S-CHB高于HB-ACLF患者(P=0.030)。HB-ACLF患者的IL-21+IL-17A+CD4+T细胞亚群频数显著高于M-CHB患者(P=0.004)和HC (P<0.001), S-CHB患者亦高于HC(P=0.012),而其余各组间比较无差异。
HB-ACLF存活与未存活患者IL-21分泌细胞及CD4+T细胞亚群的比较。入院时存活与死亡患者外周分泌IL-21. IFN-γ. IL-10或IL-17A的CD4+T细胞频数均无显著差异。进一步分析IL-21与IFN-γ、IL-10和IL-17A共表达的CD4+T细胞频数在两组间亦无显著差异。
HB-ACLF恢复患者外周IL-21分泌细胞及CD4+T细胞亚群的动态变化。6例恢复期HB-ACLF患者外周IL-21+CD4+T细胞频数随病情缓解而逐渐下降,前后变化具有显著意义(P<0.001)；而分泌IFN-γ、IL-10或IL-17A的CD4+T细胞亚群频数在病程中的变化无统计学差异。进一步对IL-21与IFN-γ、IL-10或IL-17A共表达CD4+T细胞亚群频数的动态变化进行分析,发现IL-21+IL-10+(P=0.007)和IL-21+IFN-γ-IL-10-IL-17A-(P<0.001) CD4+T细胞频数前后比较具有统计学差异,均随疾病恢复而下降；余无显著差异。
外周IL-21分泌细胞及CD4+T细胞亚群与疾病严重程度的相关性。对进行ICS检测的53例处于不同疾病状态(HB-ACLF. S-CHB或M-CHB)的HBV感染者的外周IL-21分泌细胞及CD4+T细胞亚群的频数与反映疾病严重程度的临床参数进行相关性分析。IL-21CD4+T细胞频数与MELD积分(r=0.605,P<0.001)、TBIL (r=0.656, P<0.001)、PTA (r=-0.322, P=0.019)和ALB (r=-0.587, P<0.001)均显著相关,而与HBVDNA、ALT和CR的无显著相关性。IFN-y+CD4+T细胞频数与ALT、TBIL和MELD积分呈负相关,而与ALB、CR和PTA呈正相关。IL-10+CD4+T细胞频数与TBIL和MELD积分呈正相关,而与ALB呈负相关。IL-17A+CD4+T细胞频数与各临床指标均无显著相关性。
对IL-21与IFN-γ、IL-10和IL-17A的共表达CD4+T细胞与疾病严重程度的相关性进行分析发现：在排除上述三种共表达细胞的干扰后,IL-21+IFN-γ-IL-10-IL-17A-CD4+T细胞频数与TBIL (r=0.733, P<0.001)、MELD积分(1-0.649, P<0.001)、ALB (r=-0.674, P<0.001)和PTA (r=-0.377, P=0.005)的相关性更为显著,相关系数更高。三种共表达细胞,IL-21+IFN-γ+CD4+T细胞频数与MELD积分呈正相关；IL-21+IL-10+CD4+和IL-21+IL-17A+CD4+T细胞频数与TBIL和MELD积分呈正相关,而与ALB呈负相关；其余各指标间无显著相关性。
血清IL-21. IFN-γ、IL-10、IL-17A浓度与外周相应因子分泌细胞频数的相关性。共有55例研究对象同时接受了CBA检测血清中IL-21、IFN-γ、IL-10、IL-17A蛋白浓度和ICS检测外周分泌四种细胞因子的CD4+T细胞频数。对两种检测的结果进行Spearman相关性分析,发现只有血清IL-21浓度与IL21+CD4+T细胞频数呈正相关(r=0.482, P<0.001);IFN-y与IFN-y+CD4+T细胞(r=-0.067, P=0.627)、IL-10与IL-10+CD4+T细胞(r=0.114,P=0.406). IL-17A与IL-17A+CD4+T细胞(r=0.073,P=0.596)均无显著相关性。说明外周血中的IL-21主要由CD4+T细胞分泌,而IFN-γ、IL-10和IL-17A的来源则相对广泛。
第三部分：IL-21对PBMC分泌其它细胞因子的影响。
血清IL-21水平与其它细胞因子浓度的相关性分析。对第一部分CBA检测103份血清中各细胞因子的浓度进行相关性分析,发现IL-21与IL-1p(r=0.252,P=0.010)、IL-2(r=0.426, P<0.001)、IL-4(r=0.278, P=0.004)、IL-6(r=0.532, P<0.001)、IL-8(r=0.2424, P<0.001)、IL-10(r=0.207, P=0.036)、IL-12p70(r=0.451, P<0.001)、IL-17A (r=0.330, P=0.001)、IFN-γ (r-0.362, P<0.001)和TNF-α (r=0.303,P=0.002)均呈正相关,但与IL-12/IL-23p40(r=-0.207, P=0.785)无显著相关性。
IL-21体外对PBMC分泌其它细胞因子的影响。无论是对HC还是CHB患者的PBMC, rhIL-21均能显著上调其IL-1p(P=0.009和P<0.001)、IL-6(P=0.006和P<0.001)、IL-10(P=0.002和P<0.001)、IFN-γ(P=0.006和P=0.001)和TNF-α(均P=0.042)的表达,并呈现剂量依赖效应。rhIL-21对IL-2和IL-12p70的分泌也有轻微促进作用,而对IL-8则有轻微抑制作用,尽管两者均无统计学差异。
结论与讨论
相比于M-CHB和S-CHB患者,HB-ACLF患者血清IL-21水平显著升高,且未存活患者高于存活患者,而随着病情恢复,血清IL-21水平又逐渐下降,说明IL-21参与HB-ACLF的病理生理过程。血清IL-21水平与反映肝脏疾病严重程度的临床指标(如TBIL. PTA和MELD积分等)显著相关,并且对HB-ACLF的预后具有一定的判断价值,提示血清IL-21有可能作为反映HB-ACLF肝脏疾病严重性和判断预后的新的生物学指标。
HB-ACLF和S-CHB患者外周IL-21分泌细胞的频数显著高于M-CHB和HC,随病情恢复而逐渐减少,并且与反映肝脏疾病严重程度的临床指标相关,进一步说明IL-21分泌细胞可能与肝脏严重的炎症活动有关。
体外rhIL-21能够促进IL-1β、IL-6、IL-10、IFN-γ和TNF-α的分泌,提示IL-21可能通过调节其它免疫细胞功能、上调前炎症因子的表达的方式参与肝脏炎症活动。
上述证据表明,IL-21参与HB-ACLF的病理生理过程,血清IL-21水平有可能作为评估疾病严重性和判断预后的良好指标,阻断IL-21通路或许是早期干预HB-ACLF发生发展的一个新的治疗靶点。
Bacground and Aims
Acute-on-chronic liver failure (ACLF) is an acute hepatic insult, manifesting as jaundice and coagulopathy, and complicated within four weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. In china, more than90%of ACLF cases were infected with HBV, known as hepatitis B-related ACLF (HB-ACLF). HB-ACLF patients are usually severe, with high cost of treatment and poor prognosis. The three-month survival rate of them is no more than62%. Currently, the only effective treatment is orthotopic liver transplantation (OLT).
Until now, the pathogenesis of HB-ACLF is considered to dependent on two aspects, namely, viral factors and host factors. Studies suggested that, nucleotide mutations in BCP and PC regions of HBV genome may cause changes of viral replication and antigen expression, resulting in the occurrence of hepatitis and liver failure. More researchs focused on the inflammation and immune status changes in HB-ACLF, and they found that the monocytes-macrophages, dendritic cells, natural killer cells, CD4+and CD8+T lymphocytes, regulatory T cells, Th17cells and many kinds of cytokines may participate in the pathophysiological process of HB-ACLF.
Interleukin-21(IL-21) is produced by activated CD4+T cells and NKT cells, belonging to the type I cytokines family. IL-21participates in the regulation of immune activity broadly and it can influence the development, activation, expansion and function of a variety of immune cell. Studies found that IL-21was associated with inflammatory bowel disease, systemic lupus erythematosus, type I diabetes, psoriasis, rheumatoid arthritis, and other inflammatory, autoimmune or rheumatic diseases. More and more evidence suggested that IL-21was involved in the anti-infection immunity, such as the human immunodeficiency virus (HIV) infection, by promoting the survival of NK and CD8+T cells, and enhance their cytotoxic activity. Recently, a study on HBV transgenic mouse model showed that the production of IL-21was age-dependent. However, the role of IL-21in the pathogenesis of liver inflammatory injury caused by HBV infection remains largely unknown.
By assaying the serum levels of IL-21and other cytokines, the frequencies of peripheral IL-21-secreting cells and CD4+Th subsets in HB-ACLF, severe and moderate chronic hepatitis B (S-CHB and M-CHB) patients and health controls (HC), and the effects of IL-21on the production of other inflammatory factor, this study was designed to explore the potential role of IL-21in the pathogenesis of HB-ACLF and its association with disease severity and prognosis.
Materials and Methods
Measurement of serum IL-21and other cytokines
Thirty-nine cases of HB-ACLF,20cases of S-CHB,20cases of M-CHB and10HC subjects were included in this part of study. The HB-ACLF and S-CHB cases are hospitalized patients admitted to Nanfang Hospital or The Eighth People's Hospital of Guangzhou Branch from January2008to July2009. Followed up to June2010,22HB-ACLF patients survived,16patients died, and one underwent OLT, while all the S-CHB patients survived during that period. Seven HB-ACLF patients who got clinical improvement between week1and week5-6after admission were observed continuously. These seven patients were all treated with nucleos(t)ide analogues (Lamivudine or Entecavir), and none received glucocorticiod or plasma-exchange therapy.
The including criteria for HB-ACLF were:(1) positive for serum HBsAg more than12months; and (2) serum total bilirubin (TBIL) of>171μmol/L or rapidly rising levels of TB (TB>17.1μmol/L/day; and (3) plasma prothrombin activity (PTA) of<40%; and (4) the complications of ascites and/or hepatic encephalopathy within4weeks of the first evidence of jaundice or coagulopathy.
The including criteria for S-CHB were:(1) positive for serum HBsAg more than12months; and (2) serum alanine aminotransferase (ALT) of>600IU/L; and (3) serum TBIL of>51.3μmol/L; and (4) plasma prothrombin activity (PTA) of<60%; while not enough to be diagnosed as HB-ACLF.
The M-CHB group was comprised of20out-patients who presented to Nanfang Hospital between2008and2009. The including criteria for them were:(1) positive for serum HBsAg more than12months; and (2) elevated serum ALT levels (80-40IU/L); and (3) serum TBIL of<51.3μmol/L.
HC group was comprised of students or staff in the Laboratory of Infectious Diseases, Nanfang Hospital. The including criteria for them were:(1) negative for serum HBsAg; and (2) normal serum ALT levels (<40IU/L); and (3) normal serum TBIL levels (<17.1μmol/L).
The excluding criteria for all subjects were:(1) more than60or less than18 years old; or (2) co-infection with HIV, hepatitis A virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus; or (3) co-occurrence of hyperthyroidism, diabetes or other autoimmune diseases; or (4) co-occurrence of malignant neoplasm; or (5) received interferon, glucocorticiod, or other immune modulators; or (6) obvious cirrhosis by imaging or histology.
Serum from all subjects were isolated and stored at-80℃until use. Twelve cytokines including IL-21were detected by Cytometric Beads Array.
Assay of the peripheral IL-21-secreting cells
Twenty-one cases of HB-ACLF (14survived and7died),13cases of S-CHB,19cases of M-CHB and10HC subjects were included into the second part of study. And6recovering HB-ACLF patients were observed continuously. All the subjects were from the Part I except8S-CHB patients, who were newly recruited into this study. The including and excluding criteria were the same as Part I. Peripheral blood mononuclear cells (PBMC) from all subjects were isolated with density gradient centrifugation. The frequencies of IL-21-secreting cells and CD4+Th subsets were assayed by the method of intracellular cytokine staining.
The effects of IL-21on the production of other cytokines from PBMC
Nine CHB patients and6HC subjects were included into the third part of study for IL-21inducding experiment. The diagnosis of CHB was made according to the Chinese Guideline for Chronic Hepatitis B (2010version).PBMC were isolated and stimulated with recombinant human IL-21(rhIL-21) at different concentrations (0,10or100ng/mL) for24hours. Then11cytokines in the supernatants were measured by Bender FlowCytomix
Results
Serum levels of IL-21and other cytokines in HB-ACLF patients
Comparison of serum levels of IL-21and other cytokines in the study groups. The serum levels of IL-21increased gradually from HC to M-CHB, S-CHB and HB-ACLF, and they were significantly higher in HB-ACLF than in other three groups (P<0.001, P<0.001and P=0.001), and also higher in S-CHB than in HC (P=0.042). Serum levels of IL-6, IL-8, IL-10, IL-17A, IFN-γ and TNF-a increased in HB-ACLF as well, while the levels of IL-12/IL-23p40reached topmost in M-CHB patients. However, there were no significant differences in the serum levels of IL-1β, IL-2, IL-4or IL-12p70among the four study groups.
Comparison of serum levels of IL-21and other cytokines in HB-ACLF survivors and non-survivors. The serum levels of IL-21(P=0.024), IL-2(P=0.021), IL-6(P=0.028) and IL-10(P=0.008) were significantly higher in HB-ACLF non-survivors than in survivors. Though without significance, the IL-12p70and IL-17A levels were also elevated in the non-survivors, while the other cytokines were of no difference between the two groups.
The dynamics of serum levels of IL-21and other cytokines in seven recovering HB-ACLF patients. With clinical improvement, the serum levels of IL-21(P=0.003), IL-1β (P=0.050), IL-8(P=0.004), IL-12p70(P=0.018) and IFN-y (P=0.015) decreased significantly, while the levels of IL-12/IL-23p40increased though without significance (P=0.104).
The correlations of serum levels of IL-21and other cytokines with the severity of diseases. The serum levels of IL-21correlated with MELD score (r=0.573, P<0.001), TBIL (r=0.566, P<0.001) and creatinine (CR, r=0.327, P=0.012) positively, while correlated with PTA (r=-0.520, P<0.001) and albumin (ALB, r=-0.369, P=0.001) negatively. There were no significant correlations between IL-21and HBV DNA load or ALT levels. For the other cytokines, IL-6, IL-8, IL-12p70, IL-17A, IL-12/12-23p40and IFN-y levels all correlated with TBIL significantly; IL-6, IL-8and IL-17A levels all correlated with MELD score, PTA and ALB; and the IL-4, IL-10and IFN-y levels correlated with CR values significantly.
The predictive value of serum cytokines on the outcome of HB-ACLF. The Logistic regression was carried out in HB-ALCF patients to build a diagnostic model with the outcome (survival or not) as dependent variables and the cytokines concentration as co-variables. There were six cytokines entered the regression equation with IL-21, IL-2, IL-10and TNF-a as risk factors and IL-17A and IL-12/IL-23p4protective factors for the poor outcome of HB-ACLF. With this new model, the outcome of84.6%(33/39) of HB-ALCF patients can be correctly discriminated, which was lower than that of MELD score (97.4%,38/39). The receptor's operating characteristics (ROC) curves was constructed for each cytokine, and the under-line area (A) was calculated. However, only IL-21(A=0.695, P=0.039), IL-2(A=0.695, P=0.039), IL-6(A=0.707, P=0.028) and IL-10(A-0.749, P=0.008) had diagnostic significances (P<0.005), though their power was still not comparable with MELD score (A=0.953, P<0.001).
Frequency changes of peripheral IL-21-secreting cells and CD4+Th subsets in HB-ACLF patients.
Comparison of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets among the four study groups. Globally, there were significant differences in the total frequencies of CD4+T cells secreting IL-21, IFN-y, IL-10and IL-17A. The frequencies of IL-21+CD4+T cell in HB-ACLF, IFN-γ+CD4+T cells in M-CHB, IL-10+CD4+T cells and IL-17A+CD4+T cells in S-CHB group reached topmost, respectively. And the multiple comparisons found that:the frequencies of IL-21+CD4+T cells were significantly higher in HB-ACLF and S-CHB when compared to M-CHB (P<0.001and P=0.002) and HC (P<0.001and P=0.001), but no significance achieved between HB-ACLF and S-CHB. M-CHB patients had significantly increased frequencies of IFN-y+CD4+T cells than S-CHB (P<0.001) and HB-ACLF (P=0.004) patients, though there was no significance between M-CHB and HC. The frequencies of IL-10+CD4+T cells elevated significantly in HB-ACLF and S-CHB when compared to M-CHB (both P<0.001) and HC (P=0.001and P=0.002), and they were even higher in S-CHB than in HB-ACLF (P<0.028). There were no significant differences in IL-17A+CD4+T cell frequencies in the HB-ACLF, S-CHB and M-CHB patients, but they were all higher than in the HC subjects (P=0.003, P=0.004, and P=0.017).
We analyzed the co-expression of IL-21with IFN-y, IL-10or IL-17A in CD4+T cells. Generally, the frequencies of IL-21+IFN-y+, IL-21+IL-17A+, IL-21+IL-10+and IL-21+IFN-γ-IL-10-IL-17A-CD4+T cells were significantly different among the four subject groups. In particular, the frequencies of IL-21+IL-10+CD4+T cells reached topmost in S-CHB group, while the other three IL21+CD4+T cell populations were all highest in HB-ACLF. The interclass comparisons showed that:the frequencies of IL-21+IFN-γ TL-10-IL-17A-CD4+T cells were significantly higher in HB-ACLF and S-CHB when compared to M-CHB (both P<0.001) and HC (P<0.001and P=0.001), however, no significance achieved between S-CHB and HC groups. The frequencies of IL-21+IFN-y+CD4+T increased gradually from HC to M-CHB, S-CHB and HB-ACLF, they were significantly lower in HC than in other three HBV infected groups (P=0.018, P=0.006and P<0.001), and they were also lower in M-CHB than in HB-ACLF (P=0.021), but no significance appeared between S-CHB and HB-ACLF groups. The frequencies of IL-21+IL-10+CD4+T cells increased significantly in S-CHB and HB-ACLF patients when compared to M-CHB patients (P<0.001and P=0.001) and HC subjects (P<0.001and P=0.002), and they were even higher in S-CHB than in HB-ACLF (P=0.030). The frequencies of IL-21+IL-17A+CD4+T cells elevated significantly in HB-ACLF when compared to M-CHB (P=0.004) and HC (P<0.001), and they were also higher in S-CHB than in HC (P=0.012).
Comparison of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets between HB-ACLF survivors and non-survivors. There were no significant differences in the frequencies of peripheral CD4+T cells secreting IL-21, IFN-γ, IL-10and IL-17A in HB-ACLF patients with different outcomes, neither the frequencies of IFN-γ, IL-10or IL-17A co-expressed IL-21+CD4+T cells.
The dynamics of frequencies of peripheral IL-21-secreting cells and CD4+Th subsets in recovering HB-ACLF patients. With clinic improvement, the frequencies of IL-21+CD4+T cells decreased significantly in six recovering HB-ACLF patients (P<0.001), while the frequencies of IFN-γ+, IL-10+or IL-17A+CD4+T cells showed no significant changes during the first5-6weeks after admission. For the IFN-γ, IL-10or IL-17A co-expressed IL-21+CD4+T cells, we found the frequencies of IL-21+IL-10+(P=0.007) and IL-21+IFN-γ IL-10-IL-17A-(P<0.001) CD4+T cells went down significantly with recovering of HB-ACLF.
The correlations of peripheral IL-21-secreting cells and CD4+Th subsets with disease severity. The linear correlations of the frequencies of peripheral IL-21-secreting cells and CD4+Th subsets with clinic parameters reflecting the severity of disease were analyzed in53HBV infected (HB-ACLF, S-CHB or M-CHB) patients. The frequencies of IL-21+CD4T cells correlated with MELD score (r=0.605, P<0.001) and TBIL (r=-0.322, P=0.019) positively, while correlated with PTA (r=-0.322, P=0.019) and ALB (r=-0.587, P<0.00) negatively. For other Th subsets, the frequencies of IFN-γ+CD4+T cells correlated with ALT, CR and PTA positively, while correlated with MELD score and TBIL negatively; IL-10+CD4+T cells correlated with MELD score and TBIL positively, while correlated with ALB negatively; and IL-17A+CD4+T cells correlated with none of the parameters significantly.
The correlations of IFN-y, IL-10or IL-17A co-expressed IL-21+CD4+T cells with disease severity were further analyzed. Without the interference of co-expression, the frequencies of IL-21+IFN-γ-IL-10-IL-17A-CD4+T cells correlated with TBIL (r=0.733, P<0.001), MELD score (r=0.649, P<0.001), ALB (r=-0.674, P<0.001) and PTA (r=-0.377, P=0.005) more significantly. For the three co-expressed IL-21+CD4+T cell populations, the frequencies of IL-21+IFN-y+CD4+T cells correlated with TBIL and MELD score positively; IL-21+IL-10+CD4+and IL-21+IL-17A+CD4+T cells correlated with TBIL and MELD score positively, while correlated with ALB negatively; and no significant correlations existed between any other parameters.
The correlations of serum concentrations of IL-21, IFN-γ, IL-10and IL-17A with frequencies of related cytokine-producing cells. There were55subjects underwent both CBA measurement for the serum cytokines and ICS assay for the cytokine-producing cells. Spearman correlation was performed to find the association between these two assays. And we found only the serum concentrations of IL-21correlated with the frequencies of peripheral IL21+CD4+T cells positively (r=0.482, P<0.001). There were no significant correlations between serum IFN-y and IFN-y+CD4+cells (r=-0.067, P=0.627), serum IL-10and IL-10+CD4+T cells (r=0.114, P=0.406), serum IL-17A and IL-17A+CD4+T cells (r=0.073, P=0.596). It hints that IL-21in serum mainly produced by CD4+T cells, while the sources of IFN-γ, IL-10and IL-17A were relatively broad.
Effects of IL-21on the cytokines production from PBMC
The correlations of serum levels of IL-21with other cytokines. Cytokines in103sera samples were analyzed and we found IL-21significantly correlated with all the cytokines except IL-12/IL-23p40(r=-0.207, P=0.785). IL-21correlated with IL-1β (r=0.252, P=0.010), IL-2(r=0.426, P<0.001), IL-4(r=0.278, P=0.004), IL-6 (r=0.532, P<0.001), IL-8(r=0.2424, P<0.001), IL-10(r=0.207, P=0.036), IL-12p70(r=0.451, P<0.001), IL-17A (r=0.330, P=0.001), IFN-y (r=0.362, P<0.001) and TNF-a (r=0.303, P=0.002) positively.
In vitro effects of IL-21on the secretion of other cytokines from PBMC. With the stimuli of rhIL-21, both in HC and CHB subjects, the secretion of IL-1β (P=0.009and P<0.001), IL-6(P=0.006and P<0.001), IL-10(P=0.002and P<0.001), IFN-y (P=0.006and P=0.001) and TNF-a (both P=0.042) from PBMC increased significantly and showed a dose-dependent phenomenon. Although no significance achieved, rhIL-21could promote the production of IL-2and IL-12p70as well, while inhibit the expression of IL-8slightly.
Conclusion and Discussion
As compared to M-CHB and S-CHB patients, serum IL-21levels elevated significantly in HB-ACLF patients, and they were significantly higher in HB-ACLF non-survivors than in survivors. Further more, IL-21concentrations decreased with the recovery of HB-ACLF. It indicates that IL-21is possibly involved in the pathogenesis of HB-ACLF. Serum IL-21levels correlated significantly with clinic parameters (such as TBIL, PTA and MELD score) reflecting the severity of disease and could predict the outcome of HB-ACLF. This hints that serum IL-21may become a prospective biomarker which could reflect the severity of HB-ACLF and predict the outcome of patients.
The frequencies of peripheral IL-21-secreting cells were significantly increased in HB-ACLF and S-CHB patients when compared to M-CHB and HC subjects, they decreased with the recovery of HB-ACLF, and correlated with the severity of disease significantly. All these evidences demonstrate that IL-21-secreting cells have a causal role in sever liver inflammation.
In vitro rhIL-21could up-regulate the production of IL-1β, IL-6, IL-10, IFN-y and TNF-a significantly, which will sequentially result in the liver inflammation. It indicates that IL-21may participate in the liver inflammation by means of regulating other immune cells or up-regulating the expression of certain pro-inflammatory cytokines.
After all, we found IL-21was possibly involved in the pathogenesis of HB-ACLF, serum IL-21level could be a good index to assess the severity of disease and predict the outcome, and blocking IL-21pathway may be a potential target for the treatment of HB-ACLF.
引文
[1]中华医学会感染病学分会肝衰竭与人工肝学组.肝衰竭诊疗指南[J].中华肝脏病杂志,2006,14(9)：643-6.
[2]Sarin S, Kumar A, Almeida J, et al. Acute-on-chronic liver failure:consensus recommendations of the Asian Pacific Association for the study of the liver (APASL)[J]. Hepatol Int,2009,3(1):269-82.
[3]刘晓燕,胡瑾华,王慧芬,等.1977例急性、亚急性、慢加急性肝衰竭患者的病因与转归分析[J].中华肝脏病杂志,2008,16(10)：772-5.
[4]胡瑾华,黄坤,王慧芬,等.乙型肝炎相关慢加急性肝衰竭190例生存分析[J].传染病信息,2010,23(2)：83-6.
[5]Cui Y, Yan F, Wang Y, et al. Nucleoside analogue can improve the long-term prognosis of patients with hepatitis B virus infection-associated acute on chronic liver failure[J]. Dig Dis Sci,2010,55(8):2373-80.
[6]Ren X, Xu Z, Liu Y, et al. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis [J]. J Viral Hepat,2010,17(12):887-95.
[7]Xu Z, Ren X, Liu Y, et al. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease:an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure[J]. J Gastroenterol,2010,46(3):391-400.
[8]Yang L, Ma S, Hu X, et al. Presence of valine at position 27 of the hepatitis B virus core gene is associated with severe liver inflammation in Chinese patients[J]. J Med Virol,2011,83(2):218-24.
[9]Heydtmann M. Macrophages in hepatitis B and hepatitis C virus infections [J]. J Virol,2009,83(7):2796-802.
[10]Xing T, Li L, Cao H, et al. Altered immune function of monocytes in different stages of patients with acute on chronic liver failure[J]. Clin Exp Immunol, 2007,147(1):184-8.
[11]Zou Z, Li B, Xu D, et al. unbalanced intrahepatic cytokine expression of interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis B virus infection[J]. J Clin Gastroenterol, 2009,43(2):182-90.
[12]Shi F, Zhang J, Zeng Z, et al. Skewed ratios between CD3(+) T cells and monocytes are associated with poor prognosis in patients with HBV-related acute-on-chronic liver failure[J]. Biochem Biophys Res Commun,2010, 402(1):30-6.
[13]Lambotin M, Raghuraman S, Stoll-Keller F, et al. A look behind closed doors: interaction of persistent viruses with dendritic cells[JJ. Nat Rev Microbiol,2010, 8(5):350-60.
[14]Zhang Z, Zou Z S, Fu J L, et al. Severe dendritic cell perturbation is actively involved in the pathogenesis of acute-on-chronic hepatitis B liver failure[J]. J Hepatol,2008,49(3):396-406.
[15]Li N, Li Q, Qian Z, et al. Impaired TLR3/IFN-beta signaling in monocyte-derived dendritic cells from patients with acute-on-chronic hepatitis B liver failure:relevance to the severity of liver damage[J]. Biochem Biophys Res Commun,2009,390(3):630-5.
[16]Mondelli M U, Varchetta S, Oliviero B. Natural killer cells in viral hepatitis: facts and controversies[J]. Eur J Clin Invest,2010,40(9):851-63.
[17]Fisicaro P, Valdatta C, Boni C, et al. Early kinetics of innate and adaptive immune responses during hepatitis B virus infection[J]. Gut,2009, 58(7):974-82.
[18]Dunn C, Peppa D, Khanna P, et al. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection[J]. Gastroenterology, 2009,137(4):1289-300.
[19]Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand and NKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure[J]. J Immunol,2010,184(1):466-75.
[20]Zou Z, Xu D, Li B, et al. Compartmentalization and its implication for peripheral immunologically-competent cells to the liver in patients with HBV-related acute-on-chronic liver failure [J]. Hepatol Res,2009, 39(12):1198-207.
[21]Zhu J, Paul W E. CD4 T cells:fates, functions, and faults[J]. Blood,2008, 112(5):1557-69.
[22]Mills K H. Induction, function and regulation of IL-17-producing T cells [J]. Eur J Immunol,2008,38(10):2636-49.
[23]Xu D, Fu J, Jin L, et al. Circulating and liver resident CD4+CD25+regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B[J]. J Immunol,2006,177(1):739-47.
[24]黄海英,李树臣,于建武,等.慢性乙型重型肝炎患者外周血调节性T细胞表达[J].哈尔滨医药,2009,29(1)：2-4.
[25]Wang L, Chen H, Gong Z. High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B[J]. Hepatobiliary Pancreat Dis Int,2010,9(5):499-507.
[26]Harrington L, Hatton R, Mangan P, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[J]. Nat Immunol,2005,6(11):1123-32.
[27]Park H, Li Z, Yang X, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17[J]. Nat Immunol,2005, 6(11):1133-41.
[28]Ye P, Rodriguez F, Kanaly S, et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense[J]. J Exp Med,2001, 194(4):519-27.
[29]Korn T, Bettelli E, Oukka M, et al. IL-17 and Th17 Cells[J]. Annu Rev Immunol,2009,27(485-517.
[30]Ivanov, Ⅱ, McKenzie B S, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells[J]. Cell,2006,126(6):1121-33.
[31]Lafdil F, Miller A, Ki S, et al. Thl7 cells and their associated cytokines in liver diseases[J]. Cell Mol Immunol,2010,7(4):250-4.
[32]Ge J, Wang K, Meng Q, et al. Implication of Th17 and Thl cells in patients with chronic active hepatitis B[J]. J Clin Immunol,2010,30(1):60-7.
[33]Zhang J, Zhang Z, Lin F, et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B[J]. Hepatology,2010,51(1):81-91.
[34]Ye Y, Xie X, Yu J, et al. Involvement of Th17 and Th1 effector responses in patients with Hepatitis B[J]. J Clin Immunol,2010,30(4):546-55.
[35]Zhang J, Song C, Shi F, et al. Decreased ratio of Treg cells to Th17 cells correlates with HBV DNA suppression in chronic hepatitis B patients undergoing entecavir treatment[J]. PLoS One,2010,5(11):e13869.
[36]Li J, Wu W, Peng G, et al. HBcAg induces interleukin-10 production, inhibiting HBcAg-specific Th17 responses in chronic hepatitis B patients [J]. Immunol Cell Biol,2010,88(8):834-41.
[37]Wang L, Chen S, Xu K. IL-17 expression is correlated with hepatitis B-related liver diseases and fibrosis[J]. Int J Mol Med,2011,27(3):385-92.
[38]Zhang J, Yan J, Xu J, et al. Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients[J]. J Hepatol, 2009,50(5):980-9.
[39]Han T, Liu Y, Liu H, et al. Serum thymosin beta4 levels in patients with hepatitis B virus-related liver failure[J]. World J Gastroenterol,2010, 16(5):625-30.
[40]Zhu C, Yan W, Zhu F, et al. Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B[J]. World J Gastroenterol,2005, 11(44):6936-40.
[41]Monteleone G, Pallone F, Macdonald T. Interleukin-21 (IL-21)-mediated pathways in T cell-mediated disease[J]. Cytokine Growth Factor Rev,2009, 20(2):185-91.
[42]Taniguchi T. Cytokine signaling through nonreceptor protein tyrosine kinases[J]. Science,1995,268(5208):251-5.
[43]Muneta Y, Kikuma R, Uenishi H, et al. Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21 [J]. J Vet Med Sci, 2004,66(3):269-75.
[44]Parrish-Novak J, Dillon S R, Nelson A, et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function[J]. Nature, 2000,408(6808):57-63.
[45]Ozaki K, Kikly K, Michalovich D, et al. Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain[J]. Proc Natl Acad Sci U S A,2000, 97(21):11439-44.
[46]Habib T, Senadheera S, Weinberg K, et al. The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3[J]. Biochemistry,2002, 41(27):8725-31.
[47]Brandt K, Bulfone-Paus S, Foster D, et al. Interleukin-21 inhibits dendritic cell activation and maturation[J]. Blood,2003,102(12):4090-8.
[48]Coquet J, Kyparissoudis K, Pellicci D, et al. IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production[J]. J Immunol, 2007,178(5):2827-34.
[49]Strengell M, Matikainen S, Siren J, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells [J]. J Immunol,2003, 170(11):5464-9.
[50]Zeng R, Spolski R, Casas E, et al. The molecular basis of IL-21-mediated proliferation[J]. Blood,2007,109(10):4135-42.
[51]Konforte D, Simard N, Paige C. IL-21:an executor of B cell fate[J]. J Immunol, 2009,182(4):1781-7.
[52]Ettinger R, Kuchen S, Lipsky P. The role of IL-21 in regulating B-cell function in health and disease[J]. Immunol Rev,2008,223(60-86.
[53]Leonard W, Zeng R, Spolski R. Interleukin 21:a cytokine/cytokine receptor system that has come of age[J]. J Leukoc Biol,2008,84(2):348-56.
[54]Feldmann M. Many cytokines are very useful therapeutic targets in disease[J]. J Clin Invest,2008,118(11):3533-6.
[55]Frohlich A, Marsland B J, Sonderegger I, et al. IL-21 receptor signaling is integral to the development of Th2 effector responses in vivo[J]. Blood,2007, 109(5):2023-31.
[56]Pesce J, Kaviratne M, Ramalingam T, et al. The IL-21 receptor augments Th2 effector function and alternative macrophage activation[J]. J Clin Invest,2006, 116(7):2044-55.
[57]Monteleone G, Monteleone I, Fina D, et al. Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease[J]. Gastroenterology,2005,128(3):687-94.
[58]Fantini M, Rizzo A, Fina D, et al. IL-21 regulates experimental colitis by modulating the balance between Treg and Th17 cells[J]. Eur J Immunol,2007, 37(11):3155-63.
[59]Peluso I, Fantini M, Fina D, et al. IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes[J]. J Immunol,2007, 178(2):732-9.
[60]Comes A, Rosso O, Orengo A, et al. CD25+regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine[J]. J Immunol,2006,176(3):1750-8.
[61]Monteleone G, Pallone F, MacDonald T. Interleukin-21:a critical regulator of the balance between effector and regulatory T-cell responses[J]. Trends Immunol,2008,29(6):290-4.
[62]Yang L, Anderson D, Baecher-Allan C, et al. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells[J]. Nature,2008,454(7202):350-2.
[63]Zhou L, Ivanov, Ⅱ, Spolski R, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways[J]. Nat Immunol,2007,8(9):967-74.
[64]Korn T, Bettelli E, Gao W, et al. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells[J]. Nature,2007,448(7152):484-7.
[65]Suto A, Kashiwakuma D, Kagami S, et al. Development and characterization of IL-21-producing CD4+ T cells[J]. J Exp Med,2008,205(6):1369-79.
[66]Vogelzang A, McGuire H, Yu D, et al. A fundamental role for interleukin-21 in the generation of T follicular helper cells[J]. Immunity,2008,29(1):127-37.
[67]Zeng R, Spolski R, Finkelstein S, et al. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function[J]. J Exp Med,2005, 201(1):139-48.
[68]Moroz A, Eppolito C, Li Q, et al. IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity:comparative evaluation of IL-2, IL-15, and IL-21[J]. J Immunol,2004,173(2):900-9.
[69]Fina D, Fantini M, Pallone F, et al. Role of interleukin-21 in inflammation and allergy[J]. Inflamm Allergy Drug Targets,2007,6(1):63-8.
[70]Spolski R, Leonard W. The Yin and Yang of interleukin-21 in allergy, autoimmunity and cancer[J]. Curr Opin Immunol,2008,20(3):295-301.
[71]Pelletier M, Bouchard A, Girard D. In vivo and in vitro roles of IL-21 in inflammation[J]. J Immunol,2004,173(12):7521-30.
[72]Strengell M, Lehtonen A, Matikainen S, et al. IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells[J]. J Leukoc Biol,2006,79(6):1279-85.
[73]Brandt K, Singh P, Bulfone-Paus S, et al. Interleukin-21:a new modulator of immunity, infection, and cancer[J]. Cytokine Growth Factor Rev,2007, 18(3-4):223-32.
[74]Monteleone G, Caruso R, Fina D, et al. Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21[J]. Gut,2006, 55(12):1774-80.
[75]Jungel A, Distler J, Kurowska-Stolarska M, et al. Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis[J]. Arthritis Rheum,2004, 50(5):1468-76.
[76]Distler J, Jungel A, Kowal-Bielecka O, et al. Expression of interleukin-21 receptor in epidermis from patients with systemic sclerosis[J]. Arthritis Rheum, 2005,52(3):856-64.
[77]Gallegos A, Bevan M. Driven to autoimmunity:the nod mouse[J]. Cell,2004, 117(2):149-51.
[78]Spolski R, Kashyap M, Robinson C, et al. IL-21 signaling is critical for the development of type I diabetes in the NOD mouse[J]. Proc Natl Acad Sci U S A, 2008,105(37):14028-33.
[79]Andersson A, Feldmann M, Brennan F. Neutralizing IL-21 and IL-15 inhibits pro-inflammatory cytokine production in rheumatoid arthritis[J]. Scand J Immunol,2008,68(1):103-11.
[80]Wang X, Yuan S, Jiang L, et al. [Changes of serum BAFF and IL-21 levels in patients with systemic lupus erythematosus and their clinical significance][J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007,23(11):1041-2.
[81]Bubier J, Bennett S, Sproule T, et al. Treatment of BXSB-Yaa mice with IL-21 R-Fc fusion protein minimally attenuates systemic lupus erythematosus[J]. Ann N Y Acad Sci,2007,1110(590-601.
[82]Dutzan N, Rivas C, Garcia-Sesnich J, et al. Levels of Interleukin 21 in Untreated Chronic Periodontitis Patients[J]. J Periodontol,2011.
[83]Rodrigues L, Nandakumar S, Bonorino C, et al. IL-21 and IL-15 cytokine DNA augments HSV specific effector and memory CD8+T cell response[J]. Mol Immunol,2009,46(7):1494-504.
[84]Novy P, Huang X, Leonard W, et al. Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection[J]. J Immunol,2011,186(5):2729-38.
[85]Yi J, Du M, Zajac A. A vital role for interleukin-21 in the control of a chronic viral infection[J]. Science,2009,324(5934):1572-6.
[86]Elsaesser H, Sauer K, Brooks D. IL-21 is required to control chronic viral infection[J]. Science,2009,324(5934):1569-72.
[87]Frohlich A, Kisielow J, Schmitz I, et al. IL-21 R on T cells is critical for sustained functionality and control of chronic viral infection[J]. Science,2009, 324(5934):1576-80.
[88]Yue F, Lo C, Sakhdari A, et al. HIV-specific IL-21 producing CD4+ T cells are induced in acute and chronic progressive HIV infection and are associated with relative viral control[J]. J Immunol,2010,185(1):498-506.
[89]Iannello A, Tremblay C, Routy J, et al. Decreased levels of circulating IL-21 in HIV-infected AIDS patients:correlation with CD4+ T-cell counts[J]. Viral Immunol,2008,21(3):385-8.
[90]Iannello A, Boulassel M, Samarani S, et al. Dynamics and consequences of IL-21 production in HIV-infected individuals:a longitudinal and cross-sectional study[J]. J Immunol,2010,184(1):114-26.
[91]Williams L, Bansal A, Sabbaj S, et al. Interleukin-21-producing HTV-1-specific CD8 T cells are preferentially seen in elite controllers[J]. J Virol,2011, 85(5):2316-24.
[92]Strbo N, de Armas L, Liu H, et al. IL-21 augments natural killer effector functions in chronically HIV-infected individuals[J]. AIDS,2008, 22(13):1551-60.
[93]Iannello A, Boulassel M, Samarani S, et al. IL-21 enhances NK cell functions and survival in healthy and HIV-infected patients with minimal stimulation of viral replication[J]. J Leukoc Biol,2010,87(5):857-67.
[94]Parmigiani A, Pallin M, Schmidtmayerova H, et al. Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells[J]. Hum Immunol,2011,72(2):115-23.
[95]Chevalier M, Julg B, Pyo A, et al. HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function[J]. J Virol,2011,85(2):733-41.
[96]Zou M, Li M, Huang S et al. [The detection of anti-nuclear antibody and IL-21 in patients infected with hepatitis C virus] [J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2010,26(5):471-2.
[97]Publicover J, Goodsell A, Nishimura S, et al. IL-21 is pivotal in determining age-dependent effectiveness of immune responses in a mouse model of human hepatitis B[J]. J Clin Invest,2011.
[98]Ansari N, Kumar R, Gautam S, et al. IL-27 and IL-21 Are Associated with T Cell IL-10 Responses in Human Visceral Leishmaniasis[J]. J Immunol,2011.
[99]Hsieh C, Hsu S, Shen C, et al. Increased expression of IL-21 reduces tumor growth by modulating the status of tumor-infiltrated lymphocytes [J]. Immunobiology,2011,216(4):491-6.
[100]Skak K, Kragh M, Hausman D, et al. Interleukin 21:combination strategies for cancer therapy[J]. Nat Rev Drug Discov,2008,7(3):231-40.
[101]Sondergaard H, Galsgaard E, Bartholomaeussen M, et al. Intratumoral interleukin-21 increases antitumor immunity, tumor-infiltrating CD8+ T-cell density and activity, and enlarges draining lymph nodes[J]. J Immunother,2010, 33(3):236-49.
[102]Yu J, Sun L, Zhao Y, et al. Prediction value of model for end-stage liver disease scoring system on prognosis in patients with acute-on-chronic hepatitis B liver failure after plasma exchange and lamivudine treatment[J]. J Gastroenterol Hepatol,2008,23(8 Pt 1):1242-9.
[103]Maini M, Boni C, Lee C, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med,2000,191 (8):1269-80.
[104]Kamath P, Kim W. The model for end-stage liver disease (MELD)[J]. Hepatology,2007,45(3):797-805.
[105]Yasumi Y, Takikawa Y, Endo R, et al. Interleukin-17 as a new marker of severity of acute hepatic injury[J]. Hepatol Res,2007,37(4):248-54.
[106]Drucker C, Gewiese J, Malchow S, et al. Impact of interleukin-6 classic-and trans-signaling on liver damage and regeneration[J]. J Autoimmun,2010, 34(1):29-37.
[107]Zhang F, Yao S, Zhang M, et al. Roles of circulating soluble interleukin (IL)-6 receptor and IL-6 receptor expression on CD4+T cells in patients with chronic hepatitis B[J]. Int J Infect Dis,2011,15(4):e267-71.
[108]Hosel M, Quasdorff M, Wiegmann K, et al. Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection[J]. Hepatology, 2009,50(6):1773-82.
[109]Mahe Y, Mukaida N, Kuno K, et al. Hepatitis B virus X protein transactivates human interleukin-8 gene through acting on nuclear factor kB and CCAAT/enhancer-binding protein-like cis-elements[J]. J Biol Chem,1991, 266(21):13759-63.
[110]Tan A T, Koh S, Goh W, et al. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B[J]. J Hepatol,2010, 52(3):330-9.
[111]Blackburn S, Wherry E. IL-10, T cell exhaustion and viral persistence[J]. Trends Microbiol,2007,15(4):143-6.
[112]Wu J, Wu T, Chen C, et al. Serum levels of interleukin-10 and interleukin-12 predict early, spontaneous hepatitis B virus e antigen seroconversion[J]. Gastroenterology,2010,138(1):165-72 e1-3.
[113]Alatrakchi N, Koziel M. Regulatory T cells and viral liver disease[J]. J Viral Hepat,2009,16(4):223-9.
[114]Bluestone J, Mackay C, O'Shea J J, et al. The functional plasticity of T cell subsets[J]. Nat Rev Immunol,2009,9(11):811-6.
[115]Zhou L, Chong M, Littman D. Plasticity of CD4+ T cell lineage differentiation[J]. Immunity,2009,30(5):646-55.
[116]Wurster A, Rodgers V, Satoskar A, et al. Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon gamma-producing Thl cells[J]. J Exp Med,2002,196(7):969-77.
[117]Spolski R, Leonard W. IL-21 and T follicular helper cells[J]. Int Immunol,2010, 22(1):7-12.
[118]Lohr H, Weber W, Schlaak J, et al. Proliferative response of CD4+T cells and hepatitis B virus clearance in chronic hepatitis with or without hepatitis B e-minus hepatitis B virus mutants[J]. Hepatology,1995,22(1):61-8.
[119]Fujio K, Okamura T, Yamamoto K. The Family of IL-10-secreting CD4+T cells[J]. Adv Immunol,2010,105(99-130.
[120]中华医学会肝病学分会.慢性乙型肝炎防治指南(2010年版)[J].中华内科杂志,2011,50(2)：168-79.
[2]Sarin S, Kumar A, Almeida J, et al. Acute-on-chronic liver failure:consensus recommendations of the Asian Pacific Association for the study of the liver (APASL)[J]. Hepatol Int,2009,3(1):269-82.
[3]刘晓燕,胡瑾华,王慧芬,等.1977例急性、亚急性、慢加急性肝衰竭患者的病因与转归分析[J].中华肝脏病杂志,2008,16(10)：772-5.
[4]胡瑾华,黄坤,王慧芬,等.乙型肝炎相关慢加急性肝衰竭190例生存分析[J].传染病信息,2010,23(2)：83-6.
[5]Cui Y, Yan F, Wang Y, et al. Nucleoside analogue can improve the long-term prognosis of patients with hepatitis B virus infection-associated acute on chronic liver failure[J]. Dig Dis Sci,2010,55(8):2373-80.
[6]Ren X, Xu Z, Liu Y, et al. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis [J]. J Viral Hepat,2010,17(12):887-95.
[7]Xu Z, Ren X, Liu Y, et al. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease:an investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure[J]. J Gastroenterol,2010,46(3):391-400.
[8]Yang L, Ma S, Hu X, et al. Presence of valine at position 27 of the hepatitis B virus core gene is associated with severe liver inflammation in Chinese patients[J]. J Med Virol,2011,83(2):218-24.
[9]Heydtmann M. Macrophages in hepatitis B and hepatitis C virus infections [J]. J Virol,2009,83(7):2796-802.
[10]Xing T, Li L, Cao H, et al. Altered immune function of monocytes in different stages of patients with acute on chronic liver failure[J]. Clin Exp Immunol, 2007,147(1):184-8.
[11]Zou Z, Li B, Xu D, et al. unbalanced intrahepatic cytokine expression of interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis B virus infection[J]. J Clin Gastroenterol, 2009,43(2):182-90.
[12]Shi F, Zhang J, Zeng Z, et al. Skewed ratios between CD3(+) T cells and monocytes are associated with poor prognosis in patients with HBV-related acute-on-chronic liver failure[J]. Biochem Biophys Res Commun,2010, 402(1):30-6.
[13]Lambotin M, Raghuraman S, Stoll-Keller F, et al. A look behind closed doors: interaction of persistent viruses with dendritic cells[JJ. Nat Rev Microbiol,2010, 8(5):350-60.
[14]Zhang Z, Zou Z S, Fu J L, et al. Severe dendritic cell perturbation is actively involved in the pathogenesis of acute-on-chronic hepatitis B liver failure[J]. J Hepatol,2008,49(3):396-406.
[15]Li N, Li Q, Qian Z, et al. Impaired TLR3/IFN-beta signaling in monocyte-derived dendritic cells from patients with acute-on-chronic hepatitis B liver failure:relevance to the severity of liver damage[J]. Biochem Biophys Res Commun,2009,390(3):630-5.
[16]Mondelli M U, Varchetta S, Oliviero B. Natural killer cells in viral hepatitis: facts and controversies[J]. Eur J Clin Invest,2010,40(9):851-63.
[17]Fisicaro P, Valdatta C, Boni C, et al. Early kinetics of innate and adaptive immune responses during hepatitis B virus infection[J]. Gut,2009, 58(7):974-82.
[18]Dunn C, Peppa D, Khanna P, et al. Temporal analysis of early immune responses in patients with acute hepatitis B virus infection[J]. Gastroenterology, 2009,137(4):1289-300.
[19]Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand and NKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure[J]. J Immunol,2010,184(1):466-75.
[20]Zou Z, Xu D, Li B, et al. Compartmentalization and its implication for peripheral immunologically-competent cells to the liver in patients with HBV-related acute-on-chronic liver failure [J]. Hepatol Res,2009, 39(12):1198-207.
[21]Zhu J, Paul W E. CD4 T cells:fates, functions, and faults[J]. Blood,2008, 112(5):1557-69.
[22]Mills K H. Induction, function and regulation of IL-17-producing T cells [J]. Eur J Immunol,2008,38(10):2636-49.
[23]Xu D, Fu J, Jin L, et al. Circulating and liver resident CD4+CD25+regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B[J]. J Immunol,2006,177(1):739-47.
[24]黄海英,李树臣,于建武,等.慢性乙型重型肝炎患者外周血调节性T细胞表达[J].哈尔滨医药,2009,29(1)：2-4.
[25]Wang L, Chen H, Gong Z. High mobility group box-1 protein inhibits regulatory T cell immune activity in liver failure in patients with chronic hepatitis B[J]. Hepatobiliary Pancreat Dis Int,2010,9(5):499-507.
[26]Harrington L, Hatton R, Mangan P, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[J]. Nat Immunol,2005,6(11):1123-32.
[27]Park H, Li Z, Yang X, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17[J]. Nat Immunol,2005, 6(11):1133-41.
[28]Ye P, Rodriguez F, Kanaly S, et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense[J]. J Exp Med,2001, 194(4):519-27.
[29]Korn T, Bettelli E, Oukka M, et al. IL-17 and Th17 Cells[J]. Annu Rev Immunol,2009,27(485-517.
[30]Ivanov, Ⅱ, McKenzie B S, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells[J]. Cell,2006,126(6):1121-33.
[31]Lafdil F, Miller A, Ki S, et al. Thl7 cells and their associated cytokines in liver diseases[J]. Cell Mol Immunol,2010,7(4):250-4.
[32]Ge J, Wang K, Meng Q, et al. Implication of Th17 and Thl cells in patients with chronic active hepatitis B[J]. J Clin Immunol,2010,30(1):60-7.
[33]Zhang J, Zhang Z, Lin F, et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B[J]. Hepatology,2010,51(1):81-91.
[34]Ye Y, Xie X, Yu J, et al. Involvement of Th17 and Th1 effector responses in patients with Hepatitis B[J]. J Clin Immunol,2010,30(4):546-55.
[35]Zhang J, Song C, Shi F, et al. Decreased ratio of Treg cells to Th17 cells correlates with HBV DNA suppression in chronic hepatitis B patients undergoing entecavir treatment[J]. PLoS One,2010,5(11):e13869.
[36]Li J, Wu W, Peng G, et al. HBcAg induces interleukin-10 production, inhibiting HBcAg-specific Th17 responses in chronic hepatitis B patients [J]. Immunol Cell Biol,2010,88(8):834-41.
[37]Wang L, Chen S, Xu K. IL-17 expression is correlated with hepatitis B-related liver diseases and fibrosis[J]. Int J Mol Med,2011,27(3):385-92.
[38]Zhang J, Yan J, Xu J, et al. Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients[J]. J Hepatol, 2009,50(5):980-9.
[39]Han T, Liu Y, Liu H, et al. Serum thymosin beta4 levels in patients with hepatitis B virus-related liver failure[J]. World J Gastroenterol,2010, 16(5):625-30.
[40]Zhu C, Yan W, Zhu F, et al. Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B[J]. World J Gastroenterol,2005, 11(44):6936-40.
[41]Monteleone G, Pallone F, Macdonald T. Interleukin-21 (IL-21)-mediated pathways in T cell-mediated disease[J]. Cytokine Growth Factor Rev,2009, 20(2):185-91.
[42]Taniguchi T. Cytokine signaling through nonreceptor protein tyrosine kinases[J]. Science,1995,268(5208):251-5.
[43]Muneta Y, Kikuma R, Uenishi H, et al. Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21 [J]. J Vet Med Sci, 2004,66(3):269-75.
[44]Parrish-Novak J, Dillon S R, Nelson A, et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function[J]. Nature, 2000,408(6808):57-63.
[45]Ozaki K, Kikly K, Michalovich D, et al. Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain[J]. Proc Natl Acad Sci U S A,2000, 97(21):11439-44.
[46]Habib T, Senadheera S, Weinberg K, et al. The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3[J]. Biochemistry,2002, 41(27):8725-31.
[47]Brandt K, Bulfone-Paus S, Foster D, et al. Interleukin-21 inhibits dendritic cell activation and maturation[J]. Blood,2003,102(12):4090-8.
[48]Coquet J, Kyparissoudis K, Pellicci D, et al. IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production[J]. J Immunol, 2007,178(5):2827-34.
[49]Strengell M, Matikainen S, Siren J, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells [J]. J Immunol,2003, 170(11):5464-9.
[50]Zeng R, Spolski R, Casas E, et al. The molecular basis of IL-21-mediated proliferation[J]. Blood,2007,109(10):4135-42.
[51]Konforte D, Simard N, Paige C. IL-21:an executor of B cell fate[J]. J Immunol, 2009,182(4):1781-7.
[52]Ettinger R, Kuchen S, Lipsky P. The role of IL-21 in regulating B-cell function in health and disease[J]. Immunol Rev,2008,223(60-86.
[53]Leonard W, Zeng R, Spolski R. Interleukin 21:a cytokine/cytokine receptor system that has come of age[J]. J Leukoc Biol,2008,84(2):348-56.
[54]Feldmann M. Many cytokines are very useful therapeutic targets in disease[J]. J Clin Invest,2008,118(11):3533-6.
[55]Frohlich A, Marsland B J, Sonderegger I, et al. IL-21 receptor signaling is integral to the development of Th2 effector responses in vivo[J]. Blood,2007, 109(5):2023-31.
[56]Pesce J, Kaviratne M, Ramalingam T, et al. The IL-21 receptor augments Th2 effector function and alternative macrophage activation[J]. J Clin Invest,2006, 116(7):2044-55.
[57]Monteleone G, Monteleone I, Fina D, et al. Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease[J]. Gastroenterology,2005,128(3):687-94.
[58]Fantini M, Rizzo A, Fina D, et al. IL-21 regulates experimental colitis by modulating the balance between Treg and Th17 cells[J]. Eur J Immunol,2007, 37(11):3155-63.
[59]Peluso I, Fantini M, Fina D, et al. IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes[J]. J Immunol,2007, 178(2):732-9.
[60]Comes A, Rosso O, Orengo A, et al. CD25+regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine[J]. J Immunol,2006,176(3):1750-8.
[61]Monteleone G, Pallone F, MacDonald T. Interleukin-21:a critical regulator of the balance between effector and regulatory T-cell responses[J]. Trends Immunol,2008,29(6):290-4.
[62]Yang L, Anderson D, Baecher-Allan C, et al. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells[J]. Nature,2008,454(7202):350-2.
[63]Zhou L, Ivanov, Ⅱ, Spolski R, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways[J]. Nat Immunol,2007,8(9):967-74.
[64]Korn T, Bettelli E, Gao W, et al. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells[J]. Nature,2007,448(7152):484-7.
[65]Suto A, Kashiwakuma D, Kagami S, et al. Development and characterization of IL-21-producing CD4+ T cells[J]. J Exp Med,2008,205(6):1369-79.
[66]Vogelzang A, McGuire H, Yu D, et al. A fundamental role for interleukin-21 in the generation of T follicular helper cells[J]. Immunity,2008,29(1):127-37.
[67]Zeng R, Spolski R, Finkelstein S, et al. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function[J]. J Exp Med,2005, 201(1):139-48.
[68]Moroz A, Eppolito C, Li Q, et al. IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity:comparative evaluation of IL-2, IL-15, and IL-21[J]. J Immunol,2004,173(2):900-9.
[69]Fina D, Fantini M, Pallone F, et al. Role of interleukin-21 in inflammation and allergy[J]. Inflamm Allergy Drug Targets,2007,6(1):63-8.
[70]Spolski R, Leonard W. The Yin and Yang of interleukin-21 in allergy, autoimmunity and cancer[J]. Curr Opin Immunol,2008,20(3):295-301.
[71]Pelletier M, Bouchard A, Girard D. In vivo and in vitro roles of IL-21 in inflammation[J]. J Immunol,2004,173(12):7521-30.
[72]Strengell M, Lehtonen A, Matikainen S, et al. IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells[J]. J Leukoc Biol,2006,79(6):1279-85.
[73]Brandt K, Singh P, Bulfone-Paus S, et al. Interleukin-21:a new modulator of immunity, infection, and cancer[J]. Cytokine Growth Factor Rev,2007, 18(3-4):223-32.
[74]Monteleone G, Caruso R, Fina D, et al. Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21[J]. Gut,2006, 55(12):1774-80.
[75]Jungel A, Distler J, Kurowska-Stolarska M, et al. Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis[J]. Arthritis Rheum,2004, 50(5):1468-76.
[76]Distler J, Jungel A, Kowal-Bielecka O, et al. Expression of interleukin-21 receptor in epidermis from patients with systemic sclerosis[J]. Arthritis Rheum, 2005,52(3):856-64.
[77]Gallegos A, Bevan M. Driven to autoimmunity:the nod mouse[J]. Cell,2004, 117(2):149-51.
[78]Spolski R, Kashyap M, Robinson C, et al. IL-21 signaling is critical for the development of type I diabetes in the NOD mouse[J]. Proc Natl Acad Sci U S A, 2008,105(37):14028-33.
[79]Andersson A, Feldmann M, Brennan F. Neutralizing IL-21 and IL-15 inhibits pro-inflammatory cytokine production in rheumatoid arthritis[J]. Scand J Immunol,2008,68(1):103-11.
[80]Wang X, Yuan S, Jiang L, et al. [Changes of serum BAFF and IL-21 levels in patients with systemic lupus erythematosus and their clinical significance][J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007,23(11):1041-2.
[81]Bubier J, Bennett S, Sproule T, et al. Treatment of BXSB-Yaa mice with IL-21 R-Fc fusion protein minimally attenuates systemic lupus erythematosus[J]. Ann N Y Acad Sci,2007,1110(590-601.
[82]Dutzan N, Rivas C, Garcia-Sesnich J, et al. Levels of Interleukin 21 in Untreated Chronic Periodontitis Patients[J]. J Periodontol,2011.
[83]Rodrigues L, Nandakumar S, Bonorino C, et al. IL-21 and IL-15 cytokine DNA augments HSV specific effector and memory CD8+T cell response[J]. Mol Immunol,2009,46(7):1494-504.
[84]Novy P, Huang X, Leonard W, et al. Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection[J]. J Immunol,2011,186(5):2729-38.
[85]Yi J, Du M, Zajac A. A vital role for interleukin-21 in the control of a chronic viral infection[J]. Science,2009,324(5934):1572-6.
[86]Elsaesser H, Sauer K, Brooks D. IL-21 is required to control chronic viral infection[J]. Science,2009,324(5934):1569-72.
[87]Frohlich A, Kisielow J, Schmitz I, et al. IL-21 R on T cells is critical for sustained functionality and control of chronic viral infection[J]. Science,2009, 324(5934):1576-80.
[88]Yue F, Lo C, Sakhdari A, et al. HIV-specific IL-21 producing CD4+ T cells are induced in acute and chronic progressive HIV infection and are associated with relative viral control[J]. J Immunol,2010,185(1):498-506.
[89]Iannello A, Tremblay C, Routy J, et al. Decreased levels of circulating IL-21 in HIV-infected AIDS patients:correlation with CD4+ T-cell counts[J]. Viral Immunol,2008,21(3):385-8.
[90]Iannello A, Boulassel M, Samarani S, et al. Dynamics and consequences of IL-21 production in HIV-infected individuals:a longitudinal and cross-sectional study[J]. J Immunol,2010,184(1):114-26.
[91]Williams L, Bansal A, Sabbaj S, et al. Interleukin-21-producing HTV-1-specific CD8 T cells are preferentially seen in elite controllers[J]. J Virol,2011, 85(5):2316-24.
[92]Strbo N, de Armas L, Liu H, et al. IL-21 augments natural killer effector functions in chronically HIV-infected individuals[J]. AIDS,2008, 22(13):1551-60.
[93]Iannello A, Boulassel M, Samarani S, et al. IL-21 enhances NK cell functions and survival in healthy and HIV-infected patients with minimal stimulation of viral replication[J]. J Leukoc Biol,2010,87(5):857-67.
[94]Parmigiani A, Pallin M, Schmidtmayerova H, et al. Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells[J]. Hum Immunol,2011,72(2):115-23.
[95]Chevalier M, Julg B, Pyo A, et al. HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function[J]. J Virol,2011,85(2):733-41.
[96]Zou M, Li M, Huang S et al. [The detection of anti-nuclear antibody and IL-21 in patients infected with hepatitis C virus] [J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2010,26(5):471-2.
[97]Publicover J, Goodsell A, Nishimura S, et al. IL-21 is pivotal in determining age-dependent effectiveness of immune responses in a mouse model of human hepatitis B[J]. J Clin Invest,2011.
[98]Ansari N, Kumar R, Gautam S, et al. IL-27 and IL-21 Are Associated with T Cell IL-10 Responses in Human Visceral Leishmaniasis[J]. J Immunol,2011.
[99]Hsieh C, Hsu S, Shen C, et al. Increased expression of IL-21 reduces tumor growth by modulating the status of tumor-infiltrated lymphocytes [J]. Immunobiology,2011,216(4):491-6.
[100]Skak K, Kragh M, Hausman D, et al. Interleukin 21:combination strategies for cancer therapy[J]. Nat Rev Drug Discov,2008,7(3):231-40.
[101]Sondergaard H, Galsgaard E, Bartholomaeussen M, et al. Intratumoral interleukin-21 increases antitumor immunity, tumor-infiltrating CD8+ T-cell density and activity, and enlarges draining lymph nodes[J]. J Immunother,2010, 33(3):236-49.
[102]Yu J, Sun L, Zhao Y, et al. Prediction value of model for end-stage liver disease scoring system on prognosis in patients with acute-on-chronic hepatitis B liver failure after plasma exchange and lamivudine treatment[J]. J Gastroenterol Hepatol,2008,23(8 Pt 1):1242-9.
[103]Maini M, Boni C, Lee C, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[J]. J Exp Med,2000,191 (8):1269-80.
[104]Kamath P, Kim W. The model for end-stage liver disease (MELD)[J]. Hepatology,2007,45(3):797-805.
[105]Yasumi Y, Takikawa Y, Endo R, et al. Interleukin-17 as a new marker of severity of acute hepatic injury[J]. Hepatol Res,2007,37(4):248-54.
[106]Drucker C, Gewiese J, Malchow S, et al. Impact of interleukin-6 classic-and trans-signaling on liver damage and regeneration[J]. J Autoimmun,2010, 34(1):29-37.
[107]Zhang F, Yao S, Zhang M, et al. Roles of circulating soluble interleukin (IL)-6 receptor and IL-6 receptor expression on CD4+T cells in patients with chronic hepatitis B[J]. Int J Infect Dis,2011,15(4):e267-71.
[108]Hosel M, Quasdorff M, Wiegmann K, et al. Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection[J]. Hepatology, 2009,50(6):1773-82.
[109]Mahe Y, Mukaida N, Kuno K, et al. Hepatitis B virus X protein transactivates human interleukin-8 gene through acting on nuclear factor kB and CCAAT/enhancer-binding protein-like cis-elements[J]. J Biol Chem,1991, 266(21):13759-63.
[110]Tan A T, Koh S, Goh W, et al. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B[J]. J Hepatol,2010, 52(3):330-9.
[111]Blackburn S, Wherry E. IL-10, T cell exhaustion and viral persistence[J]. Trends Microbiol,2007,15(4):143-6.
[112]Wu J, Wu T, Chen C, et al. Serum levels of interleukin-10 and interleukin-12 predict early, spontaneous hepatitis B virus e antigen seroconversion[J]. Gastroenterology,2010,138(1):165-72 e1-3.
[113]Alatrakchi N, Koziel M. Regulatory T cells and viral liver disease[J]. J Viral Hepat,2009,16(4):223-9.
[114]Bluestone J, Mackay C, O'Shea J J, et al. The functional plasticity of T cell subsets[J]. Nat Rev Immunol,2009,9(11):811-6.
[115]Zhou L, Chong M, Littman D. Plasticity of CD4+ T cell lineage differentiation[J]. Immunity,2009,30(5):646-55.
[116]Wurster A, Rodgers V, Satoskar A, et al. Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon gamma-producing Thl cells[J]. J Exp Med,2002,196(7):969-77.
[117]Spolski R, Leonard W. IL-21 and T follicular helper cells[J]. Int Immunol,2010, 22(1):7-12.
[118]Lohr H, Weber W, Schlaak J, et al. Proliferative response of CD4+T cells and hepatitis B virus clearance in chronic hepatitis with or without hepatitis B e-minus hepatitis B virus mutants[J]. Hepatology,1995,22(1):61-8.
[119]Fujio K, Okamura T, Yamamoto K. The Family of IL-10-secreting CD4+T cells[J]. Adv Immunol,2010,105(99-130.
[120]中华医学会肝病学分会.慢性乙型肝炎防治指南(2010年版)[J].中华内科杂志,2011,50(2)：168-79.