HBcAg特异性辅助性T淋巴细胞22在HBV感染者中的变化和意义

Change and significance of HBcAg-specific Th22 cells in patients with hepatitis B virus infection

目的观察HBV感染者中辅助性T淋巴细胞(Th) 22、IL-22和转录因子芳香烃受体(AhR)的变化及与临床指标的相关性。方法选择2018年3月-2019年3月在西安市第八医院就诊的急性乙型肝炎(AHB)患者11例、慢性乙型肝炎(CHB)患者38例和健康对照者(HC) 16例,CHB患者使用替诺福韦酯(TDF)抗病毒治疗,收集AHB患者基线和出院6个月后的外周血,收集CHB患者基线、治疗6个月和治疗12个月的外周血,分离外周血单个核细胞(PBMC)和血浆,分别使用佛波酯+伊乌诺霉素和重组HBcAg刺激PBMC,流式细胞术检测非特异性CD3+CD4+IL-22+的Th22和HBcAg特异性Th22。ELISA检测血浆IL-22水平。实时定量PCR检测PBMC中AhR mRNA水平。符合正态分布的计量资料两组间比较采用t检验、配对t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验;非正态分布的计量资料多组间比较采用Kruskal-Wallis H检验。计数资料采用χ~2检验。采用Spearman相关性分析进行相关性检验。结果 AHB患者非特异性Th22细胞比例(2.86%±0.45%)高于CHB患者(1.39%±0.33%)和HC(0.80%±0.13%),差异均有统计学意义(t值分别为11.80、17.30,P值均<0.001),CHB患者非特异性Th22细胞比例亦高于HC(t=6.825,P <0.001)。AHB患者HBcAg特异性Th22细胞比例高于CHB患者(2.97%±0.52%vs 1.22%±0.22%,t=16.58,P <0.001)。AHB患者血浆IL-22水平[(130.7±39.97) pg/ml]高于CHB患者[(66.59±20.83) pg/ml]和HC[(50.63±11.07) pg/ml],差异均有统计学意义(t值分别为7.176、7.662,P值均<0.001),CHB患者血浆IL-22水平亦高于HC(t=2.887,P=0.006)。AHB患者PBMC中AhR mRNA(11.45±3.03)高于CHB患者(4.81±1.25)和HC(1.10±0.17),差异均有统计学意义(t值分别为10.85、13.75,P值均<0.001),CHB患者AhR mRNA亦高于HC(t=11.77,P <0.001)。HBcAg特异性Th22细胞比例在AHB患者和CHB患者中均与ALT水平呈显著正相关(r值分别为0.638、0.830,P值分别为0.035、0.002),血浆IL-22水平在AHB患者和CHB患者中亦均与ALT水平呈显著正相关(r值分别为0.552、0.431,P值分别为0.001、0.007)。AHB患者出院6个月后随访观察,HBcAg特异性Th22细胞比例较基线显著降低(2.79%±0.56%vs 2.97%±0.52%,t=3.055,P=0.012),血浆IL-22水平亦较基线显著降低[(105.8±25.23) pg/ml vs (130.7±39.97) pg/ml,t=2.362,P=0.040]。所有CHB患者均接受TDF抗病毒治疗,分别于治疗6、12个月时进行随访。HBcAg特异性Th22细胞比例在治疗6、12个月均显著低于基线(t值分别为4.353、3.927,P值均<0.001),血浆IL-22水平在治疗6、12个月均显著低于基线(t值分别为4.426、4.810,P值均<0.001)。结论HBcAg特异性Th22细胞和IL-22与HBV感染炎症应答密切相关。

ObjectiveTo investigate the changes of Th22 cells,interleukin-22(IL-22),and transcription factor aryl hydrocarbon receptor(AhR)in patients with hepatitis B virus(HBV)infection and their correlation with clinical indices.Methods A total of 11 patients with acute hepatitis B(AHB)and 38 patients with chronic hepatitis B(CHB)who attended Eighth Hospital of Xi’an from March 2018 to March 2019 were enrolled as AHB group and CHB group,respectively,and 16 healthy controls were enrolled as HC group.The patients with CHB received tenofovir disoproxil fumarate(TDF)antiviral therapy.Peripheral blood samples were collected for AHB patients at baseline and 6 months after discharge,and peripheral blood samples were collected for CHB patients at baseline and at months 6 and 12 of treatment;peripheral blood mononuclear cells(PBMCs)and plasma were isolated,then PBMCs were stimulated with phorbol ester+ionomycin or recombinant HBcAg,and flow cytometry was used to measure nonspecific CD3+CD4+IL-22+Th22 cells and HBcAg-specific Th22 cells.ELISA was used to measure the plasma level of IL-22,and quantitative real-time PCR was used to measure the mRNA expression of AhR in PBMCs.The t-test and the paired t-test were used for comparison of normally distributed continuous data between two groups;a one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups;the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups.The chi-square test was used for comparison of categorical data between groups.A Spearman correlation analysis was used to investigate correlation.Results The AHB group had a significantly higher percentage of nonspecific Th22 cells than the CHB group(2.86%±0.45%vs 1.39%±0.33%,t=11.80,P<0.001)and the HC group(2.86%±0.45%vs 0.80%±0.13%,t=17.30,P<0.001),and the CHB group also had a significantly higher percentage of nonspecific Th22 cells than the HC group(t=6.825,P<0.001).The AHB group had a significantly higher percentage of HBcAg-specific Th22 cells than the CHB group(2.97%±0.52%vs 1.22%±0.22%,t=16.58,P<0.001).The AHB group had a significantly higher plasma level of IL-22 than the CHB group(130.7±39.97 pg/ml vs 66.59±20.83 pg/ml,t=7.176,P<0.001)and the HC group(130.7±39.97 pg/ml vs 50.63±11.07 pg/ml,t=7.662,P<0.001),and the CHB group also had a significantly higher plasma level of IL-22 than the HC group(t=2.887,P=0.006).The AHB group had significantly higher mRNA expression of AhR than the CHB group(11.45±3.03 vs 4.81±1.25,t=10.85,P<0.0001)and the HC group(11.45±3.03 vs 1.10±0.17,t=13.75,P<0.001),and the CHB group also had significantly higher mRNA expression of AhR than the HC group(t=11.77,P<0.001).In both AHB and CHB patients,the percentage of HBcAg-specific Th22 cells was positively correlated with alanine aminotransferase(ALT)level(r=0.638 and 0.830,P=0.035 and 0.002),and the plasma level of IL-22 was also positively correlated with ALT level(r=0.552 and 0.431,P=0.001 and 0.007).The AHB patients were followed up at 6 months after discharge,and there were significant reductions in the percentage of HBcAg-specific Th22 cells(2.79%±0.56%,t=3.055,P=0.012)and the plasma level of IL-22(105.8±25.23 pg/ml,t=2.362,P=0.040)from baseline.All CHB patients received TDF antiviral therapy and were followed up at months 6 and 12 of treatment,and there were significant reductions in the percentage of HBcAg-specific Th22 cells(t=4.353 and 3.927,all P<0.001)and the plasma level of IL-22(t=4.426 and 4.810,both P<0.0001)from baseline to months 6 and 12 of treatment.Conclusion HBcAg-specific Th22 cells and IL-22 are closely associated with inflammatory response to HBV infection.