CD8^(+)Treg细胞在慢性鼻窦炎中的表达及其与嗜酸粒细胞浸润的研究

Expression of CD8^(+)Treg cells in chronic rhinosinusitis and its correlation with eosinophilic infiltration

目的明确CD8^(+)调节性T细胞(Treg细胞)在慢性鼻窦炎(CRS)患者鼻黏膜组织和外周血中的表达情况,及其与鼻黏膜组织中嗜酸粒细胞(Eos)浸润的关系。方法对2017年3月至2018年10月于中山大学附属第三医院的33例慢性鼻窦炎伴鼻息肉(CRSwNP)、26例慢性鼻窦炎不伴鼻息肉(CRSsNP)及27例对照组患者采集鼻黏膜组织及外周血,其中男59例,女27例,年龄18~72岁。苏木精-伊红染色检测鼻黏膜组织中Eos计数,将CRS患者分为嗜酸粒细胞性CRS(eosinophilic CRS,ECRS)和非嗜酸粒细胞性CRS(non-eosinophilic CRS,Non-ECRS)两组类型。采用流式细胞术检测各组患者鼻黏膜组织及外周血中CD4^(+)及CD8^(+)T细胞的比例、细胞毒性T细胞(cytotoxic T lymphocytes,Tc)1、Tc2及Tc17细胞的比例、CD8^(+)Foxp3^(+)和CD8^(+)Foxp3^(-)IL-10^(+)两种CD8^(+)Treg细胞的比例,以及产转化生长因子β(TGF-β)及白细胞介素(IL)-10的CD8^(+)Foxp3^(+)Treg细胞的比例。采用Graphpad 7.0进行作图,所有数据采用M(IQR)表示,采用SPSS 16.0软件进行统计学分析。结果CD8^(+)T细胞在所有患者鼻黏膜组织淋巴细胞中的比例高于CD4^(+)T细胞[37.75%(17.35%)比4.72%(4.29%),Z=-5.70,P<0.001],而在外周血淋巴细胞中的比例低于CD4^(+)T细胞[23.60%(9.33%)比44.05%(10.93%),t=9.72,P<0.001]。CRSwNP患者鼻黏膜组织中Tc2细胞和Tc17细胞比例显著高于CRSsNP患者及对照组[1.82%(1.22%)比0.84%(0.79%)比1.09%(0.92%),1.93%(2.32%)比0.54%(1.04%)比0.47%(0.51%),P值均<0.05]。与CRSsNP[0.43%(1.45%)]和对照组[0.48%(0.83%)]相比,CRSwNP患者鼻黏膜组织中CD8^(+)Foxp3^(+)Treg细胞占淋巴细胞比例最低[0.10%(0.32%),Z值分别为-2.24、-2.22,P值分别为0.025、0.027];CRSwNP患者鼻黏膜组织中产TGF-β和IL-10的CD8^(+)Foxp3^(+)Treg细胞占总CD8^(+)T细胞的比例显著低于对照组(Z值分别为1.46、0.49,P值均为0.001)。CRSwNP患者鼻黏膜组织中CD8^(+)Foxp3^(-)IL-10^(+)Treg细胞占总CD8^(+)T细胞的比例显著低于CRSsNP患者[0.14%(0.28%)比0.89%(0.81%),Z=0.61,P=0.030]。CD8^(+)Foxp3^(+)Treg细胞和CD8^(+)Foxp3^(-)IL-10^(+)Treg细胞在ECRS患者鼻黏膜淋巴细胞中的占比显著低于Non-ECRS患者[0.07%(0.44%)比0.53%(0.75%),0.13%(0.21%)比0.29%(0.76%),t值分别为2.14、2.78,P值均<0.05],并与CRS患者鼻黏膜组织中Eos百分比呈负相关(R^(2)值分别为0.56、0.78,P值均<0.001)。外周血中CD8^(+)Fxop3^(+)Treg和CD8^(+)Fxop3^(-)IL-10^(+)Treg细胞在各组之间的分布无明显差异。结论CD8^(+)Treg细胞在CRSwNP患者尤其是ECRS患者鼻黏膜组织中所占比例降低,与Tc2、Tc17趋势相反,与Eos计数呈负相关。提示CD8^(+)Treg比例降低可能与CRS患者鼻黏膜免疫失衡及Eos浸润有关。

Objective To detect the percentages of CD8^(+)Treg cells in the nasal mucosa and peripheral blood of chronic rhinosinusitis(CRS)and to explore their correlation with eosinophilic infiltration.Methods Thirty-three chronic rhinosinusitis with polyp(CRSwNP),26 chronic rhinosinusitis without polyp(CRSsNP)and 27 control patients who were collected with the nose mucosal tissue and peripheral blood in the Third Affiliated Hospital of Sun Yat-sen University from March 2017 to October 2018 were selected,including 59 males and 27 females,aging from 18 to 72 years.Hematoxylin and eosin(HE)staining was used to observe the number of eosinophils in the nasal tissues and to classify the CRS into eosinophilic CRS(ECRS)and non-eosinophilic CRS(Non-ECRS).Flow cytometry was used to detect the percentages of CD4^(+)and CD8^(+)T cells in lymphocytes of nasal mucosa and peripheral blood.The percentages of CD8^(+)Foxp3^(+)Treg cells,CD8^(+)Foxp3^(-)IL-10^(+)Treg cells,CD8^(+)IFN-γ+T cells(Tc1),CD8^(+)IL-4^(+)T cells(Tc2)and CD8^(+)IL-17A+T cells(Tc17)in lymphocytes of nasal mucosa and peripheral blood were also tested.Besides,the percentages of Foxp3^(+)TGF-β+Treg cells and Foxp3^(+)IL-10^(+)Treg cells in CD8^(+)T cells were determined.All data were represented by M(IQR).GraphPad 7.0 and SPSS 16.0 were used for illustration and statistical analysis.Results The percentage of CD8^(+)T cells(37.75%(17.35%))was higher than that of CD4^(+)T cells(4.72%(4.29%))in nasal mucosa(Z=-5.70,P<0.001),while lower(23.60%(9.33%))than that of CD4^(+)T cells(44.05%(10.93%))in peripheral blood(t=9.72,P<0.001).CRSwNP patients possessed the highest Tc2(1.82%(1.22%))and Tc17(1.93%(2.32%))percentages than CRSsNP(Tc2:0.84%(0.79%);Tc17:0.54%(1.04%))and control(Tc2:1.09%(0.92%);Tc17:0.47%(0.51%),both P<0.05)patients.While,CRSwNP patients possessed the lowest CD8^(+)Foxp3^(+)Treg cells percentage(0.10%(0.32%))than CRSsNP(0.43%(1.45%))and control(0.48%(0.83%),Z value was-2.24,-2.22,respectively,P value was 0.025,0.027,respectively).The percentages of Foxp3^(+)TGF-β+Treg cells and Foxp3^(+)IL-10^(+)Treg cells of CD8^(+)T cells in nasal mucosa in CRSwNP were also lower than controls(Z value was 1.46,0.49,respectively,both P=0.001).Moreover,the percentage of CD8^(+)Foxp3^(-)IL-10^(+)Treg cells of CD8^(+)T cells was decreased in nasal mucosa of CRSwNP patients(0.14%(0.28%))when compared with that of CRSsNP(0.89%(0.81%),Z=0.61,P=0.03).ECRS patients had the lower percentages of CD8^(+)Foxp3^(+)Treg cells(0.07%(0.44%))and CD8^(+)Foxp3^(-)IL-10^(+)Treg cells(0.13%(0.21%))than Non-ECRS patients(CD8^(+)Foxp3^(+)Treg cells:0.53%(0.75%);CD8^(+)Foxp3^(-)IL-10^(+)Treg cells:0.29%(0.76%),t value was 2.14,2.78,respectively,both P<0.05).The percentage of CD8^(+)Foxp3^(+)Treg cells and the ratio of CD8^(+)Foxp3^(-)IL-10^(+)T per CD8^(+)T cells were negatively correlated with the percentage of eosinophils in CRS patients(R^(2) value was 0.56,0.78,respectively,both P<0.001).There was no significant difference in the distribution of CD8^(+)Fxop3^(+)Treg cells and CD8^(+)Fxop3^(-)IL-10^(+)Treg cells in peripheral blood among different groups.Conclusion The percentages of CD8^(+)Treg cells decrease in CRSwNP patients,especially in ECRS patients,which are opposite to that of Tc2 and Tc17,and negatively correlate with the eosinophils percentage.This indicates that the decrease in the ratio of CD8^(+)Treg cell may be associated with the immune-imbalance and eosinophilic infiltration in nasal mucosa of CRS patients.