糖蛋白A为主重复序列蛋白通过调节性T细胞参与调控结核病发病的初步研究

Preliminary study on the role of glycoprotein A repetitions predominant in regulating tuberculosis pathogenesis through regulatory T cells

目的:探讨糖蛋白A为主重复序列蛋白(glycoprotein A repetitions predominant,GARP)通过调节性T细胞(regulatory T cell,Treg)参与调控结核病发病的作用,以期为结核病治疗提供新靶点。方法:纳入2021年1月至9月复旦大学附属华山医院和无锡市第五人民医院收治的60例活动性肺结核患者,同时招募6例结核潜伏感染患者和16名健康对照者。应用流式细胞术检测入组对象外周血淋巴细胞中Treg的占比,以及Treg上GARP和转化生长因子-β1(transforming growth factor-β1,TGF-β1)的表达。统计学分析采用曼-惠特尼 U检验。 结果:60例活动性肺结核患者中,23例未进行抗结核药物治疗,17例患者治疗<3个月,10例患者治疗3~<6个月,10例患者治疗≥6个月。活动性肺结核未治疗组CD4 +CD25 +叉头样转录因子3(forkhead box protein 3,Foxp3) +Treg占外周血淋巴细胞总数的7.50%(5.67%,9.00%),高于健康对照组的5.57%(5.03%,6.09%),差异有统计学意义( U=95.00,P=0.010)。活动性肺结核未治疗组中,表达GARP的CD4 +CD25 +Foxp3 + Treg占CD4 +CD25 +Foxp3 + Treg总数的10.37%(7.79%,12.90%),分别高于结核潜伏感染组的7.02%(5.15%,8.81%)和健康对照组的5.33%(4.26%,6.67%),差异均有统计学意义( U=31.00, P=0.040;U=36.00, P<0.001);而结核潜伏感染组与健康对照组之间差异无统计学意义( U=25.00, P=0.095)。活动性肺结核未治疗组中,表达TGF-β1的CD4 +CD25 +Foxp3 + Treg占Treg总数的7.13%(4.25%,8.89%),高于健康对照组的3.59%(2.10%,5.17%),差异有统计学意义( U=71.00, P=0.001)。抗结核治疗<3个月组、治疗3~<6个月组、治疗≥6个月组活动性肺结核患者的CD4 +CD8 -CD25 +Foxp3 +Treg中GARP的表达量分别为7.82%(3.94%,13.17%)、6.92%(5.61%,9.47%)和7.26%(5.82%,9.64%);3组中在CD4 +CD8 -CD25 +Foxp3 +Treg中TGF-β1表达量分别为11.16%(7.91%,15.23%)、8.66%(5.43%,12.54%)和7.82%(6.01%,9.53%),其中治疗<3个月组TGF-β1的表达量高于治疗≥6个月组,差异有统计学意义( U=37.50, P=0.024)。 结论:Foxp3/GARP/TGF-β1通路可能参与Treg调控结核病发病的免疫机制,GARP有可能成为抗结核治疗的新型靶点。

Objective To investigate the role of glycoprotein A repetitions predominant(GARP)in the pathogenesis of tuberculosis through regulatory T cell(Treg),in order to provide new targets for the treatment of tuberculosis.Methods Sixty patients with active pulmonary tuberculosis(ATB)admitted to Huashan Hospital,Fudan University and Wuxi Fifth People′s Hospital from January to September 2021 were included.And six individuals with latent tuberculosis infection(LTBI),and 16 healthy controls(HC)were recruited during the same period.Flow cytometry was performed to detect the proportion of Treg in the peripheral blood,and the expressions of GARP and transforming growth factor-β1(TGF-β1)on Treg in different groups.Mann-Whitney U test was used for statistical analysis.Results Among the 60 patients with ATB,23 patients did not receive anti-tuberculosis drug therapy,17 patients were treated for less than three months,ten patients were treated for three to less than six months,and ten patients were treated for greater than or equal to six months.The percentage of CD4+CD25+forkhead box protein 3(Foxp3)+Treg in untreated ATB patients was 7.50%(5.67%,9.00%),which was higher than that in HC(5.57%(5.03%,6.09%)),and the difference was statistically significant(U=95.00,P=0.010).The percentage of GARP expressing in CD4+CD25+Foxp3+Treg in untreated ATB patients was 10.37%(7.79%,12.90%),which was higher than that in LTBI(7.02%(5.15%,8.81%))and HC(5.33%(4.26%,6.67%)),respectively,and the differences were both statistically significant(U=31.00,P=0.040;U=36.00,P<0.001,respectively),while there was no significant difference between LTBI and HC(U=25.00,P=0.095).The percentage of CD4+CD25+Foxp3+Treg expressing TGF-β1 in untreated ATB patients was 7.13%(4.25%,8.89%),which was higher than that in HC(3.59%(2.10%,5.17%)),and the difference was statistically significant(U=71.00,P=0.001).The expressions of GARP in CD4+CD8-CD25+Foxp3+Treg in patients with ATB treated for less than three months group,three to less than six months group and greater than or equal to six months group were 7.82%(3.94%,13.17%),6.92%(5.61%,9.47%)and 7.26%(5.82%,9.64%),respectively.The expressions of TGF-β1 in CD4+CD8-CD25+Foxp3+Treg in the above three treatment groups were 11.16%(7.91%,15.23%),8.66%(5.43%,12.54%)and 7.82%(6.01%,9.53%),respectively,and the expression of TGF-β1 in CD4+CD8-CD25+Foxp3+Treg in the patients with ATB treated for less than three months group was higher than that in the greater than or equal to six months group,the difference was statistically significant(U=37.50,P=0.024).Conclusions Foxp3/GARP/TGF-β1 pathway may be involved in the immune mechanism of Treg regulating the pathogenesis of tuberculosis,and GARP may be a new target for anti-tuberculosis therapy.