奥布替尼在分枝杆菌感染宿主巨噬细胞中的免疫调控作用  

Immunomodulatory effect of orelabrutinib in host macrophages infected with mycobacterium

作　　者：王艺霖 吴潇 逄宇 李姗姗 Wang Yilin;Wu Xiao;Pang Yu;Li Shanshan(Beijing Key Laboratory on Drug-Resistant Tuberculosis Research/Beijing Chest Hospital,Capital Medical University/Bacterial Immunology Laboratory,Beijing Tuberculosis and Thoracic Tumor Institute,Beijing 101149,China;The First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310003,China)

机构地区：[1]首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所细菌免疫学实验室/耐药结核病研究北京市重点实验室,北京101149 [2]浙江大学医学院附属第一医院,杭州310003

出　　处：《中国防痨杂志》2024年第9期1063-1068,共6页Chinese Journal of Antituberculosis

基　　金：国家自然科学基金(82202530);北京市科技新星计划创新新星(20220484169、20230484295)

摘　　要：目的:探讨新型布鲁顿酪氨酸激酶(Bruton tyrosine kinase,BTK)抑制剂奥布替尼在结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染宿主巨噬细胞中的免疫调控作用。方法:通过人急性单核白血病细胞(human acute monocytic leukemia cells,THP-1)培养与分化,用耻垢分枝杆菌感染分化后的THP-1细胞,并以不同浓度的奥布替尼处理,比较其对巨噬细胞吞噬和清除耻垢分枝杆菌的能力。利用siRNA干扰技术敲低BTK蛋白表达,通过蛋白免疫印迹法验证敲除效果,并使用菌落形成单位(CFU)计数法检测奥布替尼对巨噬细胞胞内耻垢分枝杆菌的清除能力。结果:奥布替尼能够明显抑制BTK的酪氨酸磷酸化而不改变BTK蛋白的表达量,同时奥布替尼处理后的巨噬细胞胞内耻垢分枝杆菌菌落计数从130.0(120.3~137.5)×10^(3)个/ml下降至59.0(55.8~65.3)×10^(3)个/ml,差异有统计学意义(U=0.000,P=0.014)。在敲低BTK蛋白表达后,巨噬细胞内耻垢分枝杆菌的载菌量由52.0(44.0~61.5)×10^(3)个/ml降低至25.0(22.0~28.0)×10^(3)个/ml,差异有统计学意义(U=9.000,P=0.002)。结论:BTK在巨噬细胞功能调节中起关键作用,奥布替尼作为新型BTK抑制剂,不仅能够抑制BTK酪氨酸磷酸化,还能明显增强巨噬细胞对耻垢分枝杆菌的清除能力,为其潜在抗结核应用提供了新的思路。Objective:To investigate the immunomodulatory effect of a novel Bruton tyrosine kinase(BTK)inhibitor orelabrutinib,in host macrophages infected with Mycobacterium tuberculosis(MTB).Methods:Human acute monocytic leukemia(THP-1)cells were cultured and differentiated.The differentiated THP-1 cells were infected with Mycobacterium smegmatis(M.smeg)and then treated with different concentrations of orelabrutinib to compare the ability of macrophages to engulf and clear M.smeg.BTK protein expression was knocked down by using siRNA interference technology,and the knockdown effect was verified by Western blotting.The colony-forming unit(CFU)assay was used to measure the clearance ability of macrophages against M.smeg by treated with orelabrutinib.Results:Orelabrutinib significantly inhibited the tyrosine phosphorylation of BTK without changing the level of BTK protein expression.Additionally,the CFU count of M.smeg in macrophages treated with orelabrutinib decreased from 130.0(120.3-137.5)×10^(3)/ml to 59.0(55.8-65.3)×10^(3)/ml,with statistical significance(U=0.000,P=0.014).After knocking down BTK protein expression,the bacterial load of M.smeg in macrophages decreased from 52.0(44.0-61.5)×10^(3)/ml to 25.0(22.0-28.0)×10^(3)/ml,with statistical significance(U=9.000,P=0.002).Conclusion:BTK plays a crucial role in macrophage functional regulation.As a novel BTK inhibitor,orelabrutinib not only suppresses BTK tyrosine phosphorylation but also significantly enhances the clearance ability of macrophage against M.smeg,providing a new perspective for its potential anti-tuberculosis applications.

关 键 词：分枝杆菌 结核 蛋白酪氨酸激酶类 巨噬细胞 奥布替尼 

分 类 号：R516[医药卫生—内科学] R378.91[医药卫生—临床医学]