伪结核棒状杆菌感染影响巨噬细胞IL-1α表达、成熟及分泌的机制研究  

Mechanism of IL-1αexpression,maturation and secretion in macrophages infected with Corynebacterium pseudotuberculosis

作　　者：王晓涵 田尚全 邹安龙 谭静梅 易文毅 王芝英[1,2,3] 周作勇 WANG Xiaohan;TIAN Shangquan;ZOU Anlong;TAN Jingmei;YI Wenyi;WANG Zhiying;ZHOU Zuoyong(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;Veterinary Science Engineering Research Center of Chongqing,Chongqing 402460,China;Immunology Research Center,Medical Research Institute,Southwest University,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]重庆市兽医科学工程研究中心,重庆402460 [3]西南大学医学院免疫研究中心,重庆402460

出　　处：《微生物学报》2022年第10期3947-3956,共10页Acta Microbiologica Sinica

基　　金：重庆市自然科学基金(cstc2021jcyj-msxmX0884);中央高校基本科研业务费专项基金(XDJK2020B016);重庆市技术创新与应用示范项目(cstc2018jscx-msybX0216)。

摘　　要：【目的】伪结核棒状杆菌(Corynebacterium pseudotuberculosis,Cp)感染动物常引起内脏和体表淋巴结脓肿,以被感染部位炎性细胞因子大量增加为特征。研究Cp感染小鼠巨噬细胞对IL-1α成熟分泌的影响及机制。【方法】采用荧光定量PCR、ELISA和Western blotting等方法,首先检测Cp(ATCC19410、XH02)及其磷脂酶D基因(pld)缺失株(ATCC19410Δpld、XH02Δpld)感染巨噬细胞对IL-1α表达及成熟分泌的影响,进而以Ca^(2+)螯合剂(EDTA、EGTA+Mg^(2+)和BAPTA-AM)、calpain抑制剂(calpain inhibitorⅢ、calpain inhibitorⅣ和EST)处理巨噬细胞,观察对Cp感染介导IL-1α成熟分泌的影响。【结果】Cp感染巨噬细胞后,IL-1αmRNA表达及分泌显著增加,与ATCC19410、XH02感染巨噬细胞相比,ATCC19410Δpld、XH02Δpld感染细胞IL-1α表达及分泌显著下降。Cp感染引起巨噬细胞内Ca^(2+)浓度显著增加,calpain活性增强,而缺失pld的Cp感染巨噬细胞内Ca^(2+)浓度显著下降,calpain活性降低。EDTA、EGTA+Mg^(2+)、BAPTA-AM、calpain inhibitorⅢ、calpain inhibitorⅣ处理Cp感染的巨噬细胞,巨噬细胞的IL-1α表达及分泌显著下降。【结论】Cp感染可激活巨噬细胞IL-1α表达和成熟分泌,磷脂酶D参与Cp感染巨噬细胞介导的IL-1α成熟分泌。Cp感染巨噬细胞介导IL-1α成熟与分泌与胞内Ca^(2+)浓度增加、激活钙蛋白酶有关。[Objective] Corynebacterium pseudotuberculosis(Cp) infection of animals often causes visceral and superficial lymph node abscesses,which is characterized by a large increase in inflammatory cytokines at the infected site.This study aimed to investigate the effect of Corynebacterium pseudotuberculosis(Cp) infection on IL-1α maturation and secretion in macrophages and the underlying mechanism.[Methods] The IL-1α expression,maturation and secretion in macrophages infected with Cp(ATCC19410,XH02) and phospholipase D gene(pld) deficient strains(ATCC19410Δpld,XH02Δpld) were evaluated by real-time quantitative PCR,ELISA and Western blotting.Further,the effects of Ca^(2+) chelates(EDTA,EGTA+Mg^(2+) and BAPTA-AM) and calpain inhibitors(calpain inhibitor Ⅲ,calpain inhibitor Ⅳ and EST) on IL-1α maturation and secretion in macrophages infected with Cp were tested.[Results] Cp infection induced increasing of IL-1α mRNA expression and secretion in macrophages,and the IL-1α mRNA expression and secretion in ATCC19410Δpld-infected and XH02Δpld-infected macrophages were significantly decreased compared with those of ATCC19410-infected and XH02-infected macrophages.The intracellular Ca^(2+) concentration and calpain activity were elevated in macrophages after Cp infection,while they were lowered in those infected with pld deficient Cp.Treatment of EDTA,EGTA+Mg,BAPTA-AM,calpain inhibitor Ⅲ and calpain inhibitor Ⅳ resulted in remarkable decrease of IL-1α expression and secretion in Cp infected macrophages.[Conclusion] Cp infection induced the IL-1α expression,maturation and secretion in macrophages,and phospholipase D was involved in this process.The IL-1αmaturation and secretion in macrophages mediated by Cp were related to the increase of intracellular Ca^(2+) and the activation of calpain.

关 键 词：伪结核棒状杆菌 巨噬细胞 IL-1Α 磷脂酶D 钙蛋白酶 

分 类 号：S852.61[农业科学—基础兽医学]