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-1α expression, maturation and secretion in macrophages infected with Corynebacterium pseudotuberculosis

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Ca²⁺螯合剂(EDTA、EGTA+Mg²⁺和BAPTA-AM)、calpain抑制剂(calpain inhibitor Ⅲ、calpain inhibitor Ⅳ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-1α mRNA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Ca²⁺浓度显著增加，calpain活性增强，而缺失pld的Cp感染巨噬细胞内Ca²⁺浓度显著下降，calpain活性降低。EDTA、EGTA+Mg²⁺、BAPTA-AM、calpain inhibitor Ⅲ、calpain inhibitor Ⅳ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Ca²⁺浓度增加、激活钙蛋白酶有关。

Abstract:

[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 Ca²⁺ chelates (EDTA,EGTA+Mg²⁺ 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 Ca²⁺ 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+Mg²⁺,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-1α maturation and secretion in macrophages mediated by Cp were related to the increase of intracellular Ca²⁺and the activation of calpain.