Mascarenhas Danielle P A, Zamboni Dario S
Department of Cell Biology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.
Methods Mol Biol. 2019;1921:305-319. doi: 10.1007/978-1-4939-9048-1_20.
Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1β and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.
嗜肺军团菌是一种革兰氏阴性细菌，可在淡水环境中感染多种单细胞原生动物。人类感染是偶然发生的，而且这种细菌可能尚未进化出绕过哺乳动物巨噬细胞中固有免疫信号传导的策略。因此，嗜肺军团菌会触发许多固有免疫途径，包括炎性小体激活。炎性小体是在宿主细胞质中组装的多分子平台，可导致炎性半胱天冬酶激活。炎性小体激活会导致炎性细胞因子（如IL-1β和IL-18）的分泌，以及一种称为细胞焦亡的炎性细胞死亡形式，细胞焦亡始于巨噬细胞膜上孔的诱导。在本章中，我们提供了详细的方案来评估巨噬细胞中军团菌诱导的炎性小体激活，包括实时孔形成测定、用于检测炎性半胱天冬酶激活（切割和下拉）的蛋白质印迹，以及炎性细胞因子的测量。
