Legionella pp 305-319 | Cite as
Inflammasome Activation in Legionella-Infected Macrophages
Protocol
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Abstract
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.
Key words
Macrophages Inflammasome Caspase-1 Caspase-11 LegionellaNotes
Acknowledgments
We thank Dr. Alexandre L.N. Silva and Talita D. Fernandes for details and discussions about the protocols detailed in this manuscript. Work in our laboratory is funded by grants from FAPESP and CNPq. We are grateful to technical assistance of Maira Nakamura, Catarina Horta, Leticia Corsi, Victoria Maria dos Santos, Vitoria Turin, and Laís de Castro Batista.
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