Legionella pp 305-319 | Cite as

Inflammasome Activation in Legionella-Infected Macrophages

  • Danielle P. A. Mascarenhas
  • Dario S. ZamboniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)


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 Legionella 



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.


  1. 1.
    Mascarenhas DP, Zamboni DS (2017) Inflammasome biology taught by Legionella pneumophila. J Leukoc Biol 101(4):841–849CrossRefGoogle Scholar
  2. 2.
    Newton HJ, Ang DK, van Driel IR, Hartland EL (2010) Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev 23(2):274–298CrossRefGoogle Scholar
  3. 3.
    Swanson MS, Hammer BK (2000) Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54:567–613CrossRefGoogle Scholar
  4. 4.
    Massis LM, Zamboni DS (2011) Innate immunity to legionella pneumophila. Front Microbiol 2:109CrossRefGoogle Scholar
  5. 5.
    Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10(2):417–426CrossRefGoogle Scholar
  6. 6.
    Berger KH, Isberg RR (1993) Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7(1):7–19CrossRefGoogle Scholar
  7. 7.
    Berger KH, Merriam JJ, Isberg RR (1994) Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol 14(4):809–822CrossRefGoogle Scholar
  8. 8.
    Isberg RR, O’Connor TJ, Heidtman M (2009) The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nat Rev Microbiol 7(1):13–24CrossRefGoogle Scholar
  9. 9.
    Roy CR, Berger KH, Isberg RR (1998) Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol 28(3):663–674CrossRefGoogle Scholar
  10. 10.
    Zamboni DS, Kobayashi KS, Kohlsdorf T, Ogura Y, Long EM, Vance RE et al (2006) The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection. Nat Immunol 7(3):318–325CrossRefGoogle Scholar
  11. 11.
    Amer A, Franchi L, Kanneganti TD, Body-Malapel M, Ozoren N, Brady G et al (2006) Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf. J Biol Chem 281(46):35217–35223CrossRefGoogle Scholar
  12. 12.
    Lightfield KL, Persson J, Brubaker SW, Witte CE, von Moltke J, Dunipace EA et al (2008) Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin. Nat Immunol 9(10):1171–1178CrossRefGoogle Scholar
  13. 13.
    Molofsky AB, Byrne BG, Whitfield NN, Madigan CA, Fuse ET, Tateda K et al (2006) Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J Exp Med 203(4):1093–1104CrossRefGoogle Scholar
  14. 14.
    Ren T, Zamboni DS, Roy CR, Dietrich WF, Vance RE (2006) Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog 2(3):e18CrossRefGoogle Scholar
  15. 15.
    Tenthorey JL, Haloupek N, Lopez-Blanco JR, Grob P, Adamson E, Hartenian E et al (2017) The structural basis of flagellin detection by NAIP5: a strategy to limit pathogen immune evasion. Science 358(6365):888–893CrossRefGoogle Scholar
  16. 16.
    Silveira TN, Zamboni DS (2010) Pore formation triggered by Legionella spp. is an Nlrc4 inflammasome-dependent host cell response that precedes pyroptosis. Infect Immun 78(3):1403–1413CrossRefGoogle Scholar
  17. 17.
    Case CL, Kohler LJ, Lima JB, Strowig T, de Zoete MR, Flavell RA et al (2013) Caspase-11 stimulates rapid flagellin-independent pyroptosis in response to Legionella pneumophila. Proc Natl Acad Sci U S A 110(5):1851–1856CrossRefGoogle Scholar
  18. 18.
    Cerqueira DM, Pereira MS, Silva AL, Cunha LD, Zamboni DS (2015) Caspase-1 but not Caspase-11 is required for NLRC4-mediated pyroptosis and restriction of infection by Flagellated legionella species in mouse macrophages and in vivo. J Immunol 195(5):2303–2311CrossRefGoogle Scholar
  19. 19.
    Mascarenhas DPA, Cerqueira DM, Pereira MSF, Castanheira FVS, Fernandes TD, Manin GZ et al (2017) Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome. PLoS Pathog 13(8):e1006502CrossRefGoogle Scholar
  20. 20.
    Casson CN, Copenhaver AM, Zwack EE, Nguyen HT, Strowig T, Javdan B et al (2013) Caspase-11 activation in response to bacterial secretion systems that access the host cytosol. PLoS Pathog 9(6):e1003400CrossRefGoogle Scholar
  21. 21.
    Cunha LD, Silva ALN, Ribeiro JM, Mascarenhas DPA, Quirino GFS, Santos LL et al (2017) AIM2 engages active but unprocessed Caspase-1 to induce noncanonical activation of the NLRP3 inflammasome. Cell Rep 20(4):794–805CrossRefGoogle Scholar
  22. 22.
    Ge J, Gong YN, Xu Y, Shao F (2012) Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking. Proc Natl Acad Sci U S A 109(16):6193–6198CrossRefGoogle Scholar
  23. 23.
    Pereira MS, Morgantetti GF, Massis LM, Horta CV, Hori JI, Zamboni DS (2011) Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo. J Immunol 187(12):6447–6455CrossRefGoogle Scholar
  24. 24.
    Feeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC et al (1979) Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 10(4):437–441PubMedPubMedCentralGoogle Scholar
  25. 25.
    Hori JI, Zamboni DS (2013) The mouse as a model for pulmonary legionella infection. Methods Mol Biol 954:493–503CrossRefGoogle Scholar
  26. 26.
    Englen MD, Valdez YE, Lehnert NM, Lehnert BE (1995) Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells. J Immunol Methods 184(2):281–283CrossRefGoogle Scholar
  27. 27.
    Marim FM, Silveira TN, Lima DS Jr, Zamboni DS (2010) A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells. PLoS One 5(12):e15263CrossRefGoogle Scholar
  28. 28.
    Cunha LD, Ribeiro JM, Fernandes TD, Massis LM, Khoo CA, Moffatt JH et al (2015) Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA. Nat Commun 6:10205CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Danielle P. A. Mascarenhas
    • 1
  • Dario S. Zamboni
    • 1
    Email author
  1. 1.Department of Cell Biology, School of Medicine of Ribeirão PretoUniversity of São PauloRibeirão PretoBrazil

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