Measuring Inflammasome Activation in Response to Bacterial Infection

  • Petr Broz
  • Denise M. Monack
Part of the Methods in Molecular Biology book series (MIMB, volume 1040)


Inflammasomes are multi-protein signaling platforms assembled in response to viral and bacterial pathogens as well as endogenous danger signals. Inflammasomes serve as activation platforms for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. The hallmarks of inflammasome activation are the processing of caspase-1, the maturation and release of interleukin-1β (IL-1β) and the induction of pyroptosis, a lytic inflammatory cell death. This protocol describes methods for studying inflammasome activation in response to bacterial pathogens in bone-marrow derived murine macrophages (BMDMs). In particular, we outline the protocols to measure cytokine maturation by ELISA and pyroptosis by the release of Lactate Dehydrogenase (LDH). In addition, we describe methods to visualize endogenous ASC specks or foci in infected cells and to study the release of processed caspase-1, caspase-11 and mature cytokines into the cell supernatant by Western blotting. General considerations are discussed to design and optimize the infection protocol for the study of inflammasome activation by other bacterial pathogens.

Key words

Inflammasome Caspase-1 Caspase-11 Pyroptosis Interleukin-1 LDH release NLRs Bacterial infections ASC speck 



We would like to thank Maikke B. Ohlson and Jens Kortmann for critical reading of the manuscript. This work was supported by awards AI095396 and AI08972 from the National Institute of Allergy and Infectious Diseases to D.M., a Stanford Digestive Disease Center (DDC) pilot grant to P.B. and a long-term fellowship from the Human Frontiers in Science Program (HFSP) to P.B.


  1. 1.
    Schroder K, Muruve DA, Tschopp J (2009) Innate immunity: cytoplasmic DNA sensing by the AIM2 inflammasome. Curr Biol 19(6):R262–R265. doi: S0960-9822(09)00677-0 [pii]  10.1016/j.cub.2009.02.011 PubMedCrossRefGoogle Scholar
  2. 2.
    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):417–426. doi: S1097276502005993 [pii] PubMedCrossRefGoogle Scholar
  3. 3.
    Lamkanfi M (2011) Emerging inflammasome effector mechanisms. Nat Rev Immunol 11(3):213–220. doi: nri2936 [pii]  10.1038/nri2936 PubMedCrossRefGoogle Scholar
  4. 4.
    Keller M, Ruegg A, Werner S, Beer HD (2008) Active caspase-1 is a regulator of unconventional protein secretion. Cell 132(5):818–831. doi: S0092-8674(08)00111-6 [pii]  10.1016/j.cell.2007.12.040 PubMedCrossRefGoogle Scholar
  5. 5.
    Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73(4):1907–1916. doi: 73/4/1907 [pii]  10.1128/IAI.73.4.1907-1916.2005 PubMedCrossRefGoogle Scholar
  6. 6.
    Boyden ED, Dietrich WF (2006) Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat Genet 38(2):240–244. doi: ng1724 [pii]  10.1038/ng1724 PubMedCrossRefGoogle Scholar
  7. 7.
    Hornung V, Latz E (2010) Critical functions of priming and lysosomal damage for NLRP3 activation. Eur J Immunol 40(3):620–623. doi: 10.1002/eji.200940185 PubMedCrossRefGoogle Scholar
  8. 8.
    Skeldon A, Saleh M (2011) The inflammasomes: molecular effectors of host resistance against bacterial, viral, parasitic, and fungal infections. Front Microbiol 2:15. doi: 10.3389/fmicb.2011.00015 PubMedCrossRefGoogle Scholar
  9. 9.
    Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S, Dixit VM (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430(6996):213–218. doi: 10.1038/nature02664 nature02664 [pii] PubMedCrossRefGoogle Scholar
  10. 10.
    Amer A, Franchi L, Kanneganti TD, Body-Malapel M, Ozoren N, Brady G, Meshinchi S, Jagirdar R, Gewirtz A, Akira S, Nunez G (2006) Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf. J Biol Chem 281(46):35217–35223. doi: M604933200 [pii]  10.1074/jbc.M604933200 PubMedCrossRefGoogle Scholar
  11. 11.
    Franchi L, Amer A, Body-Malapel M, Kanneganti TD, Ozoren N, Jagirdar R, Inohara N, Vandenabeele P, Bertin J, Coyle A, Grant EP, Nunez G (2006) Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages. Nat Immunol 7(6):576–582. doi: ni1346 [pii]  10.1038/ni1346 PubMedCrossRefGoogle Scholar
  12. 12.
    Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW, Miller SI, Aderem A (2006) Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat Immunol 7(6):569–575. doi: ni1344 [pii]  10.1038/ni1344 PubMedCrossRefGoogle Scholar
  13. 13.
    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):e18. doi: 10.1371/journal.ppat.0020018 PubMedCrossRefGoogle Scholar
  14. 14.
    Miao EA, Mao DP, Yudkovsky N, Bonneau R, Lorang CG, Warren SE, Leaf IA, Aderem A (2010) Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci USA 107(7):3076–3080. doi: 0913087107 [pii]  10.1073/pnas.0913087107 PubMedCrossRefGoogle Scholar
  15. 15.
    Kofoed EM, Vance RE (2011) Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477(7366):592–595. doi: 10.1038/nature10394 nature10394 [pii] PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao Y, Yang J, Shi J, Gong YN, Lu Q, Xu H, Liu L, Shao F (2011) The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477(7366):596–600. doi: 10.1038/nature10510 nature10510 [pii] PubMedCrossRefGoogle Scholar
  17. 17.
    Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J, Datta P, McCormick M, Huang L, McDermott E, Eisenlohr L, Landel CP, Alnemri ES (2010) The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat Immunol 11(5):385–393. doi: ni.1859 [pii]  10.1038/ni.1859 PubMedCrossRefGoogle Scholar
  18. 18.
    Rathinam VA, Jiang Z, Waggoner SN, Sharma S, Cole LE, Waggoner L, Vanaja SK, Monks BG, Ganesan S, Latz E, Hornung V, Vogel SN, Szomolanyi-Tsuda E, Fitzgerald KA (2010) The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol 11(5):395–402. doi: ni.1864 [pii]  10.1038/ni.1864 PubMedCrossRefGoogle Scholar
  19. 19.
    Jones JW, Kayagaki N, Broz P, Henry T, Newton K, O’Rourke K, Chan S, Dong J, Qu Y, Roose-Girma M, Dixit VM, Monack DM (2010) Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc Natl Acad Sci USA 107(21):9771–9776. doi: 1003738107 [pii]  10.1073/pnas.1003738107 PubMedCrossRefGoogle Scholar
  20. 20.
    Kim S, Bauernfeind F, Ablasser A, Hartmann G, Fitzgerald KA, Latz E, Hornung V (2010) Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome. Eur J Immunol 40(6):1545–1551. doi: 10.1002/eji.201040425 PubMedCrossRefGoogle Scholar
  21. 21.
    Warren SE, Armstrong A, Hamilton MK, Mao DP, Leaf IA, Miao EA, Aderem A (2010) Cutting edge: cytosolic bacterial DNA activates the inflammasome via Aim2. J Immunol 185(2):818–821. doi: jimmunol.1000724 [pii]  10.4049/jimmunol.1000724 PubMedCrossRefGoogle Scholar
  22. 22.
    Wu J, Fernandes-Alnemri T, Alnemri ES (2010) Involvement of the AIM2, NLRC4, and NLRP3 inflammasomes in caspase-1 activation by Listeria monocytogenes. J Clin Immunol 30(5):693–702. doi: 10.1007/s10875-010-9425-2 PubMedCrossRefGoogle Scholar
  23. 23.
    Tsuchiya K, Hara H, Kawamura I, Nomura T, Yamamoto T, Daim S, Dewamitta SR, Shen Y, Fang R, Mitsuyama M (2010) Involvement of absent in melanoma 2 in inflammasome activation in macrophages infected with Listeria monocytogenes. J Immunol 185(2):1186–1195. doi: jimmunol.1001058 [pii]  10.4049/jimmunol.1001058 PubMedCrossRefGoogle Scholar
  24. 24.
    Sauer JD, Witte CE, Zemansky J, Hanson B, Lauer P, Portnoy DA (2010) Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe 7(5):412–419. doi: S1931-3128(10)00110-1 [pii]  10.1016/j.chom.2010.04.004 PubMedCrossRefGoogle Scholar
  25. 25.
    Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N, Hidaka E, Katsuyama T, Higuchi T, Sagara J (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274(48):33835–33838PubMedCrossRefGoogle Scholar
  26. 26.
    Broz P, Newton K, Lamkanfi M, Mariathasan S, Dixit VM, Monack DM (2010) Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J Exp Med 207(8):1745–1755. doi: jem.20100257 [pii]  10.1084/jem.20100257 PubMedCrossRefGoogle Scholar
  27. 27.
    Fernandes-Alnemri T, Wu J, Yu JW, Datta P, Miller B, Jankowski W, Rosenberg S, Zhang J, Alnemri ES (2007) The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9):1590–1604. doi: 4402194 [pii]  10.1038/sj.cdd.4402194 PubMedCrossRefGoogle Scholar
  28. 28.
    Bryan NB, Dorfleutner A, Rojanasakul Y, Stehlik C (2009) Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J Immunol 182(5):3173–3182. doi: 182/5/3173 [pii]  10.4049/jimmunol.0802367 PubMedCrossRefGoogle Scholar
  29. 29.
    Broz P, von Moltke J, Jones JW, Vance RE, Monack DM (2010) Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing. Cell Host Microbe 8(6):471–483. doi: S1931-3128(10)00380-X [pii]  10.1016/j.chom.2010.11.007 PubMedCrossRefGoogle Scholar
  30. 30.
    Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A, Warren SE, Wewers MD, Aderem A (2010) Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol 11(12):1136–1142. doi: ni.1960 [pii]  10.1038/ni.1960 PubMedCrossRefGoogle Scholar
  31. 31.
    Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J, Newton K, Qu Y, Liu J, Heldens S, Zhang J, Lee WP, Roose-Girma M, Dixit VM (2011) Non-canonical inflammasome activation targets caspase-11. Nature 479(7371):117–121. doi: 10.1038/nature10558 nature10558 [pii] PubMedCrossRefGoogle Scholar
  32. 32.
    Broz P, Ruby T, Belhocine K, Bouley DM, Kayagaki N, Dixit VM, Monack DM (2012) Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature 490(7419):288–291. doi: 10.1038/nature11419 nature11419 [pii] PubMedCrossRefGoogle Scholar
  33. 33.
    Rathinam VA, Vanaja SK, Waggoner L, Sokolovska A, Becker C, Stuart LM, Leong JM, Fitzgerald KA (2012) TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150(3):606–619. doi: S0092-8674(12)00825-2 [pii]  10.1016/j.cell.2012.07.007 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+business media, New York 2013

Authors and Affiliations

  • Petr Broz
    • 1
  • Denise M. Monack
    • 1
  1. 1.Department of Microbiology and Immunology, Stanford School of MedicineStanford UniversityStanfordUSA

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