Skip to main content
SpringerLink
Log in

Measuring Innate Immune Responses to Bacterial Viability

  • Protocol
  • First Online:
Innate Immune Activation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1714))

Abstract

The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1β) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Blander JM, Sander LE (2012) Beyond pattern recognition: five immune checkpoints for scaling the microbial threat. Nat Rev Immunol 12(3):215–225. https://doi.org/10.1038/nri3167

    Article  CAS  PubMed  Google Scholar 

  2. Sander LE, Davis MJ, Boekschoten MV, Amsen D, Dascher CC, Ryffel B, Swanson JA, Muller M, Blander JM (2011) Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature 474(7351):385–389. https://doi.org/10.1038/nature10072

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Moretti J, Blander JM (2014) Insights into phagocytosis-coupled activation of pattern recognition receptors and inflammasomes. Curr Opin Immunol 26:100–110. https://doi.org/10.1016/j.coi.2013.11.003

    Article  CAS  PubMed  Google Scholar 

  4. 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. https://doi.org/10.1016/j.cell.2012.07.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S, Miyake K, Zhang J, Lee WP, Muszynski A, Forsberg LS, Carlson RW, Dixit VM (2013) Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341(6151):1246–1249. https://doi.org/10.1126/science.1240248

    Article  CAS  PubMed  Google Scholar 

  6. Hagar JA, Powell DA, Aachoui Y, Ernst RK, Miao EA (2013) Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341(6151):1250–1253. https://doi.org/10.1126/science.1240988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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. https://doi.org/10.1038/nature10558

    Article  CAS  PubMed  Google Scholar 

  8. McWhirter SM, Barbalat R, Monroe KM, Fontana MF, Hyodo M, Joncker NT, Ishii KJ, Akira S, Colonna M, Chen ZJ, Fitzgerald KA, Hayakawa Y, Vance RE (2009) A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP. J Exp Med 206(9):1899–1911. https://doi.org/10.1084/jem.20082874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Woodward JJ, Iavarone AT, Portnoy DA (2010) c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328(5986):1703–1705. https://doi.org/10.1126/science.1189801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kailasan Vanaja S, Rathinam VA, Atianand MK, Kalantari P, Skehan B, Fitzgerald KA, Leong JM (2014) Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 111(21):7765–7770. https://doi.org/10.1073/pnas.1400075111

    Article  PubMed  PubMed Central  Google Scholar 

  11. Mankan AK, Schmidt T, Chauhan D, Goldeck M, Honing K, Gaidt M, Kubarenko AV, Andreeva L, Hopfner KP, Hornung V (2014) Cytosolic RNA:DNA hybrids activate the cGAS-STING axis. EMBO J 33(24):2937–2946. https://doi.org/10.15252/embj.201488726

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rigby RE, Webb LM, Mackenzie KJ, Li Y, Leitch A, Reijns MA, Lundie RJ, Revuelta A, Davidson DJ, Diebold S, Modis Y, MacDonald AS, Jackson AP (2014) RNA:DNA hybrids are a novel molecular pattern sensed by TLR9. EMBO J 33(6):542–558. https://doi.org/10.1002/embj.201386117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vanaja SK, Russo AJ, Behl B, Banerjee I, Yankova M, Deshmukh SD, Rathinam VA (2016) Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation. Cell 165(5):1106–1119. https://doi.org/10.1016/j.cell.2016.04.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Premaratne RJ, Lin WJ, Johnson EA (1991) Development of an improved chemically defined minimal medium for Listeria monocytogenes. Appl Environ Microbiol 57(10):3046–3048

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Julien Moretti and Nicolas Vabret contributed equally to this work.

Authors and Affiliations

  1. Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, 10021, USA

    Julien Moretti & J. Magarian Blander

  2. Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, 10021, USA

    Julien Moretti & J. Magarian Blander

  3. Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nicolas Vabret

  4. Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, 10021, USA

    J. Magarian Blander

Authors
  1. Julien Moretti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicolas Vabret
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Magarian Blander
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Magarian Blander .

Editor information

Editors and Affiliations

  1. Inflammation Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Dominic De Nardo

  2. Chemical Biology Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Christine M De Nardo

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Moretti, J., Vabret, N., Blander, J.M. (2018). Measuring Innate Immune Responses to Bacterial Viability. In: De Nardo, D., De Nardo, C. (eds) Innate Immune Activation. Methods in Molecular Biology, vol 1714. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7519-8_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7519-8_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7518-1

  • Online ISBN: 978-1-4939-7519-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation