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Methods to Activate the NLRP3 Inflammasome

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NLR Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2696))

Abstract

The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors.

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Authors and Affiliations

  1. Faculty of Medicine, Institute of Neuropathology, Medical Center – University of Freiburg, Freiburg, Germany

    Benedikt S. Saller, Emilia Neuwirt & Olaf Groß

  2. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Benedikt S. Saller & Emilia Neuwirt

  3. Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

    Emilia Neuwirt & Olaf Groß

  4. Faculty of Medicine, Center for Basics in NeuroModulation (NeuroModulBasics), University of Freiburg, Freiburg, Germany

    Olaf Groß

Authors
  1. Benedikt S. Saller
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  2. Emilia Neuwirt
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  3. Olaf Groß
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Corresponding author

Correspondence to Olaf Groß .

Editor information

Editors and Affiliations

  1. Molecular Inflammation Group, Biomedical Research Institute of Murcia, El Palmar, Spain

    Pablo Pelegrín

  2. Department of Morphology, University of Ferrara, Ferrara, Ferrara, Italy

    Francesco Di Virgilio

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Saller, B.S., Neuwirt, E., Groß, O. (2023). Methods to Activate the NLRP3 Inflammasome. In: Pelegrín, P., Di Virgilio, F. (eds) NLR Proteins. Methods in Molecular Biology, vol 2696. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3350-2_12

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  • DOI: https://doi.org/10.1007/978-1-0716-3350-2_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3349-6

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