title: Assessment of Inflammasome Activation by Cytokine and Danger Signal Detection.
abstract: The evaluation of the inflammasome activation usually addresses the presence of extracellular IL-1β and IL-18 or the secretion of danger signal proteins such as HMGB-1 through their quantification using an enzyme-linked immunosorbent assay (ELISA). The ELISA is a routine laboratory technique that uses antibodies and colorimetric changes to identify a substance of interest. ELISA uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid or wet sample. Using 96 well plates, the ELISA technique enables to quantify the concentration of a single cytokine in multiple samples. However, a limitation of IL-1β and IL-18 ELISA is the absence of discrimination between active and non-active form of the proteins, parameter critical, for example, to distinguish the biologically relevant IL-1β from its poorly active form pro-IL-1β. This issue can be solved using western blots or immunoblots (IB), a common analytical procedure to detect the presence of different proteins in biological samples. Using denaturating conditions, IB allows the visualization of different sizes of the proteins of choice and is a commonly used technique in the inflammasome field to evaluate, for instance, the maturation of pro-IL-1β, pro-IL-18, and pro-caspase-1 into mature IL-1β, mature IL-18, and mature caspase-1, respectively. Moreover inflammasome activation may lead to the release of inflammasome particles outside the cell through caspase-1- or caspase-11-dependent cell death mechanism termed pyroptosis. In this case, NLR, ASC, and caspase-1 components are detectable outside the cell using IB analysis. ELISA and IB can be performed on cell culture supernatant or cell extract and on ex vivo samples from organ homogenates or biological fluids such as serum and plasma or bronchoalveolar lavages.
authors.affiliation: Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Riteau
authors.affiliation: INEM, CNRS, UMR7355, University of Orleans, 3B rue de la Férollerie, Orleans, 45071, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Gombault
authors.affiliation: INEM, CNRS, UMR7355, University of Orleans, 3B rue de la Férollerie, Orleans, 45071, France. couillin@cnrs-orleans.fr.|Molecular and Experimental Immunology and Neurogenetics, UMR 7355, 3B, rue de la Férollerie, Orleans, 45071, France. couillin@cnrs-orleans.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Couillin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D020170:Caspase 1 / Q000378:metabolism; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D024243:HMGB1 Protein / Q000378:metabolism*; D015151:Immunoblotting; D058847:Inflammasomes / Q000378:metabolism*; D020382:Interleukin-18 / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D000069292:Pyroptosis
chemical_list: D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; D058847:Inflammasomes; D020382:Interleukin-18; D053583:Interleukin-1beta; D020170:Caspase 1
keywords: ATP; ELISA; HMGB-1; IL-18; IL-1β; Immunoblot