Abstract
In addition to several other extracellular substances, phagocytosis of amyloid-forming peptides can perturb cellular homeostasis, leading to activation of the cytoplasmic innate immune receptor NLRP3. Once triggered, NLRP3 forms an inflammasome complex that ultimately cleaves pro-IL-1β and pro-IL-18 into their mature, secreted forms. Here we describe a protocol by which one type of amyloidogenic peptide, islet amyloid polypeptide (IAPP, otherwise known as amylin) can be prepared and used to stimulate myeloid cells in vitro to engage the NLRP3 inflammasome. Methods for measuring the ensuing inflammasome activation are also described. Although initially soluble, IAPP monomers rapidly aggregate in solution to form oligomers and subsequently insoluble amyloid fibrils. More work is required to examine how this transition influences inflammasome activation for different types of amyloid. The course of amyloid formation and corresponding inflammatory capacity of these pre-fibrillar species following uptake also requires further examination, and we hope that our protocols are useful in these endeavors. While these protocols are restricted to examination of synthetic IAPP, isolation of IAPP aggregates from human and transgenic mouse pancreas will be required to definitively determine the proinflammatory effects of endogenous IAPP oligomers and fibrils.
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Acknowledgments
The authors would like to thank Ben A. Croker for initially optimizing the IL-18 ELISA protocol. S.L.M. is supported by a fellowship from the National Health and Medical Research Council of Australia (516783). C.W.R. is supported by a Vanier Canada Graduate Scholarship and C.B.V. by grants from the Canadian Institutes of Health Research (MOP-14862) and 20R66631 and the Canadian Diabetes Association (OG-3-11-3413-CV).
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Westwell-Roper, C., Dunne, A., Kim, M.L., Verchere, C.B., Masters, S.L. (2013). Activating the NLRP3 Inflammasome Using the Amyloidogenic Peptide IAPP. In: De Nardo, C., Latz, E. (eds) The Inflammasome. Methods in Molecular Biology, vol 1040. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-523-1_2
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DOI: https://doi.org/10.1007/978-1-62703-523-1_2
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