Activating the NLRP3 Inflammasome Using the Amyloidogenic Peptide IAPP

  • Clara Westwell-Roper
  • Aisling Dunne
  • Man Lyang Kim
  • C. Bruce Verchere
  • Seth L. Masters
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1040)

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.

Key words

Inflammasome Amyloid Innate immunology NLR IAPP 

Notes

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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Copyright information

© Springer Science+business media, New York 2013

Authors and Affiliations

  • Clara Westwell-Roper
    • 1
  • Aisling Dunne
    • 2
  • Man Lyang Kim
    • 3
  • C. Bruce Verchere
    • 4
  • Seth L. Masters
    • 3
  1. 1.Department of Pathology and Laboratory Medicine, Child and Family Research InstituteUniversity of British ColumbiaVancouverCanada
  2. 2.Immunology Research Centre, School of Biochemistry and ImmunologyTrinity CollegeDublin 2Ireland
  3. 3.Inflammation DivisionThe Walter and Eliza Hall Institute of Medical ResearchMelbourneAustralia
  4. 4.Department of Pathology and Laboratory Medicine and the Department of Surgery, Child and Family Research InstituteUniversity of British ColumbiaVancouverCanada

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