Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 87, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/51284
Keywords
Immunology; Issue 87; NLRP3; inflammasome; IL-1beta; Interleukin-1 beta; dendritic; cell; Nigericin; Toll-Like Receptor 8; TLR8; R848; Monocyte Derived Dendritic Cells
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Funding
- National Institute of Allergy and Infectious Disease
- NIH grants Ruth L. Kirschstein National Research Service [F31, AI089030, AI081848]
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Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control(1,2). Interleukin-1 beta is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection(1-5). Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1 beta secretion(6). Pro-IL-1 beta protein expression is limited in resting cells; therefore a priming signal is required for IL-1 beta transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multiprotein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1 beta secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin. Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.
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