title: Activation and measurement of NLRP3 inflammasome activity using IL-1β in human monocyte-derived dendritic cells.
abstract: 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-1β 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-1β secretion(6). Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β 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.
authors.affiliation: Department of Pathology, New York University School of Medicine.
authors.forename: Melissa V
authors.initials: MV
authors.lastname: Fernandez
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Miller
authors.affiliation: Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center; nina.bhardwaj@mssm.edu.
authors.forename: Nina
authors.initials: N
authors.lastname: Bhardwaj
mesh_terms: D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D003713:Dendritic Cells / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D058847:Inflammasomes / Q000737:chemistry* / Q000276:immunology; D053583:Interleukin-1beta / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D009550:Nigericin / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D058847:Inflammasomes; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D009550:Nigericin