TY  - JOUR
AU  - Cardona Gloria, Yamel
AU  - Weber, Alexander N R
TI  - Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay.
JO  - Methods in molecular biology
VL  - 2696
SN  - 1064-3745
CY  - [Heidelberg]
PB  - [Springer]
M1  - DKFZ-2023-01653
SN  - 978-1-0716-3349-6 (print)
SP  - 239-256
PY  - 2023
N1  - Second Edition
AB  - Interleukin (IL)-1β is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1β  31 kDa pro-peptide into a mature,  17 kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1β fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1β, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1β conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1β needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1β directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.
KW  - Humans
KW  - Inflammasomes
KW  - NLR Family, Pyrin Domain-Containing 3 Protein
KW  - Reproducibility of Results
KW  - Cells, Cultured
KW  - Macrophages
KW  - Interleukin-1beta
KW  - Immunoblotting
KW  - Inflammation
KW  - Caspase 1
KW  - Automated capillary-based immunoassay (Other)
KW  - Caspase-1 (Other)
KW  - Human macrophages (Other)
KW  - IL-1β (Other)
KW  - Inflammasome (Other)
KW  - NLRP3 (Other)
KW  - Inflammasomes (NLM Chemicals)
KW  - NLR Family, Pyrin Domain-Containing 3 Protein (NLM Chemicals)
KW  - Interleukin-1beta (NLM Chemicals)
KW  - Caspase 1 (NLM Chemicals)
LB  - PUB:(DE-HGF)3 ; PUB:(DE-HGF)16
C6  - pmid:37578727
DO  - DOI:DOI:10.1007/978-1-0716-3350-2_16
UR  - https://inrepo02.dkfz.de/record/278419
ER  -