Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer-Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation

Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (T...

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Bibliographic Details
Published in:The Journal of immunology (1950) 2019-02, Vol.202 (3), p.1003-1015
Main Authors: Nagar, Abhinit, DeMarco, Richard A, Harton, Jonathan A
Format: Article
Language:English
Subjects:
CARD Signaling Adaptor Proteins - analysis
Caspase 1 - analysis
Flow Cytometry - methods
Fluorescence
HEK293 Cells
Humans
Image Processing, Computer-Assisted - methods
Inflammasomes - metabolism
Macrophages
NLR Family, Pyrin Domain-Containing 3 Protein - agonists
NLR Family, Pyrin Domain-Containing 3 Protein - metabolism
Single-Cell Analysis - methods
THP-1 Cells
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Summary:Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStream instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.
ISSN:0022-1767
1550-6606
DOI:10.4049/jimmunol.1800973