A Novel Flow Cytometric Method To Assess Inflammasome Formation

Abstract

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

The Inflammasome Adaptor ASC Induces Procaspase-8 Death Effector Domain Filaments

“…3A). Although ASC can form specks spontaneously when expressed alone above a certain threshold, speck formation is greatly increased in the presence of AIM2 (39). We surmise that AIM2 clustering nucleates the formation of an ASC speck, and reciprocally, the interaction with ASC stabilizes AIM2 self-association so that it remains part of a stable speck.…”

“…The procaspase-8 DEDs (residues 1-216), ASC PYD (residues 1-96), and NLRP3 PYD (residues 1-100) were separately cloned into pEF6-HIN AIM2 -mCherry to generate pEF6-DEDs procaspase- 8 -HIN AIM2 -mCherry, pEF6-PYD ASC -HIN AIM2 -mCherry, and pEF6-PYD NLRP3 -HIN AIM2 -mCherry, respectively. pEF6-ASCeGFP (pEF6 with human ASC with a C-terminal enhanced GFP fusion) and pcDNA3-ASC (wild type and mutants) have been described previously (31,39). For fluorescence polarization assays and/or electron microscopy studies, ASC PYD (residues 1-106), ASC CARD (residues 107-195), or full-length ASC (residues 1-195) was cloned into pDB.His.MBP for expression with an N-terminal His 6 -MBP tag that was cleavable with TEV protease.…”

“…The percentage of speck-forming cells was determined in a window of moderate ASC expression, below the level at which ASC spontaneously forms specks (39).…”

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

“…Finally, inflammasome formation was confirmed by flow cytometry assessment of ASC oligomerization, where ASC polymerization in inflammasome complexes can be detected by changes in fluorescence pulse height and area ( Figure 1F). 24 MDS specimens also displayed significantly greater inflammasome assembly compared with controls, irrespective of International Prognostic Scoring System (IPSS) risk group ( Figure 1C). NLRP3 inflammasome assembly was increased 2.9-fold in LR-MDS patients (P 5 3.9 3 10 25 ) and 3.1-fold in HR-MDS patients (P 5 7.1 3 10 25 ).…”

“…To specifically distinguish pyroptosis from apoptosis, the percentage of pyroptotic cells, defined as a-caspase-1 1 /a-caspase-3/7 1 /annexin V 1 cells, was determined in phenotypically distinct hematopoietic lineages by flow cytometry. Normal (n 5 5) and LR-MDS BM-MNCs (n 5 8) were incubated with autologous BM plasma for 24 Figure 1H). Additionally, the percentage of a-caspase-1 1 cells was increased 14.2-fold in the stem cell fraction (P 5 8.0 310 23 ), 13.2-fold in progenitors, 12.9-fold in immature myeloid cells (P 5 1.3 3 10 24 ), and 13.0-fold in CD71 1 erythroid precursors (P 5 7.7 3 10 23 ) ( Figure 1I).…”