Abstract
Canonical inflammasome activation is a tightly regulated process that has been implicated in a broad spectrum of inflammatory disorders. Inflammasome formation requires assembly of a cytosolic sensor protein with the adapter, ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain). Once formed, this multimeric protein structure allows for the activation of caspase-1, responsible for IL-1ß/IL-18 release. During this process, cytoplasmic dispersed ASC molecules cluster in one condensed micrometric-sized complex named ASC “speck,” which is traditionally assessed by fluorescence microscopy and widely accepted as a readout for canonical inflammasome activation. However, equally reliable but less time-consuming quantitative methods have emerged as a significant need in order to improve clinical assessment of inflammasome-related conditions. Multispectral imaging flow cytometry (MIFC) combines the qualitative power of fluorescence microscopy with high throughput capabilities and multiplexing potential of flow cytometry into one single system. Here we explored the optimal imaging-based tools to measure ASC speck formation via imaging flow cytometry by using peripheral blood mononuclear cells (PBMCs) stimulated with the NLRP3 agonist Nigericin, as a positive control. We demonstrate that this technique is also able to detect the distribution of active caspase-1 within the ASC aggregates by incubating cells with FAM-FLICATM, a fluorochrome inhibitor of caspase-1. By applying these tools in PBMCs from patients with distinct inflammatory disorders we demonstrate that MIFC is able to assess canonical inflammasome activation in a quantitative and statistically robust manner in clinically relevant samples. Therefore, we propose that accurate assessment of specks by MIFC could help guide preventive or therapeutic strategies in an array of human inflammatory diseases in which inflammasomes play an important role.
Highlights
Inflammasomes are cytosolic protein aggregates that are assembled in order to coordinate distinct immune responses to infectious agents or physiological anomalies [1, 2]
ASC recruitment is a key event for canonical inflammasome activation, because it links activated inflammasome sensors to the effector molecule pro-caspase-1, and provides the conformational changes that are crucial to drive caspase-1 autoproteolysis and subsequent inflammatory cytokine production
We demonstrated that multispectral imaging flow cytometry provides a very simple, fast and reliable method to detect ASC specks in human monocytes, in a quantitative and qualitative manner
Summary
Inflammasomes are cytosolic protein aggregates that are assembled in order to coordinate distinct immune responses to infectious agents or physiological anomalies [1, 2]. Inflammasomes are formed when members of the NLR (nucleotide-binding oligomerization domain (NOD)like receptor) family or the HIN200 (hematopoietic interferoninducible nuclear antigens with 200 amino-acid repeats) family, sense either microbial components or host-derived molecules [2]. Upon activation, these sensors oligomerize and recruit an adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain or CARD). These effector caspases are caspase-1, −4, and −5 in humans and caspase-1, −11, and −12 in mice, caspase-1 is the most commonly inflammasomeassociated caspase [1, 19]
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