FACS-based detection of extracellular ASC specks from NLRP3 inflammasomes in inflammatory diseases

Abstract

ABSTRACTThe apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is crucial for inflammasome assembly and activation of several inflammasomes, including NLRP3. ASC aggregates are detected in human sera post pyroptotic cell death, but their inflammasome origin remains unclear. This study aimed to develop a method to detect ASC aggregates originating from NLRP3 inflammasomes. Initially, human monocytes, macrophages, and THP-1 ASC reporter cells were employed to validate the detection of ASC/NLRP3-positive events through flow cytometry. The presence of ASC/NLRP3 specks was confirmed in cell supernatants from monocytes and macrophages treated with LPS and nigericin or ATP. Flow cytometry analysis identified double-positive specks in patient sera from inflammatory conditions when compared to healthy controls. Elevated ASC/NLRP3 specks were observed in conditions such as CAPS and Schnitzler’s syndrome. We validated FACS as a reliable method for detecting ASC/NLRP3 specks in human sera, with potential diagnostic and monitoring applications in certain systemic autoinflammatory diseases.