Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1β and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer’s disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze–thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck⁺ cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck⁺ cells. Aria III, Astrios EQ, and Canto II performed best in separating ASC speck⁺ from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck⁺ cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck⁺ cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.

Staphylococcus aureus is a versatile pathogen, renowned for its arsenal of virulence and immune evasion factors. Several S. aureus virulence factors have been targeted in vaccination trials; however, so far, without success. Promising new vaccine candidates are the staphylococcal serine protease-like proteins (Spl A–F), which are involved in the pathogenesis and immune evasion of S. aureus . SplB, for instance, promotes type 2 immune responses and inactivates human complement factors. In this study, we report on the production and characterization of a murine monoclonal antibody (mAb) against SplB. The murine anti-SplB mAb α-SplB1 was produced by hybridoma technology, and its binding characteristics were investigated using enzyme-linked immunosorbent assay (ELISA), Western blot, and MicroScale Thermophoresis. Its neutralizing capacity was determined in a fluorogenic substrate assay, Western blot, and a murine vascular leakage model. α-SplB1 bound to recombinant SplB with high specificity, showing no cross-reactivity to other Spls or secreted proteins of S. aureus . MicroScale Thermophoresis revealed a K _D value of 37.9 nM for the α-SplB1:SplB interaction. α-SplB1 neutralized the enzymatic activity of SplB in vitro in a dose-dependent manner, yielding complete neutralization at a twofold molar excess of the antibody. In a murine vascular leakage model, the antibody completely abolished SplB-mediated endothelial damage. In summary, we produced a neutralizing mAb against the staphylococcal protease SplB, which merits further investigation as a candidate for the immunotherapy of SplB-induced pathologies.