Investigating the Role of Mast Cell Condensates in Regulating Macrophage Phenotype and Dynamics in Tissue Repair 2309191

Abstract Introduction The peritoneal cavity contains diverse immune cells that coordinate complex processes in response to injury and disease. During tissue repair, these cells form large repair aggregates within the cavity, however, the exact mechanism coordinating this function remains unclear. Large peritoneal macrophages (LPMs) appear to be the dominant cells that form the aggregates at the site of injury, however mast cells were seen to also be prevalent participants. Methods In-vitro assays were performed using peritoneal fluid to induce LPM aggregation via stimulation with ATP and adding back calcium. Confocal microscopy was used to visualize aggregation formation and identify extracellular granules released by mast cells during aggregation. Rheometry and fluorescence recovery after photobleaching (FRAP) were used to characterize these extracellular condensates after isolation and assess their material properties. Lattice-light sheet microscopy was used to visualize the release of these extracellular condensates by mast cells. Results Stimulation of the peritoneal fluid revealed that mast cells were responsible for the release of large (∼1 μm) avidin-positive extracellular condensates that localized between aggregating LPMs. Confocal microscopy demonstrated that the extracellular condensates were also positive for macrophage receptor with collagenous structure (MARCO), a scavenger receptor known to be involved with macrophage aggregation. These granules were further defined to be membraneless condensates using rheometry and FRAP imaging where both MARCO and avidin demonstrated recovery. Lattice light sheet microscopy revealed thin, actin-positive filapodia extending hundreds of microns in length coming from the mast cell, which grab condensates and deposit them on neighboring cells at the injury site, creating a limited sphere of influence. Conclusion These findings suggest that mast cell-released condensates play a part in macrophage aggregation and drive the tissue-repair mechanism in the peritoneal cavity. Funding Source Canadian Research Excellence Chair (CERC), Infrastructure Operating Fund (IOF), Canadian Foundation for Innovation (CFI), and Bruce Mitchell Research Program Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)