INTRODUCTION: Efferocytosis, the process by which macrophages clear apoptotic cells, is crucial for resolving inflammation and is often impaired in various inflammatory conditions. Previous studies suggest that natural products and their bioactive components can modulate macrophage functions, including activation, recruitment, polarization, and metabolism. In this context, our research explores the potential of Broccoli-Derived Nanovesicles (BDNVs) to enhance macrophage efferocytic capacity. We propose that pretreating macrophages with varying doses of BDNVs may improve their ability to remove apoptotic neutrophils.
METHODS: Human macrophages were obtained by stimulating monocytic THP1 cells with phorbol myristate acetate (PMA) for 72 hours. Human neutrophils were isolated from blood by Ficoll and their apoptosis was induced by exposing them to thermal shock at 43°C for 60 minutes, followed by a 24-hour incubation at 37°C. Broccoli-derived nanovesicles (BDNVs) were isolated from fresh Brassica oleracea L. sprouts using ultracentrifugation, and their concentration was quantified as weight per volume. PKH-26 labeled apoptotic neutrophils were then incubated with macrophages pretreated with BDNVs, and phagocytic activity was assessed using epifluorescence microscopy and flow cytometry.
RESULTS: Macrophage morphology was verified through microscopic analysis, and neutrophil apoptosis was assessed using the trypan blue exclusion method. Notably, our results demonstrated that macrophages treated with BDNVs showed a significantly higher phagocytic ability for apoptotic neutrophils compared to untreated macrophages. Specifically, epifluorescence microscopy revealed an increased intracellular PKH-26 signal that correlated with the BDNV concentration (ranging from 5 to 100 µg/mL). These findings were further supported by flow cytometry, which showed a higher percentage of PKH-26 positive macrophages following BDNV treatment, across a broad concentration range (0-5000 µg/mL).
CONCLUSIONS: Collectively, these results suggest that BDNVs promote the rapid clearance of apoptotic neutrophils through enhanced efferocytosis. BDNVs might be considered as pro-resolvent agents in the treatment of chronic inflammatory conditions.