<em>Karenia brevis</em> Extract Induces Cellular Entry through Distinct Mechanisms in Phagocytic RAW 264.7 Macrophages versus Non-Phagocytic Vero Cells

Abstract

Marine algae extracts are an important area of potential drug discovery; however, nearly all studies to date have used non-fluorescent-based methods to determine changes in target cell activity. Many of the most robust immunological and cellular analyses rely on fluorescent probes and readouts, which can be problematic when the algae extract is fluorescent itself. In this study, we identified the fluorescent spectrum of an isolated extract from the marine dinoflagellate Karenia brevis, which included two fluorescing components: chlorophyll α and pheophytin α. When excited at 405 nm and 664 nm, the extract emitted fluorescence at 676 nm and 696 nm, respectively. The extract and its fluorescing components, chlorophyll α and pheophytin α, entered phagocytic RAW 264.7 macrophages and non-phagocytic Vero kidney cells through distinct mechanisms. When incubated with the extract and its main components, both the RAW 264.7 macrophages and the Vero cells accumulated fluorescence as early as 30 min and continued through 48 h. Vero kidney cells accumulated the K. brevis fluorescent extract through a dynamin-independent and acidified endosomal-dependent mechanism. RAW 264.7 macrophages accumulated fluorescent extract through a dynamin-independent, acidified endosomal-independent mechanism, which supports accumulation through phagocytosis. Furthermore, RAW 264.7 macrophages downregulated cell-surface expression of CD206 in response to extract stimulation indicating activation of phagocytic responses and potential immunosuppression of these immune cells. This study represents the first characterization of the cellular update of K. brevis extracts in phagocytic versus non-phagocytic cells. The data suggest the importance of understanding cellular uptake of fluorescing algae extracts and their mechanism of action for future drug discovery efforts.

Keywords

• flow cytometry
• autofluorescence
• immunofluorescence
• macrophages
• Vero cells