Karenia brevis Extract Induces Cellular Entry through Distinct Mechanisms in Phagocytic RAW 264.7 Macrophages versus Non-Phagocytic Vero Cells.

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.

Immune Modulating Brevetoxins: Monocyte Cytotoxicity, Apoptosis, and Activation of M1/M2 Response Elements Is Dependent on Reactive Groups.

Brevetoxins are a suite of marine neurotoxins that activate voltage-gated sodium channels (VGSCs) in cell membranes, with toxicity occurring from persistent activation of the channel at high doses. Lower doses, in contrast, have been shown to elicit neuroregeneration. Brevetoxins have thus been proposed as a novel treatment for patients after stroke, when neuron regrowth and repair is critical to recovery. However, findings from environmental exposures indicate that brevetoxins may cause inflammation, thus, there is concern for brevetoxins as a stroke therapy given the potential for neuroinflammation. In this study, we examined the inflammatory properties of several brevetoxin analogs, including those that do and do not bind strongly to VGSCs, as binding has classically indicated toxicity. We found that several analogs are toxic to monocytes, while others are not, and the degree of toxicity is not directly related to VGSC binding. Rather, results indicate that brevetoxins containing aldehyde groups were more likely to cause immunotoxicity, regardless of binding affinity to the VGSC. Our results demonstrate that different brevetoxin family members can elicit a spectrum of apoptosis and necrosis by multiple possible mechanisms of action in monocytes. As such, care should be taken in treating "brevetoxins" as a uniform group, particularly in stroke therapy research.