COMPARATIVE STUDY OF THE IMMUNOMODULATING EFFECTS OF HOLOTHURIAN EXTRACT AND MACROPHAGE POLARIZATION DRIVERS ON THE MODEL OF PHAGOCYTES OF INJURED EUPENTACTA FRAUDATRIX

AbstractHolothurians (sea cucumbers) are known for their high regenerative ability. Extract from holothurian tissues (EH) can accelerate wound healing. In vertebrates, two types of macrophages display different roles in regulation of healing at its different stages. In holothurians, two enriched fractions of phagocytes that are analogs of macrophages, P1 and P2, are also identified. The mechanisms of EH influence on separate types of phagocytes in the wound healing in model experiments remained unexplored. It was of interest to study the effect of EH in comparison with substances known as inducers of macrophage polarization The aim of this work was to study the effect of EH on the number and phenotype of phagocytes P1 and P2 in the wounded holothurian Eupentacta fraudatrix in comparison with that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α.    Experimental animals (n=4-6) received a superficial wound with a scalpel. Some animals were additionally injected with GM-CSF, TNF-α or EH. After 0 min and 1 day phagocytes P1 and P2 were isolated by centrifugation of coelomic fluid in a density gradient of ficoll-verographin. The concentration of coelomocytes and content of phagocytes P1 and P2 were determined by light microscopy. The binding of cells to FITC-labeled lectins from Dolichos biflorus (DBA), Canavalia ensiformis (conA) and Glycin max (SBA) was assessed using a fluorescence microscope. Wound length measurements were performed after 0 min and 72 h.    Surface damage of the holothurian body caused an increase in the concentration of coelomocytes and the content of phagocytes P1, but a decrease in the content of P2 phagocytes. Changes in the number of cells in the coelomic fluid of wounded animals and decrease in their differentiation, judged by DBA lectin binding, during the exposure to the substances studied indicate that cytokines apparently caused a shift in the ratio of phagocytes of two types in tissues towards the predominance of P1 phagocytes, while EH - P2 phagocytes. Evaluation of conA cell binding revealed that cytokines suppressed the functional activity of P1 phagocytes during wounding. EH not only suppressed the functional activity of P1 phagocytes, but also stimulated the activity of P2 phagocytes, and accelerated wound healing compared to cytokines.     Acceleration of wound healing under the influence of EH is associated with the preferential activation of P2 phagocytes and their recruitment into tissues.This indicates an important role of P2 phagocytes in tissue repair.