Comparison of immunomodulatory effects induced by holothurian extract and macrophage polarization drivers using model of phagocytes of injured <i>Eupentacta fraudatrix</i>

Holothurians (sea cucumbers) are known for their high regenerative ability. Extract from Far-Eastern holothurian tissues (EH) is able to 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 (P1 and P2) are also identified being the analogues to macrophages. The mechanisms of EH influence on separate types of phagocytes in the wound healing remains unexplored. It was of interest to study the effect of EH in model experiments, 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 efficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNFα). The experimental animals (n = 4 to 6) received a superficial wound with a lancet. Some animals were additionally injected with GM-CSF, TNFα 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 Glycine max (SBA) was assessed using a fluorescence microscope. The wound length was measured at initial time point (0 min.) and 72 h. Surface damage of the holothurian body caused an increase in concentration of coelomocytes and contents of P1 phagocytes, but decreased the number of P2 phagocytes. Changed cell numbers in coelomic fluid of wounded animals and decrease in their differentiation, assessed by DBA lectin binding upon exposure to the test substances indicate that the cytokines apparently caused a shift in the ratio of tissue phagocytes in favor of P1 phagocytes, while EH promoted P2 phagocytic population. Evaluation of ConA cell binding revealed that cytokines suppressed the functional activity of P1 phagocytes upon wound injury. EH did not only suppress the functional activity of P1 phagocytes, but also stimulated the activity of P2 phagocytes, and promoted wound healing, as compared to cytokines. Acceleration of wound healing under the influence of EH is associated with preferential activation of P2 phagocytes and their recruitment into tissues. This finding suggests an important role of P2 phagocytes in tissue repair.