Crotoxin-induced macrophage secretory activity on fibroblast functions involved with the wound healing process. in vitro studies

Abstract

Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus Venom (CdtV), has anti-inflammatory and immunomodulatory actions in vivo and in vitro. As regards immuno-modulatory activities, CTX stimulates, particularly, the metabolism of macrophages and the production of cytokines, lipoxin A4 and analogue 15-Epi-LXA4, by these cells, leading to inhibition of the events involved with tumor progression and angiogenesis. Regarding anti-inflammatory actions, CTX induces important inhibition on the vascular and cellular events of the inflammatory response induced by phlogistic agents, presenting an effect of greater magnitude than those induced by classical anti-inflammatory drugs. The regenerative/inflammatory response involves three fundamental phases: inflammation, formation of granulation tissue and deposition of extracellular matrix and remodeling. Particularly, with the local presence of monocyte-derived macrophages and the production and release of the chemical mediators produced by them, the migration and activation of fibroblasts is intensified, leading to the deposition of large amounts of fibronectin and collagen, favoring wound healing. Therefore, adequate activation of macrophages is crucial to adequate resolution of local tissue regeneration. Thus, the objective of the present project is to investigate the importance of the crotoxin-induced macrophage secretory activity on fibroblast functions involved with the wound healing process. For this purpose, macrophage monocultures differentiated from the THP-1 human monocytic lineage will be incubated with different concentrations of CTX for 2 hours, washed and incubated with fresh RPMI 1640 medium for 24 hours and after that period. Human fibroblasts lineage will be incubated with the supernatants of these monocultures (cel: supernatant) or with macrophages (cel: cel) for in vitro determination of fibroblast events, at different periods, such as: 1) proliferation assay; 2) migration assay in the Wound healing model; 3) migration test in transwell chamber; 4) fibroblast actin cytoskeleton morphology and expression of fibronectin and type I collagen secreted by these cells, visualized by confocal microscopy; 5) alterations in the viscoelastic properties of the cytoskeleton of the fibroblasts by means of Optical Magnetic Twist Cytometry (OMTC) and 6) quantification of IL-10, TNF-±, VEGF and TGF-² by enzyme-linked immunosorbent assay (EIA). As control groups will be kept, in parallel, monocultures of the different cells treated or not with CTX or classical anti-inflammatories drugs. The data obtained with this project will contribute significantly to the broadening of the characterization of the anti-inflammatory property of CTX, evidencing the importance of the toxin modulating activity on macrophages for this property and the possible participation of fibroblasts, crucial cells for the process of regeneration and tissue healing. (AU)