Importância da galectina-3 na ação moduladora da crotoxina sobre função de monócito/macrófago

Abstract

Crotoxin (CTX) has biological effects both in experimental models and in clinical trials. Our group showed that the antitumor activity of CTX is associated with the modulating actions of the toxin on the secretory activity of inflammatory mediators and on the energy metabolism of macrophages. Furthermore, we demonstrated that CTX, in vivo, interferes with the phenotypic reprogramming of resident peritoneal macrophages and the tumor microenvironment, suggesting a reprogramming or prevalence of the M1 phenotype of macrophages. Among the mechanisms involved with the phenotypic reprogramming of monocytes/macrophages, galectin-3 has been described as an immunoregulatory protein, which may favor the classic pro-inflammatory condition (macrophage-M1) or alternative (macrophage-M2, anti-inflammatory), depending on the pathophysiological condition, with involvement of the PI3K/Akt signaling pathway. Therefore, the objective of this project was to investigate whether CTX is capable of interfering with the expression and secretion of galectin-3 by monocytes and the participation of this lectin in the modulating activity of CTX on the phenotypic condition of these cells by different stimuli. For this purpose, in vitro assays were carried out using cells of the human monocytic lineage (THP-1), pre-incubated in the absence (control) or presence of CTX (0.3; 0.6 and 1.2 μg/mL), by 2 hours and maintained in culture for 72 hours under different microenvironmental stimuli: a) only in fresh RPMI 1640 culture medium (to evaluate the per se activity of CTX on the investigated proteins); b) with culture medium containing 800 μg/mL of LPS; or c) and conditioned medium (CM) from MDA-MB-231 tumor cells. The Western Blotting assay was performed to mark the proteins: PI3Kp110a, Akt (types 1 and 2) and galectin-3. Functional/phenotypic parameters, such as H2O2 release, secretion of pro-(TNF-α) and anti-inflammatory cytokines (IL-10/TGF-β1) and Gal-3, and phenotypic markers (M1- CD80/CD86 and M2-CD163/CD206) was determined in order to associate possible changes in the functional/phenotypic behavior (M1/M2) of these cells and with the expression of signaling proteins involved in the reprogramming of monocytes/macrophages, in the face of different stimuli. The results indicate that CTX modulates the Akt signaling pathway and with the expression and secretion of Gal-3,

as well as with the release of H2O2 and against different signals in the microenvironment. Furthermore, a pro-inflammatory and pro-tumor role is attributed to Gal-3 in inflammatory and tumoral conditions and here, the results show the participation of this lectin in the immunomodulatory effect of CTX, both in the action per se of the toxin and in the inflammatory and tumoral microenvironments. Furthermore, particularly the concentration of CTX 0.6 and 1.2 μg/ml seems to promote a THP-1 mixed monocyte/macrophage profile or switch phenotype. This study contributes to knowledge about the immunomodulatory actions of CTX, showing for the first time the participation of Gal-3 in this process.