Reprogramação metabólica e de função do macrófago por crotoxina

Abstract

Macrophages (MØ) are cells with high plasticity that can be reprogrammed into two distinct phenotypes – MØ M1 (pro-inflammatory) and MØ M2 (pro-resolving) – according to the nature of the disorder detected in the organism. Experimentally, it is already possible to induce the reprogramming of macrophages to M1 and M2 and the functional and metabolic status of both phenotypes have already been characterized. Previous studies by our group have shown that Crotoxin (CTX) exerts two actions on macrophages that make them suitable in cell therapy: 1) it alters their metabolic and functional profile and 2) these effects are long-lasting. Such effects are observed after a single, short exposure to the toxin. In this context, this research sought to characterize the metabolism and functional status of MØ treated with different concentrations of CTX, with the aim of verifying whether in each concentration it is possible to identify different phenotypes and activation states of the already identified M1 and M2 profiles, in order to seek to reprogram these cells according to specific pathophysiological processes. The capacity for phenotypic reprogramming of monocytes induced by CTX was evaluated in in vitro assays, using cells of the human monocytic lineage (THP-1), previously incubated with RPMI medium (control), or LPS (1ug/mL) or tumor conditioned medium of MCF7 cells (MCT) for 48 hours. Subsequently, they were incubated in the absence (control) or presence of CTX at different concentrations (2 nM; 10 nM; 50 nM and 100 nM) for 1 hour. After the different incubation protocols with the toxin, the differentiation and phenotype markers were analyzed using the qPCR technique, and the latter were also analyzed using flow cytometry. To evaluate the effects of treatments on cellular functions, the phagocytosis capacity, the generation of reactive oxygen species (H2O2) and the production of cytokines by MØ were observed. Cellular metabolism was evaluated through gene expression and activity of key enzymes of the glycolytic pathway. The data presented show that THP-1 cells are satisfactorily differentiated by LPS (1 µg/mL) and tumor environment-MCT (vol. obtained from 1x106 /mL) and these cells have their functions modulated distinctly by CTX, depending on the concentration used. CTX had a dual action on the functions of these human macrophages, since it induced phagocytosis activity, while at the same time stimulated the production of H2O2 in control cells. However, these effects vary depending on the microenvironment stimuli, since, in a tumor and inflammatory context, phagocytosis was preserved in a concentration-dependent manner, concomitant with the increase in H2O2 release in some concentrations. Regarding the metabolic pathways, the undifferentiated THP-1 cells did not show alteration in the gene expression of any of the evaluated glycolytic enzymes, under the effect of any concentration of CTX, suggesting that the toxin does not modulate the expression of these enzymes in the THP-1 cell in monocyte condition. However, when stimulated by LPS and MCT, CTX modulated the gene expression of some enzymes: the 50 nM concentration increased the expression of enzymes from metabolic pathways that are more active in M1 (PFK, FAS and citrate synthase) and also reduced the activity of the enzyme CPT-1 (lipolysis), wich hight activity is related to M2. These effects were accompanied by an increase in pro-inflammatory cytokines. In the inflammatory environment (LPS) the concentration of 10 nM of the toxin promoted a concomitant positive marking of both M1 and M2 markers. Also, at 100 nM CTX promoted parallel release of pro- and anti-inflammatory cytokines. Taken together, the results allow us to state that CTX modulates functions and bioenergetic pathways of human macrophages, depending on the concentration used and the microenvironment stimulus involved.