High dilutions of antimony modulate cytokines production and macrophage - Leishmania (L.) amazonensis interaction in vitro

Background: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. Methodology: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. Results: Cells treated with AC 30 cH (10(-58) M) and AC 200 cH ( 10(-398) M) presented a temporary reduction of the spreading after 02 h of incubation, followed by increase after 48 h, being the most significant increase observed after the AC 200 cH treatment. However, the percentage of internalized parasites at 48, 96 and 120 h of incubation was also higher in cells treated with AC 200 cH. It is suggested that the AC 200 cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite induced reduction of cytokines production, especially IL-6, IL 12 p40 and gamma-IFN, after 48 h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120 h. A specific effect of AC 30 cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120 h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30 cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120 h of treatment with AC 200 cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30 cH and 200 cH was seen, in function of time. Conclusions: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion. (C) 2017 Elsevier Ltd. All rights reserved. (AU)