Evaluation of the ability of miltefosine associated with topic GM-CSF in modulate the immune response of patients with cutaneous Leishmaniasis

Abstract

Cutaneous leishmaniasis (CL) due to L. braziliensis is associated with an exaggerated inflammatory response and tissue damage. Miltefosine is more effective than antimony (Sbv) in the treatment of CL and here we evaluate the ability of miltefosine and GM-CSF to modify the immune response in participants of a clinical trial aimed to compare the efficacy of miltefosine plus GM-CSF vs miltefosine plus placebo vs antimony in CL caused by L. braziliensis. Patients were allocated in three groups of treatment. Miltefosine plus GM-CSF, Miltefosine plus placebo and Sbv. Mononuclear cells were obtained from those patients on day 0 and day 15 of therapy and cultured with or without soluble leishmania antigen for 72 hours. Granzyme-B, IFN-y, TNF and IL-1β, were determined in supernatants by ELISA. The lymphocyte proliferation was evaluated, utilizing Ki-67 as marker, by flow cytometry. The oxidative burst was evaluated in presence of L. braziliensis by flow cytometry, with dihydrorhodmine as marker, and monocytes were cultured with L. braziliensis (5:1) for 2, 48 and 72 hours for evaluation of infection ratio through optical microscopy. We observed that patients treated with miltefosine plus GM-CSF have decreased levels of Granzyme B, but increased levels of IL-1β during treatment compared to before therapy and higher production of IFN-γ and TNF than Sbv treated ones. There was an increase in the proliferation of CD4+ T cells in patients using miltefosine and in CD8+ T cells when GM-CSF was associated, and an increase in the oxidative burst after L. braziliensis infection in miltefosine plus GM-CSF group on day 15 of therapy. Moreover, the number of L. braziliensis in infected monocytes as well as the percentage of infected was higher after 2 hours and lower after 48 and 72 hours in cells from patients treated with miltefosine plus GM-CSF. In patients treated only with 10 miltefosine the same effects were observed after 2 and 72 hours. In 38 this study we show that in addition to the ability of miltefosine to kill leishmania, the modulation of the immune response caused by miltefosine and GM-CSF may increase the cure rate of CL patients using these drugs.