Miltefosine restores the infectivity of miltefosine resistant Leishmania parasites by attenuating the innate immune response

Tue16  Apr12:33pm(3 mins)
Renold C16


D Bulté1; L Van Bockstal1; G Caljon1; L Maes1
1 Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Belgium


INTRODUCTION: Miltefosine (MIL) is an oral drug that is used to treat Visceral Leishmaniasis (VL) but is failing to permanently clear parasites in an increasing number of patients. Especially immunocompromised patients are prone to relapse. Parasites isolated from these relapse cases do not seem to display an increased resistance, but are hypothesized to interact differently with the immune system in favor of survival inside macrophages even under drug pressure. Only four MIL resistant (MIL-R) parasites have been isolated so far, although mutations in a single gene can render parasites MIL resistant. In this study, the infection characteristics of parasites with acquired resistance were explored under conditions with and without drug pressure.

METHODOLOGY: In order to study the impact of drug treatment on survival of MIL sensitive (MIL-S) and MIL-R parasites, double reporter lines were generated expressing both the PpyRE9 (in vivo/ex vivo bioluminescent imaging) and the DsRed gene (flow cytometry, fluorescence microscopy). In vivo bioluminescent infection studies were complemented with the analysis of cellular immunological responses in the liver and spleen. Major inflammatory cytokine responses were monitored in plasma and correlated with the host infection dynamics. The role of selected immune cell types [natural killer(NK) cells, NKT cells and neutrophils] during the early stage of the infection was assessed by specific depletion protocols and by flow cytometry. The impact of MIL was evaluated by in vitro pretreatment of parasites prior to inoculation of C57Bl/6 and BALB/c mice.

RESULTS: The MIL-R parasite line showed a reduced infectivity when compared to the isogenic MIL-S line. This reduced infectivity was accompanied by an increased monocyte and neutrophil influx in the spleen and liver during the early stage of the infection. This was associated with elevated IFN-γ, TNF-α and IL-6 levels in the blood. This early cytokine storm, either directly or indirectly, lead to the rapid clearance of MIL-R parasites from the liver and abrogated further dissemination to spleen and bone marrow. Experiments where NK and/or NKT cells were depleted in C57Bl/6 and BALB/c mice, identified these cells as the main source of IFN-γ during infection onset and moreover, in the absence of these cells, infectivity of the MIL-R line was partially restored. Finally, infections with MIL-S and MIL-R parasites under drug pressure have revealed that both in vivo MIL-treatment and in vitro MIL pre-exposure significantly rescues the in vivo infectivity of the MIL-R parasite. During these infections under MIL-treatment, the early induction of IFN-γ was less prominent, indicating a reduced activation of NK and NKT cells and a reduced clearance of the MIL-R parasite. These observations emphasize the risk of MIL treatment in sustaining infections with MIL-R parasites that are attenuat

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