Miltefosine enhances infectivity of a miltefosine-resistant Leishmania infantum strain by attenuating its innate immune recognition

Abstract

Background Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. Methodology/Principal findings To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. Conclusions/Significance Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.

In Vivo Bioluminescence Imaging Reveals Differences in Leishmania infantum Parasite Killing Kinetics by Antileishmanial Reference Drugs

“…15 Our previous reports used BLI to demonstrate a striking MIL dependency of a resistant L. infantum strain, linked to lowered NK and NKT cell responses in the liver, leading to reduced IFN-γ production. 7,13 The differences in cidal dynamics between the reference drugs observed in this study corroborate previous findings in vitro, where the effect of PMM was slower than that of MIL 2 . PMM monotherapy was also found to be prone to fast relapse, originating from the bone marrow.…”

“…Although various bioluminescent Leishmania species and strains have already been used to monitor in vitro drug efficacy, , in vivo parasite fitness linked to drug resistance, , and the impact of (vaccine-induced) immune responses or interventions, , the application of BLI for in vivo drug potency has been scarce. Nevertheless, the kinetics of drug treatment have already been assessed in the hamster and the BALB/c mouse model. ,,, Álvarez-Velilla et al showed that BALB/c mice were seemingly cleared of infection after 5 days of MIL treatment at 40 mg/kg/day .…”

Long-term hematopoietic stem cells as a parasite niche during treatment failure in visceral leishmaniasis

“…The L. donovani strain MHOM/ET/67/L82 was isolated from an Ethiopian VL-patient. Both were modified to express bioluminescent (PpyRE9) and/or fluorescent (DsRed) reporter proteins (LEM3323 WT PpyRE9 , LEM3323 WT PpyRE9/DsRed and L82 WT PpyRE9/DsRed ) 81 . Promastigotes were sub-cultured twice weekly at 25 °C in hemoflagellate-modified minimal essential medium (HOMEM, Gibco®), supplemented with 10 % inactivated fetal calf serum (iFCS), 200 mM L-glutamine, 16.5 mM NaHCO 3 , 40 mg/L adenine, 3 mg/L folic acid, 2 mg/L D-biotin, and 2.5 mg/L hemin.…”

Impact of pulmonary African trypanosomes on the immunology and function of the lung

“…Cells were measured by flow cytometry using a MACSQuant® Analyzer 10 (Miltenyi Biotec) and analyses were performed using FlowLogic TM Software (Miltenyi Biotec) following specific gating strategies (Supplementary Table S2 , Supplementary Figs. S9 – 11 ) based on published methods 66 – 69 .…”