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

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. 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. These data provide an immunological basis for the MIL-R-associated attenuated phenotype and for the peculiar drug-dependency that may constitute one of the mechanisms of treatment failure. Importance Recently, our laboratory demonstrated an in vivo fitness loss of experimentally selected MIL-R parasites in both the sand fly vector and vertebrate host. These findings could explain the scarce number of MIL-R clinical isolates. Surprisingly, MIL-R parasites developed a MIL-dependency which could partially rescue their fitness loss and which may constitute a mechanism of treatment failure. This research aimed to better understand the immunological basis of the attenuated phenotype and the effect of MIL on infectivity traits. Together, this study provides new insights into the complex interplay between the parasite, drug and host and discloses an immune-related mechanism of treatment failure.