Persistent myeloid cell reprogramming despite miltefosine treatment in Leishmania-infected macaques

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites. An inflammatory immune response, associated with tissue injury, is occurring shortly after infection. In this study, using a rhesus macaque (RMs) model of VL, we evaluated the impact of miltefosine (HePC) therapy administrated during the acute phase of infection. Despite drug therapy, parasites persist in multiple tissues, including the spleen, bone marrow, peripheral (PLN) and mesenteric lymph nodes (MLNs). Parasite burden inversely correlate with cellular HePC levels. Notably, L. infantum remains detectable three months post-treatment. Single-cell transcriptomic analysis reveals significant cellular heterogeneity and reprogramming of splenic myeloid cell populations that persist post-treatment. This included the emergence of inflammatory macrophages, immature plasmacytoid dendritic cells (pDCs), and type 2 dendritic cells (DCs). Flow cytometric sorting of splenic neutrophils, macrophages, and DCs confirms the presence of L. infantum post-treatment, highlighting the challenge of parasite clearance. Collectively, our findings reveal a disrupted innate immune landscape following L. infantum infection that persists after treatment, enlightening myeloid cell reprogramming that may contribute in sustaining chronic infection and parasite persistence. Overall design: Ten female rhesus macaques (Macaca mulatta, Chinese origin, 40 months old, ~4.5 kg) were used in this study. Animals were inoculated intravenously with Leishmania infantum. One day post-infection, animals received a 21-day treatment with miltefosine (HePC, 5 mg/kg by nasogastric gavage). Spleen mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Sigma). Cell suspensions were stained with a CD45 antibody. Four samples (Naive, Infected, and two treated RMs) were pooled and loaded onto a single BD Rhapsody cartridge (~40,000 pooled cells loaded). Libraries were generated following the manufacturer's protocol, and sequencing was performed on an Illumina NovaSeq 6000 targeting ~50,000 reads per cell.