nCounter analysis of Leishmania-infected macrophages and LT-HSC

Given the discontinuation of various first-line drugs for visceral leishmaniasis (VL), large-scale in vivo drug screening, establishment of a relapse model in rodents, immunophenotyping and transcriptomics were combined to study persistent infections and therapeutic failure. Double bioluminescent/fluorescent Leishmania infantum and L. donovani reporter lines enabled the identification of long-term hematopoietic stem cells (LT-HSC) as a niche with remarkably high parasite burdens, a feature confirmed for human hematopoietic stem cells (hHSPC). LT-HSC are more tolerant to antileishmanial drug action and serve as source of relapse. A unique transcriptional “StemLeish” signature in these cells was defined byupregulated TNF/NF-kB and RGS1/TGF-β/SMAD/SKIL signalling, and a downregulated oxidative burst.Cross-species analyses demonstrated significant overlap with human VL and HIV co-infected blood transcriptomes. In summary, the identification of LT-HSC as a drug- and oxidative stress-resistant niche,undergoing a conserved transcriptional reprogramming underlying Leishmania persistence and treatment failure, may open new therapeutic avenues for leishmaniasis. Cell lysates (from approximately 10,000 LT-HSC or macrophages, triplicates for both infected and uninfected conditions) for nCounter (NanoString) analysis were prepared using RNeasy Lysis buffer (RLT buffer, Qiagen). Cell lysates were hybridized to unique capture/reporter pairs (50 bp each) targeting 784 transcripts (734 murine transcripts and 20 housekeeping genes present in the Nanostring Myeloid/Innate Immunity Panel, complemented with 20 murine and 10 Leishmania spp. transcripts from a customized panel) as well as 6 positive and 8 negative control probes (all from NanoString). 7