Dual scRNA-Seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased scRNA-seq to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogenous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils and NK cells are found unexpectedly infected. In the bone marrow, the Hematopoietic Stem Cells (HSCs) expressing phagocytic receptors Fc?R and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Ourunbiased dual scRNA-seq method identifies rare, parasitized cells, possibly implicated in pathogenesis, persistence, and protective immunity using a non-targeted approach. Overall design: Spleen and bone marrow were aseptically harvested from the mice and a single cell suspension was prepared from spleen by gentle mechanical disruption and passed through a 70µm cell strainer. For the bone marrow cells, femur and tibias were isolated and cut at both ends with scissors, and cells were flushed out from the cavity into RPMI medium-1640 supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin , and 0.1% ß-mercaptoethanol . Cells were passed through a MACS-LS column to remove the non-targeted cells by using monocyte isolation kit . Monocyte enrichment was achieved by depletion of magnetically labeled non-targeted T cells, B cells, NK cells, dendritic cells, erythroid cells, and granulocytes.