Intracellular parasite kill: Flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation

Transgenic Leishmania expressing fluorescent reporter proteins such as green fluorescent protein (GFP) have opened the way for a flow cytometry (FACS)-based method to assess the killing of Leishmania parasites inside their macrophage host. Compared with counting parasites in microscopic preparations, the assessment of anti-leishmanial effects by FACS analysis promises both strict objectivity and significant reduction of labour-per-sample while scanning thousands of cells within seconds. Compared with other semi-automated methods based on host cell lysis and biochemical quantification of released parasites, the procedure is more direct and simple, reducing handling artefacts. An assay system is described using highly pure murine bone marrow-derived macrophages infected in vitro as a suspension culture with GFP-transfected Leishmania major promastigotes. The cells were rested for 24 h, allowing intracellular promastigotes to transform into amastigotes, and then exposed to macrophage-activating agents (IFN-γ, LPS) or standard anti-leishmanial therapeutics. Within 48 h, the GFP signal from parasitized macrophages became indiscernible by FACS analysis, both in activated host cells and in cultures treated with the anti-leishmanials. In cultures activated with rIFN-γ+LPS this coincided with the release of nitric oxides, but this was not the case in cultures treated with anti-leishmanials. Furthermore, by adding propidium iodide immediately before FACS analysis, the effect of treatment on the viability of the host cell was assessed at the same time. The combination of FACS analysis, and PI and NO detection offers a rapid and objective means of testing for intracellular anti-leishmanial effects and general cytotoxicity and gives a first indication of whether the former is due to direct leishmanicidal activity or indirect functions via macrophage activation.