High Content Analysis of Macrophage-Targeting EhPIb-Compounds against Cutaneous and Visceral Leishmania Species

Abstract

An immunostimulatory glycolipid molecule from the intestinal protozoan parasite Entamoeba histolytica (Eh) and its synthetic analogs derived from its phosphatidylinositol-b-anchor (EhPIb) previously showed considerable immunotherapeutic effects against Leishmania major infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein-specific staining, enabling the detection of several Leishmania species. We validated the assay using L. major, L. braziliensis, L. donovani, and L. infantum as well as investigated the anti-leishmanial activity of six immunostimulatory EhPIb-compounds (Eh-1 to Eh-6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh-6 was found to have activity against all Leishmania species. Considering the challenges in anti-leishmanial drug discovery, we developed a multi-species screening assay capable of utilizing non-recombinant parasite strains, and demonstrated its usefulness by screening macrophage-targeting EhPIb-compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis.

Past and future of trypanosomatids high-throughput phenotypic screening

“… 66 In the context of trypanosomatid drug discovery, most reports use HCS technology to evaluate the effect of test compounds on intracellular amastigotes ( Table ). In these assays, host cells and parasites, distributed in microplates, are incubated with test compounds (one or multiple concentrations for dose-response curves), stained with one 67 , 68 , 69 , 70 or more 58 , 71 , 72 , 73 , 74 fluorescent probes and their images captured in a HCS system coupled with a 10x, 75 , 76 20x (most cases 60 , 73 , 77 , 78 ) or 40x 67 , 75 objective lens ( Fig. 3 A).…”

“…3 A). Image analysis is performed using a custom pipeline in a proprietary e.g., Operetta Imaging System Harmony Software, PerkinElmer 77 , 79 or free software e.g., Cellprofiler 58 , 80 . From this analysis, it is possible to obtain a few metrics used to measure compound antiparasitic activity, such as the number of amastigotes per cell and the percentage of infected cells.…”

“…From this analysis, it is possible to obtain a few metrics used to measure compound antiparasitic activity, such as the number of amastigotes per cell and the percentage of infected cells. 69 , 73 Additionally, an estimation of compounds’ cytotoxicity can be obtained in the same assay by counting the number of host cells i.e., their nuclei 69 , 77 , 81 , 82 ( Fig. 3 B).…”

“… 91 Most assays use THP-1 cells instead of primary cells due to several technical and logistic advantages of the former, such as lower cost, ease of cultivation, applicability to large screening campaigns and less ethical restriction. 77 , 92 However, they require external chemical stimuli for monocyte to macrophage transformation and show much less biological relevance. 76 , 92 In this context, it is advisable to test the compounds in primary cells whenever possible.…”

“… 67 , 82 , 95 , 96 These methods are simple, but they may underestimate parasitaemia when parasites are located at the same place or near a cell’s nucleus, as well as detect non-specific stained spots due to the accumulation of host cell RNA in the cytosol. 25 , 69 , 77 , 78 Moreover, they generally require cell fixation making it impossible to monitor live cells over time which could inform the time-course of drug action. 97 The use of computer algorithms to delineate cell boundaries based on nucleus position may also be prone to errors since they often consider that the nucleus is located at the centre of each cell.…”