ABSTRACT
The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a Leishmania major amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC50 values for amphotericin B shifted from 54 (51-57) nM in the static system to 70 (61-75) nM under media perfusion; EC50 values for miltefosine shifted from 12 (11-15) µM in the static system to 30 (26-34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC50 values of amphotericin B but not miltefosine (EC50 values for amphotericin B were 34.9 (31.4-38.6) nM in the 2D and 52.3 (46.6-58.7) nM in 3D; EC50 values for miltefosine were 5.0 (4.9-5.2) µM in 2D and 5.9 (5.5-6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with Leishmania, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of Leishmania studied. For L. major with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC50 values were 35.9 (33.4-38.5), 33.5 (31.5-36.5), 33.6 (30.5-not calculated (NC)) and 46.4 (45.8-47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation.
ABSTRACT
BACKGROUND: In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. METHODS: Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360µl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. RESULTS: Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10-9 m/s and 1.23 x 10-7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. CONCLUSIONS: Perfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.
