Evaluating Macrophage Effects on Parasite Survival

To determine whether addition of macrophages to the culture system enhanced parasite survival further, a monocyte-derived cell line, THP-1 (ECACC), originally derived from an acute monocytic leukaemia patient were evaluated. To differentiate THP1 cells into macrophages (Mϕ), cells were treated with 10 ng/ml of 12-O-tetradecanoylphorbol-I3-acetate (PMA; Peprotech), as previously described [22 (link)]. For polarisation into either a classically- or alternatively-activated phenotype, cells were treated with 50ng/ml recombinant interferon-gamma, (IFN-γ), or 25 ng/ml of recombinant interleukin 4 (IL-4) and interleukin 13 (IL-13), respectively (Peprotech), as previously detailed [23 (link)]. Forty-eight hours post-stimulation, Mϕ(IFN-γ), Mϕ(IL-4/IL-13), or non-polarised Mϕ(naïve) cells were washed 3 times in fresh medium and 0.4 μm pore-size transwell inserts (Corning) were added to fresh monolayers of LECs in a 6-well plate. Six millilitres of EGM-2 MV medium was then added, ready for the addition of parasites, as detailed above. Lymphatic endothelial cell monolayers with additional LEC trans-well inserts were included as controls.