Ifn γ

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.

The immortalized mouse podocytes were developed by Prof. Dr. Karlhans Endlich, University of Heidelberg, Germany and kindly provided by Prof. Dr. Niels Olsen Saraiva Camara, Institute of Biomedical Sciences, University of Sao Paulo. As previously described^{42 (link)}, the cell culture was grown in 75-cm² flasks (Corning, New York, NY, USA) coated with type I collagen and maintained in RPMI-1640 medium (Thermo Fisher Scientific INC, St Peters, MO, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 30 IU/mL IFN-γ (Cell Sciences, Newburyport, MA, USA), 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L L-glutamine (pH 7.4 ) at 33 °C in a 5% CO₂ atmosphere. The culture medium was changed every two days until 85% confluence. Cells at passages 8 to 10 were detached by incubation with 5 mL trypsin-EDTA solution (Thermo Fisher Scientific, 0.05%) for 5 min at 37 °C. The cells were seeded for differentiation at specific cell densities into cell culture dishes with a diameter of 100 mm × 20 mm (Corning) in the same conditions, except for the absence of IFN-γ. The cells were differentiated for 10–15 days. Podocyte differentiation was confirmed by the identification of podocin and synaptopodin on protein expression. Phalloidin staining was used to identify the podocyte cytoskeleton.

Conditionally immortalized mouse podocyte cell lines were kindly provided by Dr. Danesh (Baylor College of Medicine, USA) and Dr. Wei Shi (Division of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, China) and were cultured as described previously. Cells were cultured at 33°C in RPMI-1640 medium (Corning, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 1 U/ml recombinant interferon-γ (IFN-γ) (ProSpec, Israel), and penicillin and streptomycin (Hyclone, USA). After four to five days, at confluence, podocytes were reseeded and incubated at 37°C in culture dishes coated with 12 μg/ml type-I collagen (Corning, USA) in RPMI-1640 medium supplemented with 10% FBS and deprived of IFN-γ, for 8 to 14 days to allow differentiation.