Parasite culture and cell lines
The GFP or RFP-expressing PTG/ME49 T. gondii strains (type II) [33, 34] were maintained by serial 48 h passaging in human foreskin fibroblasts (HFFs; CRL-2088, American Type Culture Collection). HFFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher scientific) with 10% heat inactivated foetal bovine serum (FBS, Sigma), gentamicin (20 μg/ml, Gibco), L-glutamine (2 mM, Gibco) and HEPES (10 mM, Gibco), referred to as D10. bEnd.3 cells (CRL-2299, American Type Culture Collection) were cultured in D10. All cell cultures and parasites were grown in a humidified atmosphere containing 5% CO2 at 37 °C.
Primary dendritic cells (DCs) and macrophages
Murine bone marrow-derived DCs were generated as previously described . Briefly, cells from bone marrow of 6–10 week old male or female C57BL/6NCrl mice (Charles River) were cultivated in RPMI 1640 with 10% foetal bovine serum (FBS), gentamicin (20 μg/ml), glutamine (2 mM) and HEPES (0.01 M), referred to as complete medium (CM), and supplemented with recombinant mouse GM-CSF (20 ng/ml, Peprotech). Loosely adherent cells (DCs) were harvested after 6 or 8 days. To generate macrophages, cells from bone marrow were cultivated in CM supplemented with M-CSF (20 ng/ml, ImmunoTools). Loosely adherent cells were discarded and adherent cells were harvested after 6 or 8 days.
Primary murine brain endothelial cells (MBECs)
Six to 8 week old C57BL/6NCrl mice, housed under specific pathogen-free conditions at Stockholm University, were euthanised and brains were extracted. The brain tissue was freed from the cerebellum, hypothalamus, olfactory bulb and kept in ice-cold PBS. Preparation of cells was performed as described , with modifications . The tissue was digested in collagenase IV (1 mg/ml, Gibco) for 1 h at 37 °C, homogenised and washed with wash buffer (PBS, 0.5% FBS and 2 mM EDTA). The tissue suspension was mixed with density gradient medium. Percoll (Easycoll, Biochrom) in PBS was added to the tissue suspension at a final concentration of 30% and centrifuged for 30 min at 500 g. After removal of the myelin on the top layer, the cell pellet was washed with wash buffer. A second digestion was carried out by incubating the cell pellet with collagenase IV for 30 min at 37 °C. Positive selection of CD31-expressing cells was performed by magnetic activated cell sorting (MACS, Miltenyi Biotec) and CD31 MicroBeads (Miltenyi Biotec), according to the manufacturer’s instructions. CD31+-enriched cell suspensions were finally plated onto transwell filters (8 µm pore size; BD Biosciences) at cell yield from 1 brain per 4 transwell inserts pre-coated with 0.1% gelatin (Gibco), or in 12-well culture plates and monitored for confluence and polarisation. Cells were cultured in EBM-2/EGM-2 medium (Lonza) with 12% FBS, glutamine (200 mM, Gibco) and growth supplements (Bulletkit™, Lonza) minus the vascular endothelial growth factor (mVEGF) supplement. EBM-2/EGM-2 medium was supplemented with puromycin (1 mg/ml, Gibco) for 3 days, after which medium was changed every 2 days in absence of puromycin. The cellular monolayers had high and stable expression of PECAM1/CD31 (> 99%), TJ markers ZO-1, occludin and claudin-5, with low expression of astrocyte marker GFAP (< 1%), as previously characterised .
Polarisation parameters: permeability assay and transendothelial electrical resistance (TEER)
bEnd.3 cells were cultured to 80% confluence then seeded onto transwells (8 µm pore size; BD Biosciences) and grown for 5 days until they reached polarisation, as defined below. MBECs were seeded directly after isolation and reached a polarisation plateau after ~ 11–12 days, which was maintained beyond 14 days, as defined below. Experiments were performed on days 13–14. For evaluation of cell monolayer permeability following treatments or transmigration, FITC-dextran (3 kDa; Life tech) was added to the upper compartment of the transwell at a concentration of 12.5 μg/ml for 90 min. Medium was collected from the lower compartment, and fluorescence was measured in a fluorometer (EnSpire Multimode Plate Reader, Perkin Elmer) at 485 nm excitation 520 nm emission.
MBECs and bEnd.3 cells were cultured to form polarised monolayers defined by a TEER ≥ 250 Ω∙cm2, as measured using an Ohmmeter (Millipore, Bedford, MA) and correcting measurements with the formula:
Unit area resistance (TEER) = resistance (Ω)∙effective membrane area (cm2)
TEER was measured before and after transmigration or treatments. Values are shown as percentage (%) of TEER related to TEER prior to treatment or transmigration.
Blocking antibodies, isotype controls and inhibitor of VLA-4 were used at 1 μg/ml: LEAF™ purified anti-CD29 (clone HMβ1-1, 102209, BioLegend), LEAF™ purified anti-CD31 (clone MEC13.3, 102511, BioLegend), LEAF™ purified anti-CD18 (clone M18/2, 101409, BioLegend), LEAF™ Purified Rat IgG2a, κ Isotype Ctrl (clone RTK2758, 400515, BioLegend), Ultra-LEAF™ Purified Armenian Hamster IgG Isotype Ctrl (clone HTK888, 400969, BioLegend), BIO 5192 (R&D systems), anti-CD54 (ICAM-1; clone YN1/1.7.4; eBioscience).
T. gondii-challenged DCs were plated on coverslips coated with bovine collagen I (1 mg/ml, Life Technologies). After fixation (4% PFA, Sigma-Aldrich), cells were permeabilised (0.5% Triton X-100, Sigma-Aldrich) and stained with phalloidin Alexa Fluor 595 (Invitrogen). Micrographs were generated using a 63× objective (DMi8, Leica Microsystems). Primary MBECs were seeded on coverslips pre-coated with 0.1% gelatin (BioRad). Fixation and permeabilisation steps were carried out as for DCs, followed by blocking (5% FBS in PBS for 2 h). Cells were then incubated with primary antibodies to ZO-1 (ThermoFisher) and Occludin (ThermoFisher) ON at 4 °C at 1:500. Cells were then stained with Alexa Fluor 594-conjugated secondary antibodies (Invitrogen) and DAPI for 2 h, mounted and imaged by confocal microscopy (LSM 800, Zeiss).
Day 6–8, DCs or macrophages were challenged with freshly egressed tachyzoites (ME49/PTG-GFP or ME49-RFP, MOI 2, 4 h), resulting in 60–70% infection frequency and ~ 1,2 tachyzoites/infected cell . Cells were then transferred to transwell filters (8 μm pore size; BD Biosciences), with pre-cultured polarised monolayers of bEnd.3 cells in CM or MBECs in EBM-2/EGM-2. After 16 h, transmigrated DCs were put on ice for 1 h and macrophages trypsinised (TrypLE™ Express; Gibco) for 10 min to disassociate adherent cells. Cells were then resuspended and counted in a Bürker chamber.
Motility assays were performed as previously described . Briefly, DCs were cultured with CM ± freshly egressed T. gondii tachyzoites (ME49/PTG-GFP or ME49-RFP, MOI 3, 4 h, resulting in 70–80% infection frequency) and with soluble reagents as indicated. DCs were then added to 96-well plates pre-cultured with bEnd.3 cells or embedded in bovine collagen I (1 mg/ml, Life Technologies). Live cell imaging was performed for 1 h, 1 frame/min, at 10× magnification (Z1 Observer with Zen 2 Blue v. 4.0.3, Zeiss). Time-lapse images were consolidated into stacks and motility data was obtained from 30 cells/condition (Manual Tracking, ImageJ) yielding mean velocities (Chemotaxis and migration tool, v. 2.0). Infected cells were defined by GFP+ or RFP+ cells, as indicated.
Flow condition assays
DCs were cultured as stated in motility assay and then added to fluidic channels (μ-Slide VI0.4; Ibidi) with confluent bEnd.3 cell monolayers or pre-coated with collagen I (1 mg/ml, Life Technologies), and allowed to adhere for 10 min. Phase-contrast and fluorescence images were first captured in static condition. Fluidic shear stress was then applied by flowing CM at 0.2 dyn/cm2 through the channels. Live cell imaging was immediately initiated and images acquired every 5 s for up to 10 min, at 10× magnification. Fluidic shear stress was then increased to 1 dyn/cm2 and live cell imaging immediately initiated. Time-lapse images were consolidated into stacks and motility and path-length data obtained from 20 cells (Manual Tracking, ImageJ) yielding mean velocities and pathlengths (Chemotaxis and migration tool, v 0.2.0). The percentage of remaining adherent cells was calculated by dividing the number of cells per frame following shear stress, by the number of cells in the same frame in static condition. Representative track plots were created in Python 3.6.9 (https://www.python.org/) with pandas 1.1.5 and matplotlib 3.1.3.
Bone marrow-derived DCs were cultured in CM ± freshly egressed T. gondii tachyzoites (ME49/PTG-GFP, MOI 1) or LPS (100 ng/ml, serotype 011:B4, Sigma-Aldrich) for 4 or 24 h. Cells were stained on ice in FACS buffer (1% FBS and 1 mM EDTA in PBS) with Live/Dead Violet (L34955, Life technologies), anti-CD11c (clone N418, 25-0144-82, eBioscience) and anti-CD18 (clone M18/2, 101407, BioLegend), -CD29 (clone HMβ1-1, 102213, BioLegend) -CD54 (clone YN1/1.7.4, 116119, BioLegend) or isotype control antibodies (clone R35-95, 553930, BD Pharmingen; clone HTK888, 400924, BioLegend; clone RTK4530, 400611, BioLegend), fixed with 2% PFA and analysed on a BD LSRFortessa flow cytometer (BD Biosciences) with FlowJo software (v. 10, FlowJo LLC). Prior to staining, cells were blocked in FACS buffer supplemented with anti-CD16/CD32 antibody (Fc Block, BD Pharmingen).
Lentiviral vector production and in vitro transduction
Self-complementary hairpin DNA oligos targeting Itgb1 (shITGB1, TRCN0000066645, Genscript) or Tln1 (shTln1, TRCN0000294832, Genscript) mRNA were on self-inactivating lentiviral vectors (pLL3.7) with eGFP reporter expression (Table S1). Transfer plasmid (shRNA targeting ITGB1, Tln1 or Luc) was co-transfected with psPAX2 (12260, Addgene) packaging vector and pCMV-VSVg (8454, Addgene) envelope vector into Lenti-X 293 T cells (Clontech) using Lipofectamine 2000 (Invitrogen). The resulting supernatant was harvested 24 h and 48 h post-transfection. Supernatants were centrifuged to eliminate cell debris and filtered through 0.45-mm cellulose acetate filters. DCs (3 days post-bone marrow extraction) were transduced by adding lentiviral supernatants in the presence of DEAE dextran (8 µg/ml; Sigma-Aldrich) to cells for 4 h. After 5–7 days, transduction efficiency was examined for eGFP expression by epifluorescence microscopy (Z1 Observer with Zen 2 Blue v. 4.0.3, Zeiss) followed by expression analysis by qPCR for knock-down of targeted mRNA.
Polymerase chain reaction (PCR)
Total RNA was extracted using TRIzol reagent (Sigma-Aldrich). First-strand cDNA was synthesised with Superscript IV Reverse Transcriptase (Invitrogen). Real-time quantitative polymerase chain reaction (qPCR) was performed using SYBR green PCR master mix (Kapa biosystems), forward and reverse primers (200 nM), and cDNA (100 ng) with a QuantStudio™ 5 real-time PCR system (ThermoFisher). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene to generate ΔCt values; 2−ΔCt values were used to calculate relative knock-down efficiency. All primers (Invitrogen) were designed using the Get-prime or Primer-BLAST software (Table S2).
All statistics were performed with Prism software (v. 8, GraphPad). In all statistical tests, values of P ≥ 0.05 were defined as non-significant and P < 0.05 were defined as significant.