Ethical considerations
The Regional Animal Research Ethical Board, Stockholm, Sweden, approved experimental procedures in mice and protocols involving extraction of cells from mice (permit number 16403-2022), following proceedings described in EU legislation (Council Directive 2010/63/EU). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org).
Mice
All experiments were performed using male and female C57BL/6NCrl mice (strain code 027, Charles River), aged 4 to 10 weeks, housed in a ventilated facility, provided with unrestricted access to tap water and food, and kept under a 12-h light/dark cycle at a temperature of 20–22 °C.
Parasite culture and cell lines
T. gondii tachyzoites of type I: RH-GFP, RHΔTgWIP-GFP36, RH CPS-mCherry69, type II: ME49-RFP70, PRU-GFP50, PRUΔTgGRA15-GFP50, PRUΔTgGRA15 + GRA15-GFP50 and type III: CTG (ATCC, 50842TM) were routinely maintained by serial 48 h passaging in human foreskin fibroblasts, HFFs (ATCC, CRL-2088). HFFs and murine brain endothelial cells, bEnd.3 (ATCC, CRL-2299), were cultured in High glucose Dulbecco’s modified Eagle’s medium (DMEM, VWR, Cat# 392-0415) supplemented with 10% heat inactivated fetal bovine serum (FBS, HyClon, Cat# SV30160.03), 20 μg/ml gentamicin (Gibco, Cat# 15710-049) and 10 mM HEPES (HyClone, Cat# SH30237.01). Cell cultures and parasites were cultured at 37 °C, 5% CO2 in a humidified atmosphere. All cultures were regularly tested for Mycoplasma.
Primary DCs and splenocytes
Murine bone marrow-derived DCs were generated as previously described66. Bone marrow cells were purified from 4–8 week-old mice and cultured in RPMI 1640 medium (VWR, Cat# 392-0427) supplemented with 10% FBS, 20 μg/ml gentamicin, 10 mM HEPES and 10 ng/ml recombinant GM-CSF (PeproTech, Cat# 1479-1407). DCs (loosely adherent cells) were harvested after 6 days. Spleens were isolated from 4–8 week-old mice and mashed using a 40 µm cell strainer. Splenocytes were collected and RBC lysed with RBC-lysis buffer (Invitrogen, Cat# 00-4300-54).
Preparations of T. gondii-infected cells and extracellular tachyzoites
Inoculations with infected cells were performed as previously described37,66. Briefly, DCs or splenocytes were pre-labeled with CFSE (Invitrogen, Cat# C34554) or CMTMR (Invitrogen, Cat# C2927) and challenged with freshly egressed T. gondii tachyzoites at multiplicity of infection (MOI) 1 (type I, RH) or MOI 2 (type II, PRU, ME49; type III, CTG) for 4 h to obtain an infection frequency of ∼50%. CTG was pre-labeled with CFSE. Cell suspensions were then washed thrice and spun at 60–70 x g to minimize free tachyzoites in the inoculum. Total numbers of colony-forming units (cfu) injected into animals was confirmed by plaquing assays. Host cell number and viability was assessed by ocular hemocytometry and flow cytometry, respectively.
For inoculations with free tachyzoites, tachyzoites were collected from monolayers with maximum 50% HFF lysis, passed twice through a 27-gauge needle, washed twice by centrifugation at 870xg, kept at RT and inoculated in mice within 30–60 min from collection. Total numbers of cfu injected into animals was confirmed by plaquing assays.
Intraperitoneal (ip) and intravenous (iv) inoculations in mice
Six to 8 week-old mice were inoculated ip with 0,2–10 × 106 cfu of T. gondii tachyzoites. To assess parasite loads, animals were sacrificed at 2, 3 or 6 dpi. When indicated, anti-mo CD45 Alexa fluor 647-conjugated antibody (R&D Systems, Cat# FAB3507R, Clone 319211, Lot# 1726103) was injected iv (0,4 mg/kg) 10 min before euthanasia. For adoptive transfer assays, mice were inoculated iv with 20 × 106 cfu of T. gondi-challenged DCs and sacrificed 24 hpi.
Microsurgery, intracarotid artery inoculations and treatments
Six to 8-week-old mice were anesthetized with 2% isoflurane (Sigma-Aldrich, Cat# CDS019936) and the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed under a dissecting microscope (Leica M205 FA). The left CCA was carefully separated from the vague nerve using micro-forceps. A 6–0 silk ligature was tied around the CCA and the ECA. A new loosely ligature was placed in the CCA below the bifurcation site. Using micro-scissors, a small cut in the CCA was made between the first and loose ligatures. A 32gauge/.8Fr catheter (Instech, Cat# BTPU-010) fitted on a syringe with 100 μl of medium containing cell/parasite suspensions was inserted into the CCA, guided to the bifurcation site and tied with a new ligature. The loose ligature was opened and the fluid was slowly injected over 5 min into the ICA. Then, the loose ligature was tightened and the catheter removed. Finally, fat tissue and muscles were replaced, the skin sutured and analgesic (Temgesic, Eumedica Pharmaceuticals) administrated. When indicated, 100 U/kg of heparin (ThermoFisher Scientific) was injected iv 2 h after ICA inoculation. Alternatively, 2 mg/kg of LPS (Sigma-Aldrich) was injected ip 4 h prior the surgical procedure. For blocking assays, anti-mo CD54 (eBioscience, Cat# 14-0541-85, Clone YN1/1.7.4, Lot# 2089516), anti-mo LFA-1 beta (eBioscience, Cat# 14-0181-85, Clone M18/2, Lot# 2577324) or isotype (eBioscience, Cat# 14-4321-85, Clone eBR2a, Lot# 2777082) (all 0,2 mg/kg) were mixed with the cell/parasite suspensions 5-10 min prior to ICA inoculation.
Flow cytometry
DCs were challenged with freshly egressed tachyzoites for 5 h, washed in PBS and stained in FACS buffer (0,5% BSA, 2 mM EDTA in PBS). Challenged DCs were stained with 1/50 (v/v) PE-Cyanine7 anti-mo CD11c (eBioscience, Cat# 25-0114-82, Clone N418, Lot# 2142958), 1/50 (v/v) PE anti-mo CD18 (eBioscience, Cat# 101407, Clone M18/2. Lot# B313003) and 1/ 50 (v/v) Super Bright 702 anti-mo MHCII I-A/I-E (eBioscience, Cat# 67-5321-80, Clone M5/114.15.2, Lot# 2020037) antibodies and analyzed on a LSR Fortessa cell cytometer (BD Biosciences). Blood was collected by cardiac puncture into heparinized tubes and peripheral blood mononuclear cells (PBMCs) were purified with LymphoprepTM (STEMCELL Technologies, Cat# 07801). Cells were fixed in 4% PFA (Histolab, Cat# 02176), resuspended in FACS buffer and analyzed on LSR Fortessa. Data were analyzed with FlowJo software v10.
Quantification of T. gondii foci in cortical brain sections
To visualize cerebral blood vessels, mice were injected iv with 50 μl of PBS 3% Evans blue (Sigma, Cat# E2129-106) and 5% BSA (Sigma-Aldrich) prior brain extraction. Brains were washed in PBS and fixed in 4% PFA for 48 h at 4 °C. Then, PFA was replaced by PBS 10% sucrose (Sigma-Aldrich) for 2 d at 4 °C. Fixed brains were frozen in liquid nitrogen and 50 μm thick cryosections were collected on glass slides. To quantify the number of infected DC/T. gondii foci, the prefrontal cortex area of the brain was imaged using epifluorescence microscopy (Leica DMi8). A T. gondii foci was defined as a single replicating vacuole or a localized group of tachyzoites that was not confined inside or associated with CMTMR+ DCs. Data were expressed as the number of T. gondii foci or infected DCs per cm2 of tissue. Images were manually quantified with Fiji/ImageJ software. Data tables were exported and plotted using GraphPad Prism v.10 software.
3D Immunohistochemistry and image processing
Brain sections (50 μm thick) were transferred into 2 ml U-bottom tubes (5 sections/ tube) and washed 3 times with PBS. Then, samples were permeabilized and blocked in 1 ml of blocking buffer (1% Triton X-100 and 10% FBS in PBS) for 2 h at RT. Then, samples were incubated with 0,2 ml of primary antibodies anti-NeuN (Abcam, Cat# ab177487, Clone EPR12763, Lot# GR3275122-10) and anti-GPAF (Invitrogen, Cat# 13-0300, Clone 2.2B10, Lot# VH307339) diluted 1/250 (v/v) in blocking buffer for 48 h at 4 °C with gentle shaking. Sections were washed in PBS 0.1% Triton X-100 (3 times x 1 h) and incubated with 0,2 ml of Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Cat# A21442, Lot# 2906378; Molecular probes, Cat# A2147, Lot# 52521 A) diluted 1/1000 (v/v) in blocking buffer for 24 h at 4 °C with gentle shaking. After washing with PBS 0,1% Triton X-100, samples were mounted for imaging with LSM 800 Airyscan, Zeiss confocal microscope.
To quantify the localization of T. gondii in relation to blood vessels, DCs, neurons and astrocytes, brain sections were imaged using laser lines 488 nm (for GFP and CFSE), 561 nm (for RFP, CMTMR and Alexa 594) and 640 nm (For Evans blue and Alexa 647). Z-stacks from brain sections were collected with 40x objective from 0 to 50 µm in depth with 0.5-1 µm interval in the vertical z-axis and processed with IMARIS v.10.1 software. The Surface rendering tool was used to automatically define brain vasculature, DCs, T. gondii, neurons and astrocytes. The diameter (μm) of the vessels, distance to the branching points, and distance to the nearest vessel were manually defined and data tables were exported and plotted with GraphPad Prism v.10 software. Infected DCs/neurons/astrocytes were defined with the Surface-Surface Colocalization tool.
RT-Quantitative Polymerase Chain Reaction (PCR)
Total genomic DNA was purified from brain, liver and lung using DNeasy blood & Tissue kit (Qiagen, Cat# 69506) according to manufacturer’s protocols. Organs were homogenized in 4 ml of sterile PBS on 70 µm cell strainers. 0.5-1 ml of tissue homogenate was pelleted in 1.5 ml tubes for gDNA purification. gDNA concentration was measured using a spectrophotometer (Nano Drop 1000, ThermoFisher Scientific). Real-time PCR was performed using 100 ng gDNA, forward and reverse primers (200 nM) targeting TgB1 gene or mGAPDH gene and SYBR green PCR master mix (Kappa Biosystem, Cat# KK4602) with a QuantStudio™ 5 real-time PCR system (ThermoFisher). GAPDH was used as a house-keeping gene to generate ∆Ct values in order to calculate relative expression (2^-ΔΔCT).
For brain micro-vessels, total RNA was extracted using RNeasy mini kit (Qiagen, Cat# 74104) according manufacture’s protocol. The RNA was quantified by spectrophotometry (NanoDrop 1000). cDNA was synthesized with Maxima first strand cDNA synthesis kit (ThermoFisher, Cat# EP0753). Real-time PCR as performed using 10–100 ng cDNA, 200 nM forward and reverse primers and SYBR green PCR master kit (Kapa Biosystem). Primers are listed in Table S1. GAPDH and HRT were used as house-keeping genes to generate ∆Ct values in order to calculate relative expression (2^-ΔΔCT).
Isolation of cerebral micro-vessels
Brain micro-vessels were isolated as previously described39. Brains were homogenized by passage through a 23-gauge needle. Homogenates were diluted 1:1 (v/v) with 30% dextran solution (MW 70.000; Sigma-Aldrich, Cat# 31390-25) and centrifuged at 10.000xg for 15 min at 4 °C. The myelin layer was discarded and the pellet resuspended in DMEM 10% FBS. The suspension was passed through a 40-µm cell strainer and the vessels fragments retrieved were washed with PBS. Finally, the cell strainer was back-flushed with PBS and the micro-vessels pelleted by centrifugation at 4500 x g for 30 min at 4 °C.
Plaquing and adhesion assays
For plaquing assays66, brains, livers, and spleens from infected mice were extracted and homogenized using cell strainers (70 μm pore size). Homogenates were diluted in DMEM 10% FBS at 1/10 to 1/1000 (v/v) and added to confluent HFF monolayers in 24-well plates. After 24 h, medium was replaced. The numbers of viable parasites per g of tissue were determined by counting plaque formation after 3–5 days. To assess parasite viability, freshly egressed tachyzoites were resuspended in DMEM with 10% FBS (800 tachyzoites/ml), 250 µl were added to confluent HFF monolayers in 24-well plates at 0, 2, and 4 h after harvest and the number of viable parasites was determined by plaque formation.
For adhesion assays, DCs were pre-labeled with CMTMR or CFSE and challenged with freshly egressed GFP- or RFP-expressing tachyzoites (MOI 1 for type I, MOI 2 for type II). 1–5 × 103 cfu of T. gondii challenged DCs were added to confluent bEnd.3 monolayers for 1 h. Then, monolayers were washed 5 times x 5 min with PBS under shaking (300 rpm), fixed 5 min in PFA 4%, washed with PBS and the number of infected DCs per well was quantified by epifluorescence microscopy. When indicated, monolayers were pre-treated with LPS (100 ng/ml) overnight. Before the adhesion assay, LPS-treated monolayers were washed 5 times with DMEM 10% FBS. Alternatively, heparin (100 U/ml) was mixed with the challenged DCs immediately before adding them to the monolayers. For blocking assays, anti-mo ICAM-1 or isotype (0,5 g/ ml) were mixed with the cell suspensions and immediately added to the monolayers.
Statistical analyses
Statistical analyses were performed with GraphPad Prism v. 10. For multiple sample comparisons, one-way ANOVA followed Bonferroni’s multiple comparison test were carried out on data sets with normal distribution, and Kruskal–Wallis test followed Dunn’s post-hoc test was performed on data sets with non-normal distribution. For data sets from 2 samples with normal distribution, two-tailed Student’s t-test was performed. For data sets from 2 samples with non-normal distribution, Mann–Whitney U-test was performed. A p-value < 0.05 was defined as significant differences for all statistical tests.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
