Mice and infections in vivo

Experiments were conducted with female C57BL/6 J mice (8–14 weeks old, purchased from Janvier, Cedex, France). All animals were group-housed in a 12-h day/night cycle at 22 °C with free access to food and water under specific pathogen-free conditions and according to institutional guidelines approved by the Animal Studies Committee of Saxony-Anhalt. In order to investigate early T. gondii infection, mice were infected by intraperitoneal (i.p.) injection of either cysts or tachyzoites. For cyst infection, 2 cysts of the type II ME49 strain harvested from the brains of female NMRI mice infected i.p. with T. gondii cysts 6–12 months earlier were used as previously described [39]. For tachyzoites infection, type II T. gondii reporter parasites of the PTG-GFPS65T strain were grown in monolayers of human foreskin fibroblast (HFF) with DMEM medium (FG0435, Biochrom, Germany), supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Germany), 1% penicillin/streptomycin (Pen/Strep; Sigma, USA) and 1% non-essential amino acids (NEEA) (ThermoFisher, Germany) as previously described [40]. Freshly egressed parasites were filtered through a 5 μm Millex-SV syringe filter (Millipore, Germany), and the number of living tachyzoites determined by counting under a bright-field microscope using Trypan Blue 0.4%. Mice were infected i.p. with 1 × 105 reporter parasites in 200 µl PBS.

Organ isolation

Mice were deeply anaesthetized by isoflurane inhalation (Baxter), the CSF was collected and thereafter animals were transcardially perfused with 60 ml PBS. For immunofluorescence samples, perfusion was additionally done with 20 ml of 4% paraformaldehyde (PFA) in PBS. Brain, spleen and spinal cord were removed and stored in sterile ice-cold PBS or RNAlater (Sigma) for further processing. Samples stored in RNAlater were kept at 4 °C overnight and afterwards transferred to − 80 °C.

Cerebrospinal fluid collection

CSF was collected by the cisterna magna puncture technique as described elsewhere [41]. In short, deeply anaesthetized animals were immobilized in a prone position with the head forming a 135° angle with the body, and a sagittal incision of the skin was made inferior to the occiput. Using a stereomicroscope (Stemi 305; ZEISS), the subcutaneous tissue and muscles were dissected, and a glass capillary tube was introduced into the cisterna magna through the dura matter, lateral to the arteria dorsalis spinalis. An average of 15 µl of CSF per animal were collected, and kept on ice until further processing. Samples were macroscopically assessed for blood contamination, and discarded when contamination was detected.

Choroid plexus and brain tissue isolation

Isolated brains were placed under a stereomicroscope in dissection buffer containing HBSS (ROTI®Cell, Roth), and 10 mM HEPES (Gibco, ThermoFisher). CPs were isolated from the lateral, third and fourth brain ventricles, and were either processed for total RNA/DNA isolation, or placed in digestion buffer for further cell isolation. Cerebellum, olfactory bulbs, and adjacent brain meninges were removed and discarded. The remaining brain tissue was used for cell isolation followed by flow cytometric analysis, or further utilized for brain microvessels isolation.

Brain microvessels isolation

Brain microvessels were isolated as previously described [42] with a few modifications, and used for immunofluorescence, or total RNA/DNA isolation. Briefly, brain hemispheres were minced with a scalpel, and homogenized in digestion buffer (HBSS, with 6.75 g/l glucose, 20 mM HEPES) containing 1 mg/ml DNAse I. After incubation (10 min, 37 °C), homogenate was washed in FACS buffer (PBS w/o Ca/Mg, 2 mM EDTA, 2% v/v FBS, 10 mM HEPES). The resultant pellet was resuspended and separated by successive centrifugations in 20% (w/v) bovine serum albumin (BSA)-DMEM/F12 solution. To remove remaining myelin debris, the pellet containing microvessels was resuspended in PBS, fractioned on 22% (v/v) Percoll® (Sigma, #GE17089101) gradient solution and centrifuged for 10 min, 600 × g, w/o brake. The microvessels pellet was recovered and extensively washed in PBS/HEPES.

Cell isolation

Isolated CP, the remaining brain tissue, liver and lymph nodes (inguinal and mesenteric) were further processed in order to obtain single cell suspensions. CP samples were incubated (20 min, 37 °C) in digestion buffer (PBS w/ Ca/Mg, 2% v/v FBS, 10 mM HEPES) containing 1 mg/ml DNAse I (Sigma, #DN25) and 1 mg/ml Collagenase/Dispase (Sigma, #11097113001). Digested tissues were mechanically dissociated using syringes connected to 22- and 26G needles, then cells were washed with FACS buffer, and used for further analysis. For the isolation of brain cells, liver and lymph nodes, tissues were minced with a scalpel, and homogenized in digestion buffer (HBSS, with 6.75 g/l glucose, 20 mM HEPES) containing DNAse I and Collagenase/Dispase as previously mentioned. Homogenate was incubated (40 min, 37 °C, 200 rpm), and filtered through a 70-μm cell strainer (Falcon®, #352350). The cell suspension was centrifuged (400 × g, 10 min, 4 °C), and the cell pellet separated in a 25–70% discontinuous Percoll® gradient for 20 min without brake. Cells were recovered from the gradient interface, washed with FACS buffer, and used for further analysis.

Flow-cytometric analysis

Cells were resuspended in FACS buffer, and stained as previously described [39, 40]. In short, cells were incubated with an anti-mouse CD16/32 unconjugated antibody (clone 93, BioLegend) and stained with fixable viability dye Zombie NIR (BioLegend), for 20 min at 4 °C. Subsequently, cells were stained (30 min, 4 °C) using fluorochrome-conjugated antibodies. CD11b (PerCP-Cy5.5, clone M1/70) purchased from eBioscience, CD45 (BV510, clone 30-F11), MHCII (BV711, clone M5/114.15.2), VE-cadherin (BV421, clone BV13), and gp38 (AF488, clone PMab-1) purchased from BioLegend. CD31 (APC, clone MEC13.3) purchased from BD Biosciences. Cells were washed (400 × g, 5 min 4 °C) with FACS buffer, fixed in 4% PFA for 15 min at 4 °C and re-suspended in FACS buffer. For CP, cells were additionally permeabilized with eBioscience™ Permeabilization buffer (Invitrogen), incubated with antibody rabbit-anti-TTR (Abcam) for 40 min, and stained with secondary antibody anti-rabbit AF488 (ThermoFisher). Cells were acquired using Attune NxT Flow Cytometer (ThermoFischer). Data were analyzed using FlowJo software (version 10.5.3, FlowJo LLC, OR, USA).

FITC-dextran permeability assay

BCSFB and BBB permeability were measured as previously described [34] with modifications. In short, 4 kDa FITC-dextran (Sigma, #46944) was diluted in PBS, and administered intravenously (i.v.) at 75 mg/kg body weight mice, 30 min before CSF and brain collection. CSF was collected through the cisterna magna as previously described, diluted 100-fold in PBS, and spun down (1000 × g, 5 min). The resulting supernatant was further used for analysis. After perfusion, isolated brains were weighted, minced with a scalpel, homogenized in formamide (Roth) at 0.8 ml per 100 mg tissue, and incubated overnight (37 °C, 200 rpm). Homogenates were spun down (12,000 × g, 5 min), and supernatants were collected and diluted twofold in PBS. All samples were measured in triplicates at λex/λem = 485/520 nm using SpectraMax M5e (Molecular Devices LLC).

Choroid plexus epithelial cells and T. gondii in vitro infection

Primary CP epithelial cell culture was obtained as previously described [43, 44] with modification. Isolated CP from 9 to 14 mice, one-week-old, were used in each preparation. Tissues were pooled in digestion buffer (PBS w/ Ca/Mg, 2% v/v FBS, 10 mM HEPES) containing 1 mg/ml DNAse I and 2 mg/ml Collagenase/Dispase, and incubated (20 min, 37 °C). Digested tissues were mechanically dissociated and cells were washed (400 × g 5 min) with FACS buffer (PBS w/o Ca/Mg, 2 mM EDTA, 2% v/v FBS, 10 mM HEPES). Cell pellet was resuspended, and cultivated with complete medium (DMEM/F12, supplemented with 10% FBS, 1% penicillin/streptomycin, 1% ITS (insulin–transferrin–selenite), 40 mg/ml human-EGF (epidermal growth factor, PeproTech, Germany, #AF-100–15). Cells were grown until confluence, for approximately 7 to 10 days, in 12 well plates previously coated with poly-l-lysine (PLL), or on top of PLL-coated coverslips. Once confluent, cells were infected at multiplicity of infection (MOI) = 5 with type II PTG-GFPS65T tachyzoites, and incubated at 37 °C. After 6 h, culture medium was removed, cells were washed with PBS, and further used for total RNA/DNA isolation, flow cytometric analysis (validation) or immunofluorescence. Naïve controls were treated with pre-warmed fresh medium.

Immunofluorescence

Immunofluorescence staining was performed for CP whole mount, BMV mount, brain sections and cell culture coverslips. For brain sections, isolated brains were post-fixed (4 h, at 4 °C), soaked in 30% sucrose in PBS (2 days, at 4 °C), and frozen in cryo media (OCT Compound, Tissue Tek). Coronal sections (20 µm) were obtained (Thermo Scientific CryoStar NX50) and only sections containing CP were stained. BMV mount, brain sections and coverslips were stained directly on glass slides. CP whole mount staining were performed utilizing a free-floating approach. All samples from different origins were stained and mounted with the same protocol unless otherwise stated. In short, samples were fixed (4% PFA in PBS, 20 min, 4 °C), washed twice with washing solution (PBS 0.1% (v/v) Triton X-100), and blocked/permeabilized with PBS 0.3% (v/v) Triton X-100 5% normal-goat-serum (NGS) and unconjugated F(ab′)2-goat anti-mouse IgG (H + L) antibody (1:500, Thermo Scientific), for 2 h at 4 °C. Next, samples were incubated with antibody solution (PBS 0.1% (v/v) Triton X-100 2% NGS) containing the primary antibodies of interest: anti-SAG1 (1:50, clone D61S, ThermoFisher), anti-GFP (1:1000, #132004, SYSY), anti-E-cadherin (1:500, #PA5-85088, ThermoFisher), anti-PDGFRβ (1:200, clone R.140.4, ThermoFisher), anti-ZO-1(1:50, clone R26.4C, ThermoFisher), anti-IBA-1(1:500, #234004, SYSY), anti-CD31 (1:50, clone MEC13.3, BioLegend), anti-claudin-2 (1:500, clone MH44, ThermoFisher), VE-cadherin (1:40, clone BV13, ThermoFisher). Primary antibodies were incubated overnight at 4 °C, then samples were washed twice, and incubated (30 min, RT) in antibody solution with secondary antibodies (1:1000) tagged with AF488, AF555, AF594 and AF647 according to each antibody host. Next, samples were washed twice, and mounted with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher). In some samples, anti-CD45 (clone 30-F11) conjugated antibody was used, and an additional final staining step was added. For brain sections, antigen retrieval (10 mM citrate buffer, pH 6.0, 0.1% Tween-20) was performed at 96 °C for 30 min, before the blocking and permeabilization step. Images were generated using a Leica TCS SP8 microscope, and analyzed using the ImageJ software (ImageJ 1.52p).

DNA and RNA isolation

Total DNA and RNA were isolated from CP, BMV, spleen, spinal cord, brain and CP epithelial cell culture. Samples from CP, BMV and CP epithelial cell culture were isolated with Quick-DNA/RNA Miniprep kit (Zymo Research, Germany) according to the manufacturer’s instructions. Brain, spinal cord, and spleen samples were first homogenized with TriFast (Peqlab, 30-2010) using tubes containing Zirconium oxide beads (Precellys, P000926-LYSK0-A) in a BeadBug 6 homogenizer (Biozym). DNA was isolated from the homogenate according to the manufacturer’s instructions. RNA was isolated from the homogenate by isopropanol precipitation or using peqGOLD total RNA kit (Peqlab) following the manufacturer’s instructions. The concentration and purity of DNA and RNA samples were determined using NanoDrop 2000 spectrophotometer (ThermoFisher; Germany), and samples were stored at − 80 °C until further use.

qPCR

Parasite burden was assessed in triplicates using 40 ng of isolated DNA, FastStart Essential DNA Green Master and LightCycler® 96 System (both Roche, Germany), as previously described [40]. Thermal-cycling parameters were set as follows: initial activation (95 °C, 10 min), 55 amplification cycles consisting of denaturation (95 °C, 15 s), annealing (60 °C, 15 s) and elongation (72 °C, 15 s). The DNA target was the published sequence of the highly conserved 35-fold-repetitive B1 gene of T. gondii [45, 46]. Murine argininosuccinate lyase (Asl) was used as reference gene for normalization and relative DNA levels were determined by the ratio gene of interest/reference gene and subsequently normalized to mean values of control group [47]. Primers were synthetized by Tib MolBiol (Germany) and used at 200 nM final concentration. Primer sequences are described elsewhere [see Additional file 1].

RT-qPCR

Gene expression levels of cytokines, inflammatory mediators, host-defense factors, tight junctions and MMPs were assessed in triplicates using 20 ng total RNA, TaqMan® RNA-to-CT™ 1-Step Kit (Applied Biosystems, Germany) and LightCycler® 96 (Roche, Germany) as previously described [40]. Thermal-cycling parameters were set as follows: reverse transcription (48 °C, 30 min), inactivation (95 °C, 10 min) followed by 55 cycles of denaturation (95 °C, 15 s) and annealing/extension (60 °C, 1 min). Utilized TaqMan® Gene Expression Assays (Applied Biosystems, Germany) are listed elsewhere [see Additional file 1]. Hprt was chosen as reference gene and relative mRNA levels were determined by the ratio gene of interest/reference gene and subsequently normalized to mean values of control group.

For the genes analyzed using SYBR Green technology, Power SYBR® Green RNA-to-CT™ 1-Step Kit (Applied Biosystems, Germany) was used. Samples were analyzed in triplicates (20 ng of isolated mRNA per reaction) using LightCycler® 96 with the following parameters: reverse transcription (48 °C, 30 min), inactivation (95 °C, 10 min) followed by 55 cycles of denaturation (95 °C, 15 s) and annealing/extension (60 °C, 1 min) and melting curve analysis. The primer sequences are listed elsewhere [see Additional file 1] and were synthetized by Tib MolBiol and used at 100 nM final concentration. Hprt was chosen as reference gene and relative mRNA levels were determined by the ratio gene of interest/reference gene and subsequently normalized to mean values of control group.

Statistical analysis

Results were statistically analyzed using GraphPad Prism 7 (GraphPad Software Inc., USA), post-test corrections were applied according to software recommendations. Statistical significance was set to p ≤ 0.05. All data are presented as arithmetic mean and standard error of the mean (SEM) and are representative of at least two independent experiments. For parasite burden analysis, normalized data were analyzed by multiple t-test, with Holm–Šidák correction for multiple comparisons. For flow cytometric analyses, normalized data were analyzed by one-way ANOVA, with Tukey’s correction. For RT-qPCR, data were analyzed by one-way ANOVA followed by Dunnett’s correction. BCSFB permeability assay was analyzed by multiple t-test to compare between BCSFB and BBB, and one-way ANOVA followed by Dunnett’s correction was used to compare differences between day 0 and the time-point analyzed from the same barrier type. RT-qPCR data from primary cell culture were analyzed by Student’s t-test.

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