MyD88-deficient mice (MyD88−/−), obtained from Dr S Akira (Research Institute for Microbial Disease, Osaka University, Japan) were backcrossed 10 times on the wild-type BALB/c background and bred at the Institute de Transgénose (CNRS, Orleans, France) under specific pathogen free (SPR) conditions. Age-matched BALB/c mice (MyD88+/+) were purchased from Janvier (Saint Berthevin, France) and used as controls in all experiments. Adult females weighing 20 to 25 g and aged 10 to 12 weeks were orally infected with 80 cysts of the 76 K strain, obtained as described below. In some experiments, mice were treated each day with sulfadiazine in their drinking water (400 mg/l), beginning 4 days after infection.
The animal experiments complied with the French Government’s ethical and animal experiment regulations.
Parasites and T. gondii extract
Tachyzoites of the RH strain of T. gondii were harvested from infected monolayers of human foreskin fibroblast (HFF) Hs27 (ATCC CRL-1634) and were used as a source of T. gondii extract .
Cysts of the 76 K strain of T. gondii were obtained from the brains of CBA/J mice that had been orally infected with 50 cysts 1 month earlier.
Antigen-induced cytokine production in culture
Spleens and mesenteric lymph nodes (MLNs) were harvested 13 days after the infection and pressed through a stainless steel mesh. Single cell suspensions were obtained by filtration through a nylon mesh to remove tissue debris. Spleen erythrocytes were lysed by hypotonic shock, single cells of spleen, and MLNs were resuspended in RPMI 1640 supplemented with 5% FCS, HEPES (25 mM), L-glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (5 × 10-5 M) and penicillin (50 U/ml)/streptomycin (50 μg/ml).
Spleen and MLN cells were cultured in 24-well plates at 106 cells per well, in 1 ml of culture medium, alone or containing Toxoplasma extract (10 μg/ml) . The plates were incubated for 4 days in 5% CO2 at 37°C. Cell culture supernatants were harvested between 24 hours and 4 days, and kept at −20°C until cytokine ELISA.
Spleen and MLN cell supernatants were tested for IL-2, IL-4, IL-10, IL-5, IL-13, IFN-γ, IL-12 and nitric oxide (NO) activities. Cytokine productions were evaluated using commercial ELISA kits according to the manufacturer’s instructions (e-Bioscience, San Diego, CA, USA), except for IL-12 p40 (DuoSet, R&D Systems, Minneapolis, MN, USA). Concentrations were determined by reference to standard curves constructed with known amounts of mouse recombinant IL-2, IL-4, IL-10, IL-5, IL-13 and IFN-γ. The sensitivity limits for the assays were 3.1 pg/ml for IL-2, 7.8 pg/ml for IL-4, 15.6 pg/ml for IL-5, and 31.3 pg/ml for IL-10, IL-13, IL-12p 40 and IFN-γ.
To test the presence of pro- and anti-inflammatory cytokines in the brains of infected animals, brains were harvested 13 days after infection and homogenized in 5 ml of RPMI 1640 with a pestle and mortar. Organs were then centrifuged for 5 minutes at 10,000 × g and the supernatants were tested for the presence of IFN-γ, IL-12p 40, IL-10, IL-2 and IL-13 by ELISA, as described above.
The NO production was evaluated using the Griess method. Briefly, Griess reagents (sulfanimide 1% and naphtylethylenediamine 0.1%, ratio 1:1; Sigma-Aldrich, St Louis, MO, USA) were added to the culture supernatants. The absorbance was measured at 540 nm. The NO concentration was determined with a standard curve established with a 20 nM sodium nitrite solution. The sensitivity limit was 2 μM.
MLNs, spleen, liver, heart, lungs, small intestines and brains were removed from infected and uninfected mice 13 days postinfection. The organs were placed in TRIzol (Gibco-BRL, Life Technologies, Grand Island, NY, USA) and were crushed with an ULTRA-TURRAX (IKA, Staufen, Germany). The samples were centrifuged at 8,000 x g to eliminate debris and then the supernatants were stored at −80°C until further processing. RNA was extracted according to the manufacturer’s instructions until the isopropanol addition step. The mixes were then added on Qiagen mini columns (Valencia, CA, USA) and spun for 15 seconds at 8,000 x g. The flow-through was discarded and the columns were washed. The following steps were carried out according to the manufacturer’s instructions. RNA quality was estimated by agarose gel electrophoresis using ethidium bromide for staining and RNA quantification was performed by Nanodrop (Thermo Fisher Scientific, Courtaboeuf, France) measuring absorbance at 260 nm.
Analyses of inflammatory chemokines and cytokines by real-time reverse-transcription PCR (qRT-PCR)
Total RNA was reverse transcribed using oligo(dT) primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Synthesized cDNA was then amplified by PCR using Chromo4 apparatus (Bio-Rad, Hercules, CA, USA). Hypoxanthine phosphoribosyltransferase mRNA levels were used to normalize RNA quantification. We used various primers for chemokine (CCL3, CCL5, CCL7, CCL19, CCL20 and CCL21) , cytokine (IL-17 and IL23)  and stage-specific Toxoplasma antigen (tachyzoite (SAG1) and bradyzoite (BAG1))  mRNA quantification. All real-time PCR (qPCR) displayed an efficiency of between 90% and 110%. Diluted cDNA was combined with primers and iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s recommendations, and real-time assays were run on a Chromo4 (Bio-Rad). The specificity of the qPCR reactions was assessed by analyzing the melting curves of the products and size verification of the amplicons. The information was processed to calculate the relative gene expression values determined according to a standard curve.
Histology and immunohistochemistry
For CD8α, CD3 and F4/80 fluorescence staining, at day 13 postinfection, brains were removed and embedded in optimal cutting temperature (OCT) Tissue Tek compound (Miles Scientific, Naperville, IL, USA) before storage at −80°C until use. Frozen sections of the brains (8 μm thick) were performed with a cryostat (Leica CM3050S, Leica Microsystems, Buffalo Grove, IL, USA), fixed in acetone gradient and incubated with primary antibodies (rat monoclonal antibodies anti-CD8α, anti-CD3 and F4/80; AbD Serotec, Kidlington, UK) or a rabbit serum of Toxoplasma infection for 2 hours at room temperature. Sections were washed several times in PBS and were then incubated with a donkey anti-rat IgG Alexa Fluor 594 (Life Technologies) for 1 hour at 37°C or a donkey anti-rabbit FITC (DAKO, Glostrup, Denmark). Sections were counterstained with Hoechst nuclear dye, mounted in Vectashield (H1000, Vector Laboratories, Burlingame, CA, USA) and observed on a Zeiss fluorescence microscope with Axiovision software (Zeiss, Göttingen, Germany).
For CD11b fluorescence staining, adjacent coronal sections of the brains from infected MyD88−/− and MyD88+/+ mice were stained. Briefly, after deep anesthesia (sodium pentobarbital, 40 mg/kg, intraperitoneal (i.p.)), mice were perfused through the heart with 80 ml of saline followed by 200 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed, postfixed for 2 hours in the same fixative and cryoprotected in a 20% sucrose solution overnight at 4°C. Coronal sections (45 μm thick) were cut with a cryostat (Leica CM3050S) and collected every four sections. After a series of washes in 50% ethanol and 3% H2O2, free floating sections were incubated at room temperature in primary rat anti-mouse CD11b antibody (clone 5C6, 1:500; AbD Serotec), in 5% normal horse serum, 0.2% Triton X-100 (Sigma-Aldrich) in PBS. Thirty-six hours later, sections were washed in 0.1 M PBS, incubated for 2 hours in a biotinylated anti-rat IgG (1:500; Jackson ImmunoResearch, West Grove, PA, USA) followed by ABC Kit (1:100, 1 hour; Vector Laboratories) and reacted with 3,3’-diaminobenzidine (DAB; Sigma-Aldrich) in the presence of H2O2. Sections were rinsed, mounted on gelatinized glass slides, dehydrated, cleared in Claral and coverslipped with Eukitt (Sigma-Aldrich).
Cyst counts in the brain
Thirteen days after infection, brains were harvested from surviving mice and homogenized in 5 ml of RPMI 1640 with a pestle and mortar. The cysts in each brain homogenate were counted under a microscope (10 counts, each on 10 μl). The results are expressed as means ± standard error of the mean (SEM) for each group.
Real-time PCR (qPCR) in the brain to assess parasite load
A total of 1 μg genomic DNA was prepared from the brain using a DNeasy tissue kit (Qiagen) according to the manufacturer’s instructions. Parasite burden was measured by amplifying the T. gondii B1 gene using the 2 × SYBR Green PCR Master Mix (Life Technologies) on an iCycler (Bio-Rad).
The primers used for QB1 were: forward, 5′-GGAACTGCATCCGTTCATGAG-3′; reverse, 5′-TCTTTAAAGCGTTCGTGGTC-3′. A standard curve for parasite equivalents was generated using a plasmid, as described previously .
Statistical analysis was performed using the Mann–Whitney U test (GraphPad Prism software, La Jolla, CA, USA) to analyze the observed differences in cytokines, chemokines and cyst counts. A P value <0.05 was considered significant.