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Neuronal gp130 Expression Is Crucial to Prevent Neuronal Loss, Hyperinflammation, and Lethal Course of Murine Toxoplasma Encephalitis

Open ArchivePublished:May 28, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.03.029
      The obligate intracellular parasite Toxoplasma gondii infects and persists within neurons of approximately one-third of the human population. Intracerebral control of T. gondii largely depends on interferon (IFN)-γ-producing T cells, which induce antiparasitic effector mechanisms in infected cells, as well as immunosuppressive cytokines, which prevent immunopathology. To gain further insight into the role of neurons in Toxoplasma encephalitis (TE), we generated C57BL/6 synapsin-I (Syn)-Cre gp130fl/fl mice, which lack gp130, the signal-transducing receptor for the IL-6 family of cytokines, in their neurons. On infection with T. gondii, Syn-Cre gp130fl/fl mice failed to control T. gondii infection and died of necrotizing TE before day 77. In contrast, gp130fl/fl control mice efficiently restricted parasite replication and survived the infection. TE in Syn-Cre gp130fl/fl mice was characterized by a hyperinflammatory immune response with increased numbers of IL-17- and IFN-γ-producing CD4 and CD8 T cells but reduced intracerebral production of immunosuppressive transforming growth factor (TGF)-β and IL-27. Additional in vitro experiments found that IL-6 stimulation of neurons induced gp130-dependent TGF-β1, TGF-β2, and IL-27 production. Importantly, gp130 expression and stimulation with IL-6 cytokine family members also reduced death and apoptosis of infected cultured neurons. Correspondingly, TE in Syn-Cre gp130fl/fl but not gp130fl/fl mice was characterized by progressive neuronal loss. Collectively, these findings indicate a crucial protective function of gp130-expressing neurons in a model of chronic encephalitis.
      Toxoplasma gondii is a widely distributed obligate intracellular parasite and persists intracerebrally in approximately one-third of the human population. In addition to humans, many other species, including rodents, are naturally infected by the parasite, and the murine model of toxoplasmosis is widely used to study the pathogenesis of Toxoplasma encephalitis (TE). In the central nervous system (CNS), fast-replicating tachyzoites of T. gondii infect resident cells, including microglia, astrocytes, and neurons. Under pressure of the ensuing immune response, tachyzoites convert into slowly replicating bradyzoites, which form persisting intraneuronal cysts.
      • Fagard R.
      • Van Tan H.
      • Creuzet C.
      • Pelloux H.
      Differential development of Toxoplasma gondii in neural cells.
      Immunodeficient persons are at risk of reactivation and the development of life-threatening TE.
      • Montoya J.G.
      • Liesenfeld O.
      Toxoplasmosis.
      The most important cells to control the parasite in the brain are interferon (IFN)-γ-producing CD4 and CD8 T cells, which infiltrate the brain after infection.
      • Suzuki Y.
      • Conley F.K.
      • Remington J.S.
      Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice.
      • Gazzinelli R.
      • Xu Y.
      • Hieny S.
      • Cheever A.
      • Sher A.
      Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.
      In addition to inflammatory leukocytes, microglia and astrocytes contribute to intracerebral pathogen control by the production of antiparasitic effector molecules as well as cytokines and chemokines.
      • Fischer H.G.
      • Nitzgen B.
      • Reichmann G.
      • Hadding U.
      Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells.
      • Strack A.
      • Asensio V.C.
      • Campbell I.L.
      • Schlüter D.
      • Deckert M.
      Chemokines are differentially expressed by astrocytes, microglia and inflammatory leukocytes in Toxoplasma encephalitis and critically regulated by interferon-gamma.
      The IL-6 cytokine family includes IL-6, IL-11, IL-27, ciliary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1), all of which are up-regulated in TE.
      • Drögemüller K.
      • Helmuth U.
      • Brunn A.
      • Sakowicz-Burkiewicz M.
      • Gutmann D.H.
      • Mueller W.
      • Deckert M.
      • Schlüter D.
      Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis.
      The gp130 cytokines bind to oligomeric receptors composed of cytokine-specific receptor chains and the common gp130 receptor, which leads to intracellular activation of Janus kinases (1 and 2, and tyrosine kinase 2) and subsequently results in recruitment of STAT1 and STAT3 transcription factors and activation of the rat sarcoma (Ras)-rat fibrosarcoma-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase pathway. The gp130 cytokine binding sites are cross-reactive with the ability to interact with multiple cytokines of the IL-6 family. T. gondii infection of mice with deletion of individual members of the IL-6 cytokine family and their specific receptors resulted in different disorders. IL-6−/− mice had a higher parasitic load, an insufficient antiparasitic immune response, and an increased mortality.
      • Suzuki Y.
      • Rani S.
      • Liesenfeld O.
      • Kojima T.
      • Lim S.
      • Nguyen T.A.
      • Dalrymple S.A.
      • Murray R.
      • Remington J.S.
      Impaired resistance to the development of toxoplasmic encephalitis in interleukin-6-deficient mice.
      In addition, mice with enhanced IL-6/gp130-dependent STAT3 but defective Ras signaling failed to mount a protective IL-12 and IFN-γ response and to control T. gondii.
      • Silver J.S.
      • Stumhofer J.S.
      • Passos S.
      • Ernst M.
      • Hunter C.A.
      IL-6 mediates the susceptibility of glycoprotein 130 hypermorphs to Toxoplasma gondii.
      We have shown that gp130 expression of astrocytes is important for the local containment of inflammatory lesions in TE and that stimulation of astrocytes via the IL-6 cytokine family receptor gp130 is crucial for astrocyte survival, astrogliosis, parasite control, and TE survival.
      • Drögemüller K.
      • Helmuth U.
      • Brunn A.
      • Sakowicz-Burkiewicz M.
      • Gutmann D.H.
      • Mueller W.
      • Deckert M.
      • Schlüter D.
      Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis.
      In contrast, T. gondii-infected IL-27 receptor-deficient mice effectively controlled parasites but developed a lethal T cell-mediated immunopathology.
      • Stumhofer J.S.
      • Laurence A.
      • Wilson E.H.
      • Huang E.
      • Tato C.M.
      • Johnson L.M.
      • Villarino A.V.
      • Huang Q.
      • Yoshimura A.
      • Sehy D.
      • Saris C.J.
      • O'Shea J.J.
      • Hennighausen L.
      • Ernst M.
      • Hunter C.A.
      Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.
      In conclusion, these data show that a balanced and successful control of toxoplasmosis relies on diverse gp130-dependent host responses.
      In contrast to astrocytes and microglia, the functional role of neurons in TE is largely unknown. In vitro evidence suggests that neurons do not only serve as a target cell for T. gondii tachyzoites and bradyzoites (ie, cysts) but also contribute to the intracerebral immune response by the production of IL-6 and transforming growth factor (TGF)-β1 as well as macrophage inhibitory protein 1α and 1β.
      • Schlüter D.
      • Deckert M.
      • Hof H.
      • Frei K.
      Toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon- and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of T. gondii.
      To gain further insight into the function of neurons in TE, we generated mice with a conditional deletion of the gp130 cell surface receptor in synpasin-1-expressing neurons (Syn-Cre gp130fl/fl mice). Unlike control mice, Syn-Cre gp130fl/fl mice succumbed to chronic TE because of a failure to control parasites and to limit inflammation in the CNS. Furthermore, gp130 expression of neurons was important for prevention of neuronal apoptosis and loss of neurons in TE. Because we identified a gp130-dependent production of TGF-β and IL-27 by neurons, reduced production of these immunosuppressive cytokines by gp130-deficient neurons as well as the loss of this cell population in infected Syn-Cre gp130fl/fl mice may account for the exaggerated intracerebral immune responses. In contrast to production of IL-27 and TGF-β, gp130 expression of neurons was not important to limit infection and replication of T. gondii in neurons. In conclusion, these studies establish that gp130-dependent activation of neurons is required to balance and limit intracerebral inflammation, to control the parasite, to prevent neuronal loss, and, finally, to survive TE.

      Materials and Methods

      Breeding and Genotyping of Mice

      C57BL/6 gp130fl/fl (gp130fl/fl)
      • Betz U.A.
      • Bloch W.
      • van den Broek M.
      • Yoshida K.
      • Taga T.
      • Kishimoto T.
      • Addicks K.
      • Rajewsky K.
      • Müller W.
      Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects.
      and C57BL/6 Synapsin I-Cre+/− (Synapsin I-Cre+/− gp130WT/WT)
      • Zhu Y.
      • Romero M.I.
      • Ghosh P.
      • Ye Z.
      • Charnay P.
      • Rushing E.J.
      • Marth J.D.
      • Parada L.F.
      Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.
      transgenic mice were bred to generate Synapsin I-Cre+/− (Syn-Cre) gp130fl/fl mice. The colony was maintained by breeding of female Syn-Cre gp130fl/fl mice with male gp130fl/fl control mice to avoid germline recombination in testis.
      • Rempe D.
      • Vangeison G.
      • Hamilton J.
      • Li Y.
      • Jepson M.
      • Federoff H.J.
      Synapsin I Cre transgene expression in male mice produces germline recombination in progeny.
      The genotype of offsprings was determined by PCR of tail DNA with the use of primers for floxed exon 16 of the gp130 gene as published before.
      • Betz U.A.
      • Bloch W.
      • van den Broek M.
      • Yoshida K.
      • Taga T.
      • Kishimoto T.
      • Addicks K.
      • Rajewsky K.
      • Müller W.
      Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects.
      To detect the Synapsin I-Cre transgene and deletion of exon 16 of gp130 deletion, the following primers were used: syn a, 5′-CCAGCACCAAAGGGCGGGC-3′; syn cre, 5′-TGCATCGACCGGTAATGCAG-3′; gp130spa, 5′-TGGCTTGAGCCTCAGCTTGGCTAG-3′; and gp130r2del, 5′-CAAATTAATTCAGAATGAATTACAGATGCTAGAGC-3′, respectively. Animal care and experimental procedures were performed according to European regulations and were approved by state authorities (Landesverwaltungsamt Saxony-Anhalt, Halle, Germany; AZ42502-2822).

      Parasites and T. gondii Infection

      To obtain T. gondii cysts for in vivo experiments, NMRI mice (Harlan-Winkelmann, Borchen, Germany) were orally infected with five cysts of a type II strain of T. gondii (DX strain) and sacrificed 3 to 5 months after infection. Brains were removed, and the number of cysts per brain was counted microscopically. Parasites were adjusted to a concentration of 25 cysts/mL in 0.1 mol/L PBS. The suspension (200 μL; ie, five cysts per mouse) was injected intraperitoneally into the experimental animals.
      For in vitro experiments, T. gondii of the DX strain was cultivated in human foreskin fibroblasts in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C and 5% CO2. Toxoplasma tachyzoites were purified from the supernatant fluid of partially lysed human foreskin fibroblasts. Parasites were harvested by centrifuging the tissue culture medium at 50 × g to pellet the debris, and the parasite-containing supernatant fluid was washed twice by centrifuge with 0.1 mol/L PBS (400 × g) before use in in vitro experiments. In these experiments, T. gondii was used at a multiplicity of infection of 5:1.

      Histology

      For immunohistochemistry on frozen sections, mice were perfused intracardially with 0.9% NaCl in methoxyflurane anesthesia. Brains were snap-frozen in isopentane (Fluka, Neu-Ulm, Germany) precooled on dry ice, and stored at −80°C until preparation of serial 10-μm frozen sections, and immunohistochemistry for CD45, CD4, CD8, Ly6G, F4/80, inducible nitric oxide synthase, neuron-specific nuclear protein (NeuN), and T. gondii was performed as described previously.
      • Schlüter D.
      • Löhler J.
      • Deckert M.
      • Hof H.
      • Schwendemann G.
      Toxoplasma encephalitis of immunocompetent and nude mice: immunohistochemical characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products.
      • Schlüter D.
      • Deckert-Schlüter M.
      • Lorenz E.
      • Meyer T.
      • Röllinghoff M.
      • Bogdan C.
      Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T-gondii-resistant BALB/c mice.
      For the monoclonal anti-mouse NeuN antibody (clone A60; Chemicon-Millipore, Schwalbach, Germany) the Animal Research Kit (Dako, Hamburg, Germany) was used for detection according to the manufacturer's instructions.
      For histology and immunohistochemistry on paraffin sections, anesthetized mice were perfused with 4% paraformaldehyde in PBS, brains were processed, and serial sagittal sections were prepared and stained with H&E and H&E plus luxol fast blue, respectively. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament protein L, and myelin basic protein was performed as described previously.
      • Haroon F.
      • Drögemüller K.
      • Händel U.
      • Brunn A.
      • Reinhold D.
      • Nishanth G.
      • Mueller W.
      • Trautwein C.
      • Ernst M.
      • Deckert M.
      • Schlüter D.
      Gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation.
      The TUNEL kit (Roche, Mannheim, Germany) was applied according to the manufacturer's instructions. All main regions of the brain, including frontal, parietal, and temporal lobes, basal ganglia, cerebellum, and brainstem, were analyzed in sagittal sections in at least four animals per group and time point.

      Isolation of Cerebral Leukocytes and Flow Cytometry

      Infected and noninfected Syn-Cre gp130fl/fl and gp130fl/fl mice were perfused with 0.9% NaCl, and brains were removed. Leukocytes were isolated from the tissue and stained for CD4+ T cells, CD8+ T cells, macrophages, B cells, and granulocytes, as described previously.
      • Schlüter D.
      • Oprisiu S.B.
      • Chahoud S.
      • Weiner D.
      • Wiestler O.D.
      • Hof H.
      • Deckert-Schlüter M.
      Systemic immunization induces protective CD4+ and CD8+ T cell-mediated immune responses in murine Listeria monocytogenes meningoencephalitis.
      Dendritic cells were identified as CD11c+ CD11b+, and CD45high.
      For intracellular staining of IFN-γ, IL-17 and Foxp3 cells were incubated with GolgiPlug (1 μL/mL; BD Biosciences, Heidelberg, Germany) and stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 500 ng/mL ionomycin at 37°C for 4 hours. After incubation, cells were stained with rat anti-mouse CD4-fluorescein isothiocyanate (FITC) and rat anti-mouse phycoerythrine (PE)-Cy5, fixed, permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with rat anti-mouse IFN-γ-PE, IL-17-PE, and Foxp3-PE, respectively. Control staining was performed with isotype-matched control antibodies. All antibodies were obtained from BD Biosciences. Flow cytometry was performed on a FACScan (BD Biosciences), and the data were analyzed with WinMDI version 2.8 or FlowJow software version 7.6 (BD Biosciences).

      Quantitative PCR

      To determine the parasitic load in the brains of infected mice, DNA was extracted from perfused brains with the use of the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The highly repetitive sequence REP-529 (GenBank accession no. AF487550) of T. gondii was amplified with the use of the primers 270F (5′-AGAGACACCGGAATGCGATCT-3′) and 318R (5′-TTCGTCCAAGCCTCCGACT-3′), and Taqman probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine (5′-TCGTGGTGATGGCGGAGAGAATTGA-3′). To quantify parasites within the brain tissue, cultivated T. gondii were harvested and counted; DNA was extracted and used as a standard. Real-time PCR was performed with a Light Cycler 480 (Roche).
      Transcription of IL-27 mRNA was analyzed by quantitative RT-PCR with the use of the respective Taqman gene expression assay (Applied Biosystems, Darmstadt, Germany). As negative control, water instead of cDNA was used. PCR reactions were prepared with cDNA equivalent to 5 ng of RNA in a final volume of 20 μL. All samples were studied in triplicate. Murine TATA-box binding protein and RNA polymerase II were chosen as reference genes for normalization.
      • Frericks M.
      • Esser C.
      A toolbox of novel murine house-keeping genes identified by meta-analysis of large scale gene expression profiles.
      Normalized data were calibrated by comparison with expression values of untreated gp130fl/fl mice (d 0) with the use of the ΔΔCt method.
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.

      ELISA

      Brain tissue was homogenized in lysis buffer [(20 mmol/L Tris/HCL pH 7.3, 140 mmol/L NaCl, 0.5% Triton X-100, 2 mmol/L Na3PO4, Complete Protease Inhibitor (Roche)], and protein content was determined with a Nanodrop 1000 spectrophotometer (Peqlab, Erlangen, Germany) with the use of the Bradford assay. Equal amounts of protein were analyzed by IL-27, TGF-β1 (R&D Systems, Abingdon, UK), and TGF-β2 (Promega, Madison, WI) enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.
      For determination of IL-27 protein, cultivated primary gp130-deficient and control neurons were left unstimulated or were treated with IL-6 and DX T. gondii for 24 hours, respectively. Secreted IL-27 was measured by using a commercially available ELISA kit (R&D Systems).

      In Vitro Culture and Isolation of DNA and Proteins

      For cell culture experiments, brains were isolated from pups on embryonic day 18.5. Cortices were excised without meninges, and tissue was enzymatically and mechanically dissociated with the use of trypsin and syringe needles. Cells of each embryonic brain were plated onto poly-D-lysine-coated cell culture dishes in Neurobasal media, supplemented with 2% B27 (both Invitrogen, Karlsruhe, Germany), 1% penicillin/streptomycin (PAA, Pasching, Austria), and 500 μmol/L l-glutamine. Every 4 days, two-thirds of the medium was changed. The purity of neurons was ≥98% as determined by immunofluorescence staining for neuron-specific β-III tubulin and GFAP to detect contaminating astrocytes.
      Astrocytes were isolated from 1- to 2-day-old pups as published by Frei et al
      • Frei K.
      • Bodmer S.
      • Schwerdel C.
      • Fontana A.
      Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages.
      and cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mmol/L N-acetyl-L-alanine-l-glutamine (Biochrom, Berlin, Germany), and 20 ng/mL gentamicin (Sigma-Aldrich, St. Louis, MO). To separate microglia from astrocytes, cells were detached, and microglial cells were stained with allophycocyanin (APC)-labeled rat-anti-mouse CD45 antibody (BD Biosciences). Separation was performed with the use of a FACSVantage Cell Sorter (BD Biosciences).
      DNA was isolated from trypsinated cultivated neurons and sorted astrocyes with the use of a DNA isolation kit (Qiagen). To obtain whole-cell proteins, cells were lysed with cold RIPA buffer [50 mmol/L Tris/HCl pH 7.5, 5 mmol/L EDTA, 100 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 10 mmol/L KH2PO4, 0.5% sodium deoxycholate, 1 mmol/L sodium molybdate, 20 mmol/L glycerin-2-phosphate, 2 μmol/L phenylmethylsulfonylfluoride, 1 mmol/L Na4O7P2, 1× protease inhibitor cocktail (Sigma, Steinheim, Germany), PhosStop (Roche)] at 4°C for 15 minutes. After determining the protein content, 2× SDS was added. Samples were stored at −20°C until use for Western blot analysis.

      Western Blot Analysis

      Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Schwalbach, Germany). Membranes were blocked with 5% bovine serum albumin (BSA) or 1% milk and 1% BSA in Tris-buffered saline Tween-20 (TBS-T; 140 mmol/L NaCl, 25 mmol/L Tris-HCl, 2 mmol/L KCl, pH 7.4, 0.1% Tween-20) at room temperature for 2 hours followed by overnight incubation with polyclonal rabbit anti-TGFβ (Cell Signaling Technology Inc., Danvers, MA), rabbit anti-gp130 (Santa Cruz Biotechnology Inc., Heidelberg, Germany), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology Inc.) antibodies, respectively. After washing with TBS-T, membranes were incubated with secondary swine anti-rabbit horseradish peroxidase-conjugated antibody (Dako Cytomation, Glostrup, Denmark) at room temperature for 1 hour. Western blot analyses were developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and visualized with the Lumi Imager (Roche).

      LDH Assay

      Cultivated neurons were either infected with T. gondii of the DX strain (multiplicity of infection, 5:1) alone or after stimulation with cytokines of the IL-6 family [IL-6 (20 ng/mL), IL-11 (20 ng/mL), IL-27 (20 ng/mL), LIF (50 ng/mL), OSM (50 ng/mL), CT-1 (20 ng/mL), CNTF (50 ng/mL), and CLC (20 ng/mL)] for 24 hours. Supernatant fluids were harvested, and lactate dehydrogenase (LDH) release was determined with the use of a commercially available LDH assay (CytoTox96; Promega) according to the manufacturer's instructions. Data are expressed as the percentage of cytotoxicity calculated by the following formula: experimental LDH release × 100/maximal LDH release.

      Immunostaining of Cultivated Primary Neurons

      Neurons grown on glass slides were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Cells were permeabilized with 0.5% Triton X-100 in 0.1 mol/L PBS for 1 minute. After blocking with 0.1 mol/L PBS supplemented with 10% goat serum, 2% BSA, 150 mmol/L sucrose, and 0.3% Triton X-100 at room temperature for 45 minutes, cells were incubated with mouse anti-neuronal class-III β-tubulin (Covance, Princeton, NJ) and rabbit anti-T. gondii antibody (BioGenex, Hamburg, Germany) at 4°C overnight and, thereafter, with Cy 2-coupled goat anti-mouse and Cy 3-coupled goat-anti-rabbit antibodies (Jackson ImmunoResearch, Suffolk, UK) at room temperature for 1 hour. Nuclei were visualized by DAPI staining. TUNEL staining was performed with the In Situ Cell Death Detection Kit TMR Red (Roche). Slides were embedded in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) supplemented with glycerol (1:1) and 2% 1.4-diazabicyclo[2.2.2]octane. Images were acquired with the use of a fluorescence microscope (Leica DMRE7; Leica, Wetzlar, Germany) equipped with a CCD camera (Spot RT; Diagnostic Instruments, Burroughs, MI). Separate images were taken in the corresponding channels and were merged with the use of the ImageJ software (NIH, Bethesda, MD). Neuronal class-III β-tubulin–positive cells, TUNEL-positive cells, and intracellular T. gondii were counted.

      Statistics

      To test for statistical differences in cell numbers, ELISA, and quantitative PCR data, the two-tailed Student's t-test was used. Statistical differences in LDH release and apoptosis of neurons were analyzed with the Kruskal-Wallis test. TE survival was determined with the Kaplan-Meier method, followed by calculation of statistic differences of survival with the log-rank test. P values of < 0.05 were accepted as significant. All experiments were performed at least twice.

      Results

      gp130 Surface Expression Is Exclusively Deleted in Neurons of Syn-Cre gp130fl/fl Mice

      To analyze neuronal gp130 deletion in Syn-Cre gp130fl/fl mice, PCR and Western blot analyses were performed. Brain, heart, liver, lung, spleen, intestine, muscle, kidney, thymus, and bladder of Syn-Cre gp130fl/fl mice carried the loxP flanked exon 16 of gp130, whereas the Synapsin-I Cre transgene was only detectable in the brain. Consequently, deletion of exon 16 was confined to their brains (Figure 1A). PCR analysis of cultivated neurons, astrocytes, and microglia found that exon 16 was exclusively deleted from neurons (Figure 1B). Successful deletion of the extracellular part of gp130 of Syn-Cre gp130fl/fl neurons was confirmed at the protein level by Western blot analysis (Figure 1C). In contrast, gp130 was never deleted in gp130fl/fl control mice (Figure 1, A–C).
      Figure thumbnail gr1
      Figure 1Organ and cell-type-specific deletion of gp130 in Syn-Cre gp130fl/fl mice. A: Deletion of exon 16 of gp130 from organs of Syn-Cre gp130fl/fl and gp130fl/fl mice was analyzed by PCR. The gp130Δ product has a size of 400 bp. B: Deletion of exon 16 of gp130 from cultivated neurons, astrocytes, and microglia of Syn-Cre gp130fl/fl (1) and gp130fl/fl (2) mice which had undergone fluorescence-activated cell sorting. C: Western blot analysis of lysates from cultivated neurons of Syn-Cre gp130fl/fl (1) and gp130fl/fl (2) mice probed with antibodies to gp130 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
      Syn-Cre gp130fl/fl mice were born in the expected Mendelian ratio and did not show any physiological or behavioral abnormalities. Neuropathologic analysis of serial H&E and immunostained sections of Syn-Cre gp130fl/fl and gp130fl/fl control mice found normal brain architecture without obvious alterations of the cellular composition as shown by histology, in particular, of the gray substance (cortex, basal ganglia). Thus, Syn-Cre gp130fl/fl mice are a suitable tool to study the role of neuronal gp130 in TE.

      Increased Mortality of T. gondii-Infected Syn-Cre gp130fl/fl Mice

      To determine whether gp130 expression of neurons affects the course of TE, mortality rates of Syn-Cre gp130fl/fl and gp130fl/fl mice after infection with T. gondii were monitored. In two independent experiments, Syn-Cre gp130fl/fl mice showed a significantly higher mortality rate than control mice (P < 0.05). In both experiments, 100% (10 of 10) Syn-Cre gp130fl/fl mice died up to day 80 after infection, whereas 70% (7 of 10) of control mice survived beyond day 120 after infection (Figure 2).
      Figure thumbnail gr2
      Figure 2Reduced survival of Toxoplasma gondii-infected Syn-Cre gp130fl/fl mice. Syn-Cre gp130fl/fl and gp130fl/fl mice were infected with T. gondii and survival rates were monitored. Survival of Syn-Cre gp130fl/fl mice was significantly reduced at day 77 after infection (p.i.) (P < 0.05). One of two representative experiments with 10 mice per experimental group and experiment, respectively, is shown.

      Impaired Intracerebral Parasite Control of Syn-Cre gp130fl/fl Mice

      To further assess the role of neuronal gp130 expression in TE, a detailed histologic analysis was performed on serial sections of H&E-stained and immunostained sections throughout the brain. Neuropathology found increased numbers of parasites in the brains of Syn-Cre gp130fl/fl mice throughout TE. At late stages of TE, that is, day 70 after infection, gp130fl/fl control mice confined parasites to cysts and, accordingly, did not show parasite-induced tissue necrosis (Figure 3A). In contrast, cysts as well as tachyzoites, which had caused necrosis, were scattered throughout the brain of Syn-Cre gp130fl/fl mice (Figure 3B). In accordance with histology, quantitative real-time PCR analysis showed that parasite DNA was significantly increased in infected Syn-Cre gp130fl/fl mice at days 35, 49, and 70 after infection (Figure 3C).
      Figure thumbnail gr3
      Figure 3Necrotizing Toxoplasma encephalitis (TE) and increased intracerebral parasite burden in Syn-Cre gp130fl/fl mice. A: At day 70 after infection (p.i.), a single Toxoplasma gondii cyst is present in the cortex of a gp130fl/fl mouse in the absence of inflammatory leukocytes. B: In contrast to gp130fl/f mice (A), several T. gondii cysts (asterisks) as well as tachyzoites (arrows) are associated with an inflammatory infiltrate in the cortex of a Syn-Cre gp130fl/fl mouse at day 70 p.i. A and B: Anti-T. gondii immunohistochemistry was performed on six sections per mouse brain from three mice per experimental group and representative findings are shown. Slight counterstaining with H&E. Original magnification, ×400. C: Quantitative PCR of T. gondii DNA showed a significantly increased intracerebral parasite load in Syn-Cre gp130fl/fl mice at days 35, 49, and 70 p.i. *P < 0.05, **P < 0.01. Three mice per group and time point were analyzed, and data represent the mean ± SD. One of two experiments is shown.

      Increased Intracerebral Inflammatory Response in Syn-Cre gp130fl/fl Mice

      Remarkably, Syn-Cre gp130fl/fl mice developed an exaggerated intracerebral immune response despite a significantly increased parasitic load, thus, indicating a functionally significant impairment of the intracerebral immune response with subsequent failure of intracerebral parasite control. T. gondii-associated inflammatory infiltrates were more prominent in Syn-Cre gp130fl/fl and control mice, especially in the cortex and basal ganglia at all times analyzed (days 35, 49, and 70 after infection; Figure 4, A and B). Further quantitative evaluation of this hyperinflammation by fluorescence-activated cell sorting showed a 1.5-fold increase of inflammatory CD45+ leukocytes, B cells, CD4 and CD8 T cells, as well as dendritic cells in the brains of Syn-Cre gp130fl/fl mice (Figure 4, C and D; P < 0.05) at day 49 after infection. In addition, the relative and absolute numbers of regulatory Foxp3+ CD4 T cells were elevated in Syn-Cre gp130fl/fl mice (Figure 4, E and F). A strong increase was also observed for IFN-γ-producing CD4 and CD8 T cells in Syn-Cre gp130fl/fl mice, and at day 49 after infection, their numbers were threefold increased compared with control mice (Figure 4G). Numbers of CD4 and CD8 IL-17-producing T cells increased 1.5-fold to twofold in Syn-Cre gp130fl/fl mice at day 49 after infection (Figure 4H), but absolute numbers of IL-17-producing cells were much lower than IFN-γ-producing CD4 and CD8 T cells. In contrast to IFN-γ and IL-17, no significant differences were observed for IL-10 production by CD4 T cells (data not shown).
      Figure thumbnail gr4
      Figure 4Hyperinflammatory intracerebral immune response in Syn-Cre gp130fl/fl mice as opposed to gp130fl/fl mice. A: At day 49 after infection, a few CD45+ leukocytes are scattered throughout the cerebral cortex in a gp130fl/fl mouse. In addition, activated microglial cells have up-regulated the CD45 antigen (arrows). B: Severe encephalitis with large numbers of CD45+ leukocytes in the cerebral cortex in a Syn-Cre gp130fl/fl mouse. In addition to prominent perivascular infiltrates (large arrow), many leukocytes have spread all over the cortex. Ubiquitous activation of microglia with a strong up-regulation of the CD45 antigen is increased compared with gp130fl/fl mice (small arrows). A and B: Anti-CD45 immunohistochemistry, slight counterstaining with H&E. Original magnification, ×400. C and D: Leukocytes were isolated from the brain, and the numbers of intracerebral inflammatory leukocytes, CD4 and CD8 T cells, B cells, macrophages, and granulocytes (C) as well as dendritic cells (D) were determined in Toxoplasma gondii-infected Syn-Cre gp130fl/fl and gp130fl/fl mice by flow cytometry at day 49 after infection. Data represent the mean of six mice per experimental group and time point. E and F: The percentage (E) and number (F) of Foxp3+ CD4 T cells was analyzed in infected Syn-Cre gp130fl/fl and gp130fl/fl mice by flow cytometry at day 49 after infection. In D, representative dot plots of one of three mice are shown, and E shows the mean ± SD of six brains per experimental group. G and H: The number of intracerebral interferon (IFN)-γ-producing (G) and IL-17-producing (H) CD4 and CD8 T cells was determined by flow cytometry at day 49 after infection. Data show the mean ± SD of six mice per group. CH: *P < 0.05, **P < 0.01. One of two independent experiments is shown.

      Reduced IL-6-Dependent Production of TGF-β1, TGF-β2, and IL-27 by gp130-Deficient Neurons

      To analyze whether increased inflammation of Syn-Cre gp130fl/fl mice was paralleled by a reduced production of immunosuppressive cytokines, the intracerebral production of TGF-β and IL-27 was studied. In fact, production of active TGF-β1 and TGF-β2 as well as IL-27 was significantly reduced in brains of infected Syn-Cre gp130fl/fl mice (Figure 5, A–C). Additional quantitative RT-PCR of brain samples from T. gondii-infected mice found that IL-27 mRNA was significantly reduced in Syn-Cre gp130fl/fl mice compared with gp130fl/fl animals (Figure 5D).
      Figure thumbnail gr5
      Figure 5Reduced transforming growth factor (TGF)-β and IL-27 production in Syn-Cre gp130fl/fl mice. AC: Active TGF-β1 (A), active TGF-β2 (B), and IL-27 (C) were determined in brain tissue homogenates of Syn-Cre gp130fl/fl and gp130fl/fl mice by ELISA at day 0 (uninfected mice) and day 49 after infection (p.i.). Data show the mean ± SD of five mice per group. *P < 0.05, **P < 0.01, and ***P < 0.005. D: IL-27 mRNA transcripts were assessed in the brains of Toxoplasma gondii-infected Syn-Cre gp130fl/fl and gp130fl/fl mice at days 35 and 77 p.i. Although IL-27 mRNA levels are comparable at day 35 p.i., Syn-Cre gp130fl/fl mice had significantly lower intracerebral IL-27 mRNA levels than gp130fl/fl mice (*P < 0.05) at day 77 p.i. Uninfected gp130fl/fl mice were used as controls. Two mice per experimental group were analyzed. E: Inactive and active TGF-β values were determined by Western blot analysis in lysates of cultivated neurons derived from Syn-Cre gp130fl/fl and gp130fl/fl mice. Neurons were infected with T. gondii and stimulated with IL-6 as indicated. F: IL-27 was determined by ELISA in neuronal cultures of Syn-Cre gp130fl/fl and gp130fl/fl mice. Neurons were stimulated with IL-6 or infected with T. gondii as indicated. Data show the mean ± SD of six mice per experimental group used in two independent experiments. ***P < 0.005. n.d., not detectable.
      We have previously reported that unstimulated neurons are a source of TGF-β. In good agreement, we detected small amounts of active TGF-β in both gp130-expressing and -deficient cultivated neurons by Western blot analysis (Figure 5E). Interestingly, IL-6 stimulation induced an increased neuronal production of active and inactive TGF-β in gp130fl/fl but not in Syn-Cre gp130fl/fl neurons (Figure 5E). In contrast to IL-6, infection with T. gondii induced an increased production of inactive but not of active TGF-β in gp130fl/fl neurons (Figure 5E). In Syn-Cre gp130fl/fl neurons, T. gondii infection did not modulate TGF-β production (Figure 5E). In addition to TGF-β, IL-27 production was induced by IL-6 stimulation of gp130 competent but not of gp130-deficient neurons (Figure 5F). Infection with T. gondii did not result in neuronal IL-27 production irrespective of gp130 expression (Figure 5F). Altogether, the reduced levels of anti-inflammatory TGF-β and IL-27 may contribute to the increased inflammation in TE of Syn-Cre gp130fl/fl mice.

      Neuronal gp130 Expression Protects Neurons from T. gondii-Induced Apoptosis, Thereby Preventing Neuronal Loss in TE

      In Syn-Cre gp130fl/fl mice, the impaired immune response resulted in an increased intracerebral parasitic load and an increased inflammatory response in the brain. At day 21 after infection, cortical neurons were lost in the vicinity of inflammatory infiltrates as shown by NeuN staining. Although mild inflammation-induced neuronal loss, strictly confined to inflammatory infiltrates, occurred in gp130fl/fl mice, it was more severe and widespread in Syn-Cre gp130fl/fl mice, also extending into areas not infiltrated by leukocytes, resulting in a marked loss of cortical neurons in chronic TE. At day 70 after infection, gp130fl/fl mice had developed chronic TE; however, neuronal loss was not a prominent feature and there was no evidence for progressive neuronal damage and loss (Figure 6A). At this time point, neuronal loss had progressed in critically ill Syn-Cre gp130fl/fl mice, which had severe cortical damage characterized by large areas devoid of neurons (Figure 6, B and C). In Syn-Cre gp130fl/fl mice, areas of neuronal loss were in part (Figure 6B), but not exclusively (Figure 6C), associated with inflammatory infiltrates. In both strains of mice, microglial cells and astrocytes were equally strongly activated as evidenced by a marked up-regulation of the F4/80 and major histocompatibility complex class II antigen on microglial cells with a comparable expression of inducible nitric oxide synthase protein as well as a prominent up-regulation of GFAP on astrocytes, respectively (data not shown).
      Figure thumbnail gr6
      Figure 6Progressive neuronal loss in Toxoplasma gondii-infected Syn-Cre gp130fl/fl mice. A: At day 70 after infection, architecture of the frontal neocortex is normal in a gp130fl/fl mouse, in which encephalitis has been resolved. Important, neurons in the cortex are well preserved. B: There is a prominent loss of neurons in the frontal cortex of a Syn-Cre gp130fl/fl mouse, in which encephalitis is still ongoing with prominent leukocytic infiltrates (arrow points to an area with an inflammatory infiltrate and neuronal loss). C: In Syn-Cre gp130fl/fl mice, additional cortical areas without active inflammation showed a marked loss of neurons in all layers of the neocortex (arrow points to an area with neuronal loss without an inflammatory infiltrate). AC: Anti-NeuN immunohistochemistry at day 70 after infection, slight counterstaining with H&E. Original magnification, ×200 (AC).
      Thus, these observations do not indicate a decisive role of microglial cells and astrocytes in the induction of irreversible neuronal damage, raising the question whether T. gondii may directly induce cell death of gp130-deficient neurons. To answer this question, in vitro studies were performed. Isolated cortical neurons were infected with T. gondii and neuronal death was monitored by measuring LDH release. Both uninfected gp130-deficient and control neurons showed a low spontaneous cell death rate (<2%; Figure 7A). Infection with T. gondii led to an increased cell death in both groups. Interestingly, neuronal death of gp130-deficient neurons was significantly increased compared with gp130-competent neurons (Figure 7A).
      Figure thumbnail gr7
      Figure 7Increased cell death and apoptosis of Toxoplasma gondii-infected gp130-deficient neurons. A: Lactate dehydrogenase (LDH) was measured by ELISA in tissue culture supernatant fluids of uninfected and T. gondii-infected Syn-Cre gp130fl/fl and gp130fl/fl neurons. Infected neurons were stimulated with cytokines as indicated, and LDH release was analyzed 24 hours after infection. Data show the mean ± SD of six mice per experimental group used in two independent experiments. B: Uninfected and infected neurons were stimulated with cytokines as indicated. The number of TUNEL+ neurons was microscopically determined 24 hours after infection. Neurons were counterstained with DAPI. TUNEL positivity of ≥100 DAPI+ neurons per experimental group was evaluated. Data show the mean ± SD of six mice per experiment used in two independent experiments. A and B: *P < 0.05, **P < 0.01, and ***P < 0.005. C and D: Infected gp130fl/fl (C) and Syn-Cre gp130fl/fl (D) neurons were stained with DAPI (blue), anti-T. gondii antiserum (green) and TUNEL (red). Original magnification, ×100. CLC, cardiotrophin-like cytokine; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin-1; IFN, interferon; LIF, leukemia inhibitory factor; OSM, oncostatin M; TNF, tumor necrosis factor.
      To analyze whether IL-6 cytokines regulate death of T. gondii-infected neurons, cells were infected and stimulated with CLC, CNTF, CT-1, IL-11, IL-27, IL-6, LIF, and OSM, respectively. Stimulation with all IL-6 cytokine family members significantly reduced T. gondii-induced LDH release of gp130-expressing neurons (Figure 7A). In contrast, stimulation with IL-6 cytokines had no effect on cytotoxicity of infected gp130-deficient neurons. However, stimulation with both IFN-γ and tumor necrosis factor (TNF) significantly reduced LDH release of both types of neurons (Figure 7A).
      TUNEL staining showed that T. gondii infection induced apoptosis in both Syn-Cre gp130fl/fl and gp130fl/fl neurons (Figure 7B). However, the percentage of apoptotic neurons was significantly lower in gp130fl/fl neurons. Corresponding to the improved survival of infected gp130fl/fl neurons on stimulation with members of the IL-6 cytokine family, all of these cytokines significantly reduced apoptosis of infected gp130fl/fl neurons compared with nonstimulated gp130fl/fl neurons. Apoptosis of infected Syn-Cre gp130fl/fl neurons was not reduced on stimulation with IL-6 cytokines (Figure 7B). In contrast, both IFN-γ and TNF significantly reduced apoptosis of gp130fl/fl and Syn-Cre gp130fl/fl neurons. Interestingly, combined T. gondii and TUNEL staining found that infected neurons from both groups of mice were protected from apoptosis, whereas noninfected cells underwent apoptosis (Figure 7, C and D).
      In contrast to survival of neurons, no differences between gp130-negative and -positive neurons were determined for neuronal invasion and replication of T. gondii irrespective of treatment with IL-6 cytokines, IFN-γ, and TNF (see Supplemental Figure S1at http://ajp.amjpathol.org).

      Discussion

      Parasite control and TE survival requires both production of protective cytokines, including IFN-γ by T cells, and the production of immunosuppressive cytokines that balance the intracerebral immune response and prevent lethal immunopathology. The present study extends this concept and identifies neuronal gp130 expression as an important regulator in TE; Syn-Cre gp130fl/fl mice had hyperinflammation with elevated numbers of intracerebral CD4 and CD8 T cells. Stimulation of neurons by IL-6 induced their production of the immunosuppressive cytokines TGF-β and IL-27. This novel observation that cortical neurons are a source of IL-27 is of particular importance, because this cytokine is crucial to balance the intracerebral immune response to prevent lethal immunopathology mediated by IL-17-producing CD4 and CD8 T cells in TE.
      • Stumhofer J.S.
      • Laurence A.
      • Wilson E.H.
      • Huang E.
      • Tato C.M.
      • Johnson L.M.
      • Villarino A.V.
      • Huang Q.
      • Yoshimura A.
      • Sehy D.
      • Saris C.J.
      • O'Shea J.J.
      • Hennighausen L.
      • Ernst M.
      • Hunter C.A.
      Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.
      In addition, the reduced production of TGF-β may foster an overshooting intracerebral immune response of Syn-Cre gp130fl/fl mice, because this cytokine limits IFN-γ production of T cells in TE.
      • Schlüter D.
      • Bertsch D.
      • Frei K.
      • Hubers S.B.
      • Wiestler O.D.
      • Hof H.
      • Fontana A.
      • Deckert-Schlüter M.
      Interferon-gamma antagonizes transforming growth factor-beta2-mediated immunosuppression in murine Toxoplasma encephalitis.
      Thus, in toxoplasmosis, TGF-β appears to exhibit similar effects in the brain and gut, because it has also claimed to prevent T. gondii-induced ileitis.
      • Buzoni-Gatel D.
      • Debbabi H.
      • Mennechet F.J.D.
      • Martin V.
      • Lepage A.C.
      • Schwartzman J.D.
      • Kasper L.H.
      Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes.
      In good agreement with an important immunosuppressive function of TGF-β and IL-27 in TE, numbers of IFN-γ- and IL-17-producing CD4 and CD8 T cells were increased in Syn-Cre gp130fl/fl mice compared with control animals. Note that both the reduced production of TGF-β and IL-27 by IL-6-stimulated gp130-deficient neurons on a per cell basis as well as the marked loss of neurons in T. gondii-infected Syn-Cre gp130fl/fl mice may contribute to the reduced intracerebral production of TGF-β and IL-27, leading to increased T-cellular IFN-γ and IL-17 production in Syn-Cre gp130fl/fl mice.
      Interestingly, although both absolute and relative numbers of IFN-γ-producing CD4 and CD8 T cells raised in the T. gondii-infected CNS, absolute but not relative numbers of IL-17-producing CD4 and CD8 T cells increased in Syn-gp130fl/fl mice. Thus, the less immunosuppressive milieu of the CNS of Syn-Cre gp130fl/fl appears to affect T-cell IFN-γ production more prominently than IL-17 production. In accordance with studies by Stumhofer et al
      • Stumhofer J.S.
      • Laurence A.
      • Wilson E.H.
      • Huang E.
      • Tato C.M.
      • Johnson L.M.
      • Villarino A.V.
      • Huang Q.
      • Yoshimura A.
      • Sehy D.
      • Saris C.J.
      • O'Shea J.J.
      • Hennighausen L.
      • Ernst M.
      • Hunter C.A.
      Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.
      , the number of intracerebral IL-17-producing T cells was ∼10-fold lower compared with IFN-γ-producing T cells in both Syn-Cre gp130fl/fl and gp130fl/fl mice. Although IFN-γ is of crucial importance for the control of T. gondii
      • Suzuki Y.
      • Orellana M.A.
      • Schreiber R.D.
      • Remington J.S.
      Interferon-gamma: the major mediator of resistance against Toxoplasma gondii.
      , several lines of evidence report that an overshooting production of this cytokine is harmful in TE. Thus, IFN-γ- and IL-17-producing T cells contribute to the lethal cause of TE in IL27Rα−/− mice.
      • Stumhofer J.S.
      • Laurence A.
      • Wilson E.H.
      • Huang E.
      • Tato C.M.
      • Johnson L.M.
      • Villarino A.V.
      • Huang Q.
      • Yoshimura A.
      • Sehy D.
      • Saris C.J.
      • O'Shea J.J.
      • Hennighausen L.
      • Ernst M.
      • Hunter C.A.
      Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.
      • Villarino A.
      • Hibbert L.
      • Lieberman L.
      • Wilson E.
      • Mak T.
      • Yoshida H.
      • Kastelein R.A.
      • Saris C.
      • Hunter C.A.
      The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection.
      In addition, transient depletion of CD4 T cells in chronically infected mice reduced inflammation, which reversed on termination and reappearance of CD4 T cells.
      • Israelski D.M.
      • Araujo F.G.
      • Conley F.K.
      • Suzuki Y.
      • Sharma S.
      • Remington J.S.
      Treatment with anti-L3T4 (CD4) monoclonal antibody reduces the inflammatory response in toxoplasmic encephalitis.
      In addition, IFN-γ-producing CD4 T cells play an important immunopathologic role in high-dose intestinal T. gondii infection.
      • Liesenfeld O.
      • Kosek J.
      • Remington J.S.
      • Suzuki Y.
      Association of CD4+ T cell-dependent, interferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.
      This latter observation is also of particular interest in light of our results, because we also observed increased numbers of intracerebral IFN-γ-producing T cells paralleled by an increased intracerebral parasitic load in Syn-Cre gp130fl/fl mice. Furthermore, a study by Guiton et al
      • Guiton R.
      • Vasseur V.
      • Charron S.
      • Torres Arias M.
      • Van Langendonck N.
      • Buzoni-Gatel D.
      • Ryffel B.
      • Mier-Poisson I.
      Interleukin 17 receptor signaling is deleterious during Toxoplasma gondii infection in susceptible BL6 mice.
      has shown that IL-17 augments inflammation and increases the intracerebral parasitic load in chronic TE. Note that the increased inflammation may also cause higher numbers of intracerebral T. gondii in Syn-Cre gp130fl/fl mice by the significantly enhanced recruitment of dendritic cells and macrophages to the brain, because these cell populations shuttle T. gondii to the brain.
      • Courret N.
      • Darche S.
      • Sonigo P.
      • Milon G.
      • Buzoni-Gatel D.
      • Tardieux I.
      CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.
      In conclusion, the combination of deregulated intracerebral IFN-γ and IL-17 responses paralleled by an impaired intracerebral pathogen control as well as the progressive neuronal loss may cause death of Syn-Cre gp130fl/fl mice.
      The observation that expression of gp130 is important to prevent apoptosis of cultivated T. gondii-infected neurons further establishes gp130 as an important survival factor of highly differentiated neurons in TE. In Syn-Cre gp130fl/fl mice, neuronal loss progressed during TE, ultimately resulting in large cortical areas with substantial neuronal loss. The predominant loss of cortical neurons may be explained by the preferential infection of cortical neurons by T. gondii and their synapsin-I expression. Although control mice lost a few neurons from their cortex in intimate relation with inflammatory infiltrates, this neuronal loss was not progressive. The observation that T. gondii causes loss of neurons may, at least partially, underlie the well-documented altered behavior of T. gondii-infected mice.
      • Vyas A.
      • Kim S.K.
      • Giacomini N.
      • Boothroyd J.C.
      • Sapolsky R.M.
      Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors.
      • Hay J.
      • Aitken P.P.
      • Graham D.I.
      Toxoplasma infection and response to novelty in mice.
      These in vivo studies were further corroborated by in vitro experiments which found that cortical gp130-deficient neurons responded with a significantly increased apoptosis and cell death in the vicinity of T. gondii neurons compared with control neurons. Interestingly, in both gp130-deficient and -responsive neurons, T. gondii induced apoptosis of uninfected but not of infected neurons. This is in agreement with the well-documented antiapoptotic function of the parasite in infected cells
      • Goebel S.
      • Gross U.
      • Luder C.G.
      Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression.
      • Nash P.B.
      • Purner M.B.
      • Leon R.P.
      • Clarke P.
      • Duke R.C.
      • Curiel T.J.
      Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.
      and indicates that T. gondii also inhibits apoptosis of neurons, which allows encystation and persistence of the parasite. Because Toxoplasma infection of neurons induces neuronal IL-6 production
      • Schlüter D.
      • Deckert M.
      • Hof H.
      • Frei K.
      Toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon- and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of T. gondii.
      , the protective effect of gp130 on neuronal survival was already observed in unstimulated gp130fl/fl neurons. However, additional stimulation with all IL-6 cytokine family members further significantly reduced death and apoptosis of infected gp130fl/fl neuronal cultures, indicating a redundant function of these cytokines. The observation that gp130 is important to prevent apoptosis in T. gondii-infected neuronal cultures in vitro is in good agreement with experiments in astrocytes
      • Drögemüller K.
      • Helmuth U.
      • Brunn A.
      • Sakowicz-Burkiewicz M.
      • Gutmann D.H.
      • Mueller W.
      • Deckert M.
      • Schlüter D.
      Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis.
      and further finds an important antiapoptotic function of gp130 in Toxoplasma infection irrespective of the cell type. In contrast to T. gondii, the proinflammatory cytokines IFN-γ and TNF reduced neuronal cell death and apoptosis of gp130-deficient neurons. Thus, these two protective cytokines, if produced in appropriate amount in TE, exert rather a neuroprotective than a neurotoxic activity. Most likely, the induction of neuronal death by the parasite together with the limited regenerative capacity of the neurons contributes to progressive neuronal loss of infected Syn-Cre gp130fl/fl mice. In this scenario, brain regions containing parasites might be affected by ongoing neuronal loss, whereas areas with neuronal loss but devoid of T. gondii might rather represent inactive, burnt-out foci in which after irreversible neuronal loss the process of apoptosis has terminated.
      In conclusion, the present study found that gp130 expression of neurons is required to balance the interplay among the parasite, the immune system, and the resident cell populations of the CNS, in particular of highly differentiated and vulnerable neurons, to enable TE survival.

      Acknowledgments

      We thank Mariana Carstov, Elena Fischer, Nadja Schlüter, and Annette Sohnekind for expert technical assistance.

      Supplementary data

      • Supplemental Figure S1

        gp130-independent invasion and replication of Toxoplasma gondii in neurons. A and B: Neurons of Syn-Cre gp130fl/fl and gp130fl/fl mice were infected with T. gondii in vitro and stimulated with the indicated cytokines or left unstimulated. After 24 hours, cells were fixed and stained for T. gondii antigens. The percentage of T. gondii-infected cells (A) and the number of T. gondii per parasitophorous vacuole of infected neurons (B) was microscopically determined. Data show the mean ± SD of six mice per experimental group used in two independent experiments. At least 100 neurons (A) and 100 infected neurons (B) per mouse were counted. CLC, cardiotrophin-like cytokine; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin-1; IFN, interferon; LIF, leukemia inhibitory factor; OSM, oncostatin M; TNF, tumor necrosis factor.

      References

        • Fagard R.
        • Van Tan H.
        • Creuzet C.
        • Pelloux H.
        Differential development of Toxoplasma gondii in neural cells.
        Parasitol Today. 1999; 15: 504-507
        • Montoya J.G.
        • Liesenfeld O.
        Toxoplasmosis.
        Lancet. 2004; 363: 1965-1976
        • Suzuki Y.
        • Conley F.K.
        • Remington J.S.
        Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice.
        J Immunol. 1989; 143: 2045-2050
        • Gazzinelli R.
        • Xu Y.
        • Hieny S.
        • Cheever A.
        • Sher A.
        Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii.
        J Immunol. 1992; 149: 175-180
        • Fischer H.G.
        • Nitzgen B.
        • Reichmann G.
        • Hadding U.
        Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells.
        Eur J Immunol. 1997; 27: 1539-1548
        • Strack A.
        • Asensio V.C.
        • Campbell I.L.
        • Schlüter D.
        • Deckert M.
        Chemokines are differentially expressed by astrocytes, microglia and inflammatory leukocytes in Toxoplasma encephalitis and critically regulated by interferon-gamma.
        Acta Neuropathol (Berl). 2002; 103: 458-468
        • Drögemüller K.
        • Helmuth U.
        • Brunn A.
        • Sakowicz-Burkiewicz M.
        • Gutmann D.H.
        • Mueller W.
        • Deckert M.
        • Schlüter D.
        Astrocyte gp130 expression is critical for the control of Toxoplasma encephalitis.
        J Immunol. 2008; 181: 2683-2693
        • Suzuki Y.
        • Rani S.
        • Liesenfeld O.
        • Kojima T.
        • Lim S.
        • Nguyen T.A.
        • Dalrymple S.A.
        • Murray R.
        • Remington J.S.
        Impaired resistance to the development of toxoplasmic encephalitis in interleukin-6-deficient mice.
        Infect Immun. 1997; 65: 2339-2345
        • Silver J.S.
        • Stumhofer J.S.
        • Passos S.
        • Ernst M.
        • Hunter C.A.
        IL-6 mediates the susceptibility of glycoprotein 130 hypermorphs to Toxoplasma gondii.
        J Immunol. 2011; 187: 350-360
        • Stumhofer J.S.
        • Laurence A.
        • Wilson E.H.
        • Huang E.
        • Tato C.M.
        • Johnson L.M.
        • Villarino A.V.
        • Huang Q.
        • Yoshimura A.
        • Sehy D.
        • Saris C.J.
        • O'Shea J.J.
        • Hennighausen L.
        • Ernst M.
        • Hunter C.A.
        Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system.
        Nat Immunol. 2006; 7: 937-945
        • Schlüter D.
        • Deckert M.
        • Hof H.
        • Frei K.
        Toxoplasma gondii infection of neurons induces neuronal cytokine and chemokine production, but gamma interferon- and tumor necrosis factor-stimulated neurons fail to inhibit the invasion and growth of T. gondii.
        Infect Immun. 2001; 69: 7889-7893
        • Betz U.A.
        • Bloch W.
        • van den Broek M.
        • Yoshida K.
        • Taga T.
        • Kishimoto T.
        • Addicks K.
        • Rajewsky K.
        • Müller W.
        Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects.
        J Exp Med. 1998; 188: 1955-1965
        • Zhu Y.
        • Romero M.I.
        • Ghosh P.
        • Ye Z.
        • Charnay P.
        • Rushing E.J.
        • Marth J.D.
        • Parada L.F.
        Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.
        Genes Dev. 2001; 15: 859-876
        • Rempe D.
        • Vangeison G.
        • Hamilton J.
        • Li Y.
        • Jepson M.
        • Federoff H.J.
        Synapsin I Cre transgene expression in male mice produces germline recombination in progeny.
        Genesis. 2006; 44: 44-49
        • Schlüter D.
        • Löhler J.
        • Deckert M.
        • Hof H.
        • Schwendemann G.
        Toxoplasma encephalitis of immunocompetent and nude mice: immunohistochemical characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products.
        J Neuroimmunol. 1991; 31: 185-198
        • Schlüter D.
        • Deckert-Schlüter M.
        • Lorenz E.
        • Meyer T.
        • Röllinghoff M.
        • Bogdan C.
        Inhibition of inducible nitric oxide synthase exacerbates chronic cerebral toxoplasmosis in Toxoplasma gondii-susceptible C57BL/6 mice but does not reactivate the latent disease in T-gondii-resistant BALB/c mice.
        J Immunol. 1999; 162: 3512-3518
        • Haroon F.
        • Drögemüller K.
        • Händel U.
        • Brunn A.
        • Reinhold D.
        • Nishanth G.
        • Mueller W.
        • Trautwein C.
        • Ernst M.
        • Deckert M.
        • Schlüter D.
        Gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation.
        J Immunol. 2011; 186: 6521-6531
        • Schlüter D.
        • Oprisiu S.B.
        • Chahoud S.
        • Weiner D.
        • Wiestler O.D.
        • Hof H.
        • Deckert-Schlüter M.
        Systemic immunization induces protective CD4+ and CD8+ T cell-mediated immune responses in murine Listeria monocytogenes meningoencephalitis.
        Eur J Immunol. 1995; 25: 2384-2391
        • Frericks M.
        • Esser C.
        A toolbox of novel murine house-keeping genes identified by meta-analysis of large scale gene expression profiles.
        Biochim Biophys Acta. 2008; 1779: 830-837
        • Livak K.J.
        • Schmittgen T.D.
        Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
        Methods. 2001; 25: 402-408
        • Frei K.
        • Bodmer S.
        • Schwerdel C.
        • Fontana A.
        Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages.
        J Immunol. 1986; 137: 3521-3527
        • Schlüter D.
        • Bertsch D.
        • Frei K.
        • Hubers S.B.
        • Wiestler O.D.
        • Hof H.
        • Fontana A.
        • Deckert-Schlüter M.
        Interferon-gamma antagonizes transforming growth factor-beta2-mediated immunosuppression in murine Toxoplasma encephalitis.
        J Neuroimmunol. 1998; 81: 38-48
        • Buzoni-Gatel D.
        • Debbabi H.
        • Mennechet F.J.D.
        • Martin V.
        • Lepage A.C.
        • Schwartzman J.D.
        • Kasper L.H.
        Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes.
        Gastroenterology. 2001; 120: 914-924
        • Suzuki Y.
        • Orellana M.A.
        • Schreiber R.D.
        • Remington J.S.
        Interferon-gamma: the major mediator of resistance against Toxoplasma gondii.
        Science. 1988; 240: 516-518
        • Villarino A.
        • Hibbert L.
        • Lieberman L.
        • Wilson E.
        • Mak T.
        • Yoshida H.
        • Kastelein R.A.
        • Saris C.
        • Hunter C.A.
        The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection.
        Immunity. 2003; 19: 645-655
        • Israelski D.M.
        • Araujo F.G.
        • Conley F.K.
        • Suzuki Y.
        • Sharma S.
        • Remington J.S.
        Treatment with anti-L3T4 (CD4) monoclonal antibody reduces the inflammatory response in toxoplasmic encephalitis.
        J Immunol. 1989; 142: 954-958
        • Liesenfeld O.
        • Kosek J.
        • Remington J.S.
        • Suzuki Y.
        Association of CD4+ T cell-dependent, interferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.
        J Exp Med. 1996; 184: 597-607
        • Guiton R.
        • Vasseur V.
        • Charron S.
        • Torres Arias M.
        • Van Langendonck N.
        • Buzoni-Gatel D.
        • Ryffel B.
        • Mier-Poisson I.
        Interleukin 17 receptor signaling is deleterious during Toxoplasma gondii infection in susceptible BL6 mice.
        J Infect Dis. 2010; 202: 427-435
        • Courret N.
        • Darche S.
        • Sonigo P.
        • Milon G.
        • Buzoni-Gatel D.
        • Tardieux I.
        CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.
        Blood. 2006; 107: 309-316
        • Vyas A.
        • Kim S.K.
        • Giacomini N.
        • Boothroyd J.C.
        • Sapolsky R.M.
        Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors.
        Proc Natl Acad Sci U S A. 2007; 104: 6442-6447
        • Hay J.
        • Aitken P.P.
        • Graham D.I.
        Toxoplasma infection and response to novelty in mice.
        Z Parasitenkd. 1984; 70: 575-588
        • Goebel S.
        • Gross U.
        • Luder C.G.
        Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression.
        J Cell Sci. 2001; 114: 3495-3505
        • Nash P.B.
        • Purner M.B.
        • Leon R.P.
        • Clarke P.
        • Duke R.C.
        • Curiel T.J.
        Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.
        J Immunol. 1998; 160: 1824-1830