The FASEB Journal express article 10.1096/fj.03-0670fje. Published online December 19, 2003.
Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9) Asparouh I. Iliev,*,‡ Argyrios K. Stringaris,†,‡ Roland Nau,† and Harald Neumann* *Neuroimmunology Unit, European Neuroscience Institute Göttingen, Waldweg 33, 37073 Göttingen, Germany; and †Department of Neurology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. ‡The first two authors contributed equally to this work. Corresponding author: Roland Nau, Department of Neurology, University of Göttingen, RobertKoch-Str. 40, 37075 Göttingen, Germany. E-mail:
[email protected] ABSTRACT Innate immune cells express toll-like receptor-9 (TLR9) and respond to unmethylated, CG dinucleotide motif-rich DNA released from bacteria during infection or endogenous cells during autoimmune tissue injury. Oligonucleotides containing CG dinucleotide (CpG-DNA) mimic the effect of unmethylated DNA and stimulate TLR9. CpG-DNA was cytotoxic to neurons in organotypic brain cultures. Neurotoxicity of CpG-DNA was mediated via microglial cells and started primarily from neurites as determined by time-lapse imaging of enhanced green fluorescent protein (EGFP)-transfected neurons. Cultured brain microglial cells expressed TLR9 and responded to CpG-DNA by production of the inflammatory mediators nitric oxide (NO) and tumor necrosis factor-α (TNF-α). Blockade of NO synthase and TNF-α prevented damage of neurites and neurotoxicity of CpG-DNA. The data suggest that stimulation of microglia via TLR9 and subsequent release of NO and TNF-α is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury. Key words: microglia, neurons • toll-like receptor-9 • CpG-DNA • neurite damage • neuroinflammation • meningitis • TNF-α • NO
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hort segments of unmethylated, CG dinucleotide motif-rich DNA stimulate cells of the innate immune system, including dendritic cells, macrophages, and microglia (1–3). Unmethylated bacterial DNA is recognized by toll-like receptor-9 (TLR9) in a speciesspecific manner (4, 5). Furthermore, self-DNA/immunoglobulin complexes have been suggested to trigger an autoimmune cascade in systemic lupus erythematosus in a TLR9-dependent manner (6, 7). Synthetic oligonucleotides enriched in CG dinucleotide motifs (CpG-DNA) reliably mimic the effects of unmethylated DNA (8, 9). Stimulation of TLR9 via CpG-DNA injected intraperitoneally into mice caused septic shock (8). Intraarticular injection of CpG-DNA exacerbated experimental adjuvans-induced arthritis (10). Furthermore, CpG-DNA could directly regulate microglia function, both in vivo and in vitro. Isolated murine microglia and human microglial cell lines treated with CpG-DNA produced various cytokines, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6, and IL-12 (3). CpG-DNA injection into the cerebrospinal fluid (CSF) of mice stimulated microglial cells and induced meningitis (11,
12). Here we demonstrate that CpG-DNA is strongly neurotoxic via stimulation of microglial TLR9 and release of nitric oxide (NO) and TNF-α. MATERIALS AND METHODS CpG-oligodeoxynucleotide (ODN) 1668 (TCC ATG ACG TTC CTG ATG CT), phosphothioate protected form, was obtained from TIB Molbiol (Berlin, Germany). Culture and labeling of slices Hippocampal tissue slices of 350 µm thickness were obtained from postnatal Day 3 C57BL/6 mice by using tissue chopper slicing (Vibratome, St. Louis, MO) and grown on culture plate inserts (Millipore Corp., Bedford, MA) as described previously (13). Slices were cultured in BME-based medium (BME, GibcoBRL, Invitrogen GmbH, Karlsruhe, Germany), 2% B-27 neuronal supplement (GibcoBRL), 1% glucose (Sigma, Deisenhofen, Germany), and 1% fetal calf serum (PAN Biotech GmbH, Aidenbach, Germany). To visualize dead cells, the slices were incubated with propidium iodide (2 µg/ml, Sigma). Selective labeling of neurites was performed by using biotinylated dextran amine (BDA, 10 kDa, Molecular Probes, Leiden, The Netherlands) placed on the entorhinal cortex of cultured slices 24 h before fixation. Slices were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde and visualized by confocal microscopy on a Leica TCS SP2 confocal system (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) following incubation with streptavidin conjugated with fluorochrome Cy3 (1:500, Dianova, Hamburg, Germany). The total length of neurites from individual neurons was measured with Image Tool 2.0 software (University of Texas Health Science Center, San Antonio, TX). Primary neuronal and microglial cell cultures Neuronal cell cultures were prepared from embryonic C57BL/6 mice and cultured as described previously (14) using the same medium as the brain slices. Neurons were cultured 7–14 days to obtain a morphologically mature phenotype. Microglial cells were prepared from the brains of postnatal Day 3 C57BL/6 mice and cultured as previously reported (13). Microglial cells were added in a fixed ratio of 1:2 (microglia: neurons) to neurons. The following substances were applied 2 h before CpG-DNA treatment to the cells in the following final concentrations: 200 µM aminoguanidine (Sigma); 10 µg/ml TNF receptor 1 IgG fusion protein (recombinant human TNF receptor p55IgG1; gift from Werner Lesslauer and Hansruedi Loetscher, Roche Ltd., Basel, Switzerland); 10 µg/ml human control IgG (Dianova); and 10 µM MK-801 (Sigma). Immunocytochemistry and confocal imaging of dissociated cultures Immunocytochemistry was performed as described previously (14). After fixation cells were incubated with primary monoclonal antibody directed against the neuronal cytoskeleton protein β-tubulin III (1:300; Sigma) and biotinylated isolectin B4 (3 µg/ml; Sigma) as a microglial marker. Secondary antibodies were goat antibody directed against mouse immunoglobulin conjugated to fluorochrome FITC (1:300, Dianova) and streptavidin conjugated with fluorochrome Cy3 (1:500, Dianova). The cells were then incubated with 4,6-diamidino-2phenylindole dihydrochloride (1 µg/ml, DAPI, Molecular Probes) to label nuclei and mounted.
Neurite evaluation and counting of neuronal nuclei Six randomly selected areas in each dish were scanned and analyzed by confocal microscopy. βtubulin III-positive neurites, which crossed one of four 350-µm-long parallel lines (distance of 70 µm), were counted. Total number of nuclei stained with DAPI and double-labeled with antibodies directed against β-tubulin III were counted in five microscopic fields. Data are presented as percentage of untreated controls. Transfection procedure and time lapse imaging Transfection of neurons was performed during seeding with a plasmid-expressing EGFP (enhanced green fluorescent protein) under the human synapsin promoter by using the Effectene Transfection Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Following the addition of 4–8 µl enhancer (as provided by supplier), 0.5–1 µg EGFP-DNA per 106 cells were incubated with 12 µl Effectene and 85 µl transfection buffer for 30 min. Subsequently, cells were washed in ice-cold PBS, reconstituted in neuronal medium, and seeded at a density of 600,000 cells/50 mm dish or 150,000 cells/chamber in fourwell chamber slides. The average transfection efficiency was 0.1%, and the majority of transfected neurons showed plasmid expression at least until Day 14. The plasmid used was p(synapsin)-EGFP (kind gift from S. Kügler, Dept. of Neurology, University of Göttingen, Germany). The plasmid was purified by using EndoFree Maxi Kit (Qiagen GmbH, Hilden, Germany). Transfected cells in culture dishes were treated with 10 µM CpG-DNA for 48 h. Time-lapse imaging was performed at several time points (0, 24, 30, and 48 h) on an inverted luminescent microscope (Olympus Optical Co., Japan) with a CCD camera (Nikon Coolpix 995, Nikon Precision Europe GmbH, Langen, Germany). For time-lapse imaging microglia were prelabeled for 1 h with 1 µg/ml CellTracker CM-DiI (Molecular Probes). RT-PCR for TLR9, TNF-α, and iNOS gene transcripts RT-PCR was performed as described previously (14). Total RNA was extracted from cultured cells with RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and transcribed with reverse transcriptase (200 U/µl; SuperScript RT, GibcoBRL, Life Technologies GmbH). PCR was performed in a thermocycler (MultiCycler, MJ Research Inc., Waltham, MA) for 30 cycles. The primers used were for TLR9 (5′-CTGGCCTGGCTAATGGTGTGAAGT-3′ and 5′TCTGTGCCTTATCGAACACCACGA-3′), for TNF-α (5′-TCGTAGCAAACCACCAAGTG-3′ and 5′-CTGAGTTGGTCCCCCTTCTC-3′) and for iNOS (5′-AAGCTGCATGTGACATCGAC3′ and 5′-TGCTGAAACATTTCCTGTGC-3′). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard control (primers: 5′-TCCGCCCCTTCTGCCGATG-3′ and 5′CACGGAAGGCCATGCCAGTGA-3′). TNF-α ELISA Isolated microglia (200,000 cells/ml) were cultured and exposed to CpG-DNA for 24 h. Enzymelinked immunosorbent assays (ELISA) for TNF-α of supernatants were performed according to the instructions of the manufacturer (R&D Systems GmbH, Wiesbaden, Germany).
Statistics Data were analyzed by one-way ANOVA with P-value adjustment for repeated testing by the Bonferroni method, and unpaired t-test, performed by using GraphPad Prism (GraphPad Software, San Diego, CA) and Microsoft Excel Software (Microsoft Corporation, Redmond, WA). RESULTS Neuronal damage to cortico-hippocampal slice cultures by CpG-DNA Cortico-hippocampal slices containing the hippocampus and the adjacent neocortex derived from postnatal mice were cultured in a membrane dish. Brain cultures were treated with unmethylated CpG-DNA oligonucleotides, which mimic bacterial or unmethylated CG-rich endogenous DNA. Slice cultures treated with 10 µM CpG-DNA for 72 h showed cell death in the dentate gyrus and the hippocampal area CA1/CA2 as determined by the labeling of living slices with propidium iodide (Fig. 1). The morphological pattern of labeling indicated mainly neuronal damage after exposure to CpGDNA, both in the zones of the pyramidal layer of CA1/CA2 and the granular layer of the dentate gyrus. No injury of glial cells was observed following treatment with CpG-DNA. To analyze neurite integrity in the entorhinal cortex in slices, we traced neurites with biotinylated dextran amine (BDA), a molecule taken up by neuronal cell bodies and transported to axons and dendrites. Control slices labeled with BDA showed long and well-defined neurites, whereas neurite length in slices treated with 10 µM CpG-DNA for 72 h was reduced (Fig. 2). Slice cultures treated with 10 µM CpG-DNA for 72 h showed a reduced average total neurite length of 96 µm ± 24 µm (mean ± SEM), whereas neurons in untreated slices demonstrated a total neurite length of 243 µm ± 46 µm (mean ± SEM) (Fig. 2). Expression of TLR9 on microglia and stimulation of iNOS and TNF-α by CpG-DNA To study the brain cell type responding to CpG-DNA challenge, we assessesd TLR9 gene transcripts by RT-PCR in isolated cell preparations. A murine spleen cell cDNA preparation containing lymphocytes, macrophages, and dendritic cells served as a positive control. Microglial cells were isolated from postnatal hippocampal tissue and enriched to a purity of more than 96% as determined by staining with antibodies directed against CD11b and flow cytometry analysis. TLR9 gene transcripts were detected after PCR amplification in the spleen, untreated microglia and microglial cell preparations treated for 24 h with 10 µM CpG-DNA (Fig. 3). No PCR product for TLR9 was detected in cultured hippocampal neurons (Fig. 3). We then assessed the impact of CpG-DNA treatment on the expression and release of microglial cytotoxic mediators. Challenge of microglia with either 1 or 10 µM CpG-DNA for 24 h led to prominent induction of both TNF-α and inducible nitric oxide synthase (iNOS) gene transcripts (Fig. 4A). No gene transcripts for TNF-α and iNOS were detected in untreated microglia (Fig. 4A).
Furthermore, secretion of TNF-α from CpG-DNA-stimulated microglia was analyzed by enzyme-linked immunosorbent assay (ELISA). CpG-DNA treatment for 24 h led to a strong secretion of TNF-α (Fig. 4B). Treatment with 1 µM CpG-DNA stimulated microglia to release ~1000 pg/ml of TNF-α protein within 24 h, whereas no significant further increase of TNF-α secretion was achieved when 10 µM CpG-DNA was applied (Fig. 4B). CpG-DNA-induced damage to hippocampal neurons is mediated via microglia and starts at neurites Microglial cells were isolated and added to the neuronal cultures. After co-culturing for 72 h, the cells were fixed and double-labeled with microglial isolectin B4 and antibodies directed against the neuronal cytoskeleton protein β-tubulin III. CpG-DNA treatment with 10 µM CpG-DNA for 72 h was not toxic to isolated neurons, indicating that CpG-DNA was acting indirectly via microglial cells (Fig. 5A). Unstimulated microglia had no cytotoxic effects on neurons after 72 h of co-culture (Fig. 5B). However, strong neurotoxicity occurred after treatment of co-cultures with 10 µM CpG-DNA for 72 h (Fig. 5B). To examine the primary site of neuronal damage by CpG-DNA-stimulated microglia, we performed live time-lapse imaging and monitored CellTracker-labeled microglia in a co-culture with enhanced green fluorescent protein (EGFP)-transfected neurons in the presence of 10 µM CpG-DNA. Distinct morphological changes indicating primary neurite damage evolved within a few hours following treatment with 10 µM CpG-DNA. Distal neurite segments surrounded by microglia were the first to display signs of degeneration, which then spread proximally (Fig. 6). During distal degeneration of neurites, cell bodies showed no signs of damage (Fig. 6). In total, 40% of the time lapse analyzed EGFP-transfected neurons showed this pattern of primary neurite damage in the presence of microglia following CpG-DNA treatment. TNF-α and nitric oxide are effectors of CpG-DNA-induced damage to hippocampal neurons Confocal microscopic analysis suggested that microglia directly damaged neurites and subsequently neurons after CpG-DNA treatment. Indeed, neurites in close vicinity to microglia displayed bulb and spheroid formation, beading, and reduced overall length. Because NO and TNF-α are produced by microglia upon activation with CpG-DNA, we determined their contribution to neuronal injury. Co-cultures, treated with 10 µM CpG-DNA for 72 h, showed a strong reduction of neurite density, reaching 9.6 ± 2.5% (mean ± SEM) of untreated control cultures (Fig. 7). Co-cultures were then treated with the iNOS inhibitor aminoguanidine (200 µM), starting 2 h prior to stimulation with 10 µM CpG-DNA for 72 h. Blockade of iNOS protected neurons from microglial CpG-DNA toxicity. Neurons in cultures treated with aminoguanidine showed a neurite density of 70.8% ± 5.1% (mean ± SEM) in relation to untreated controls (Fig. 7). As shown above, TNF-α was secreted in large amounts following stimulation with CpG-DNA. Because TNF-α is reported to be involved in neuronal damage, we studied whether neutralization of TNF-α would preserve neurite integrity. Indeed, incubation of cocultures with TNF-R1 fusion protein (10 µg/ml) partially protected neurites from challenge with 10 µM CpG-DNA for 72 h. Relative neurite density of CpG-DNA-challenged co-cultures treated with TNF-R1 IgG-fusion protein was 54.6 ± 5.5% (mean ± SEM) compared with 17.6 ± 7.2% of those cultures treated with CpG-DNA plus control IgG (Fig. 7). Treatment of co-cultures with
the glutamate (NMDA) receptor antagonist MK-801 (10 µM) did not prevent toxicity of CpGDNA substantially (Fig. 7). DISCUSSION TLRs play an essential role in the innate recognition of “pathogen-associated molecular patterns” and trigger protective immune responses (9). During inflammatory reactions, TLRs gene transcripts are up-regulated in the central nervous system (CNS; 15, 16). It was suggested that stimulation of TLRs is one of the major triggers for neurodegeneration in the CNS (17, 18). Recently, unmethylated bacterial DNA has been shown to possess the capacity of inducing sustained inflammation via stimulation of TLR9 (4, 5). Moreover, evidence exists that selfDNA/immunoglobulin complexes may trigger autoimmune responses in a TLR9-dependent manner (6, 7). We, therefore, asked whether CpG-DNA, which stimulates TLR9, might cause neuronal damage in the presence of brain-innate immune cells. Damage to neurites and neuronal somata was visible in CpG-DNA-treated organotypic cortico-hippocampal slices. In particular, CpG-DNA-induced neuronal and neurite injury in the dentate gyrus, stratum radiatum of the CA1 area, and entorhinal cortex. No signs of glial cell injury were observed in the organotypic slice cultures after CpG-DNA treatment. However, we cannot exclude the possibility, which would require detailed electron microscopic analysis, that CpG-DNA treatment acts cytotoxically on oligodendrocytes and myelin of the perforant path in the organotypic slice cultures. Direct visualization of neurites by tracing with BDA demonstrated significant neurite loss in the entorhinal cortex, confirming axonal degeneration following CpG-DNA treatment. Time-lapse imaging of microglia-neuron co-cultures revealed distal degeneration of neurites as a characteristic pattern of injury. Damage to axons and neurites in response to inflammation is a typical feature of traumatic infectious (19) and autoimmune diseases of the CNS (20). Microglial cells ensheath neurites in inflammatory cortical lesions (21), and the number of activated microglia and macrophages positively correlate with the extent of axonal damage in multiple sclerosis (22). Here we provide new evidence that microglia can act primarily toxically on neurites and that axonal damage may result from stimulation of microglia via toll-like receptors. In this study, injury of neurons and neurites following CpG-DNA treatment depended on the presence of microglia. Microglial cells express TLR9 and up-regulate gene transcripts for the enzyme NO synthase and the cytokine TNF-α in response to CpG-DNA treatment. Activated microglia can act in a protective manner by removing damaged cells from injured sites in the CNS and by promoting tissue repair (23). However, microglial activation can also be harmful to surrounding neurons. Neurotoxicity of microglia was observed in vitro following stimulation with amyloid-β protein (24), lipopolysaccharide (LPS) together with IFN-γ (25–27), and chromogranin A (28). The neurotoxic mechanisms of microglia involve generation of NO (25, 29), other reactive oxygen species, and the synergistic actions of TNF-α and IL-1β (30). No direct toxicity of CpG-DNA treatment was observed on isolated cultured neurons (Fig. 5A), astrocytes, or microglia (unpublished observation). However, after addition of microglial cells, CpG-DNA treatment preferentially injured neurites. Using specific inhibitors, we demonstrate that neurotoxicity of CpG-DNA is mediated mainly via release of NO and TNF-α from microglia. Cultured embryonic hippocampal neurons express TNFR1 and respond to TNF-α
treatment by rhoA activation and reduced neurite outgrowth, if cultured on a monolayer of astrocytes (14). In the present study, we used hippocampal neurons that matured in vitro for 7–14 days and were then co-cultured with microglia. We do not know whether the action of TNF-α following CpG-DNA treatment is direct, indirect, or depends on the presence of additional neurotoxic factors secreted from microglia. The sole application of TNF-α on mature hippocampal neurons at doses equivalent to those measured in our cultures did not result in neurite injury or neurotoxicity (Iliev and Neumann, unpublished observation). Thus, an autocrine loop involving release of TNF-α and stimulation of microglia by TNF-α might be involved in the observed neurotoxicity of CpG-DNA treatment. Previous data suggest that challenge of neurons with NO induces rapid glutamate release (27) and NMDA receptor-mediated neurotoxicity in mixed co-cultures of cerebellar granular neurons and microglia (31). In the present study, the NMDA antagonist MK-801 did not protect neurons from CpG-DNA-mediated neurotoxicity, suggesting that excitotoxicity is not involved in TLR9mediated neuronal injury. Alternatively, differences in the cell type or culture conditions between hippocampal and cerebellar neurons might account for the distinct mechanisms of cytotoxicity. Unmethylated CG-rich DNA derived from bacterial microorganisms is released into the subarachnoid space during bacterial growth and following antibiotic therapy of bacterial CNS infections (32, 33). The exact concentration of bacterial DNA in the cerebrospinal fluid and in the extracellular fluid of the brain is difficult to determine. In vitro, 107 colony-forming units (CFU) of Streptococcus pneumoniae/ml contained ~165µg/ml of bacterial DNA [Gerber and Nau, data calculated from (32)], which is released during spontaneous growth and antimicrobial therapy with β-lactam antibiotics. Because in S. pneumoniae meningitis in humans and experimental animals bacterial concentrations in CSF can rise up to ~5 × 108 CFU/ml, the concentrations of bacterial DNA in CSF probably can be up to two orders of magnitude higher than the concentration of CpG-DNA used in this study (10 µM = 63.8µg/ml). Our data suggest that CpG-DNA released during bacterial CNS infections act on microglia, thereby facilitating neurite damage and hippocampal cell loss, both commonly observed during the course of bacterial meningitis (33). Recently, self-DNA/immunoglobulin complexes have been shown to contribute to autoimmune disease by stimulation of TLR9 (6, 7). Therefore, it is tempting to speculate that self-DNA released during autoimmune inflammation of the CNS promotes microglial stimulation and subsequent axonal damage as observed in multiple sclerosis lesions. In conclusion, stimulation of TLR9 by CpG-DNA leads to microglia-induced neurotoxicity in a NO- and TNF-α-dependent fashion. Neurites are particularly vulnerable to damage of CpGDNA-stimulated microglia. These results help to explain the pathogenesis of bacterial and possibly autoimmune brain injury. Furthermore, our data suggest a benefit from adjunctive therapy aimed at blocking TLR9 or its signaling pathway in CNS diseases involving unmethylated CG dinucleotide motif-rich DNA.
ACKNOWLEDGMENTS The work was supported by the University of Göttingen, the “Deutsche Forschungsgemeinschaft” (grants to H. N. and R. N.) and the “Hertie Stiftung” (grant to H. N.). We thank Werner Lesslauer and Hansruedi Loetscher for the gift of TNF receptor 1 IgG fusion protein. We are grateful to Alexandra Bohl and Heiko Rhöse for technical assistance and to Christine Crozier and Christian Rochford for critical reading of the manuscript. Competing interest statement: The authors declare that they have no competing financial interests. REFERENCES 1.
Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A., and Klinman, D. M. (1995) CpG motifs in bacterial DNA trigger direct Bcell activation. Nature 374, 546–549
2.
Takeshita, S., Takeshita, F., Haddad, D. E., Janabi, N., and Klinman, D. M. (2001) Activation of microglia and astrocytes by CpG oligodeoxynucleotides. Neuroreport 12, 3029–3032
3.
Dalpke, A. H., Schafer, M. K., Frey, M., Zimmermann, S., Tebbe, J., Weihe, E., and Heeg, K. (2002) Immunostimulatory CpG-DNA activates murine microglia. J. Immunol. 168, 4854–4863
4.
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745
5.
Bauer, S., Kirschning, C. J., Hacker, H., Redecke, V., Hausmann, S., Akira, S., Wagner, H., and Lipford, G. B. (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98, 9237–9242
6.
Vinuesa, C. G., and Goodnow, C. C. (2002) Immunology: DNA drives autoimmunity. Nature 416, 595–598
7.
Leadbetter, E. A., Rifkin, I. R., Hohlbaum, A. M., Beaudette, B. C., Shlomchik, M. J., and Marshak-Rothstein, A. (2002) Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603–607
8.
Sparwasser, T., Miethke, T., Lipford, G., Erdmann, A., Hacker, H., Heeg, K., and Wagner, H. (1997) Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-alpha-mediated shock. Eur. J. Immunol. 27, 1671–1679
9.
Wagner, H. (1999) Bacterial CpG DNA activates immune cells to signal infectious danger. Adv. Immunol. 73, 329–368
10. Ronaghy, A., Prakken, B. J., Takabayashi, K., Firestein, G. S., Boyle, D., Zvailfler, N. J., Roord, S. T., Albani, S., Carson, D. A., and Raz, E. (2002) Immunostimulatory DNA sequences influence the course of adjuvant arthritis. J. Immunol. 168, 51–56
11. Schluesener, H. J., Seid, K., Deininger, M., and Schwab, J. (2001) Transient in vivo activation of rat brain macrophages/microglial cells and astrocytes by immunostimulatory multiple CpG oligonucleotides. J. Neuroimmunol. 113, 89–94 12. Deng, G. M., Liu, Z. Q., and Tarkowski, A. (2001) Intracisternally localized bacterial DNA containing CpG motifs induces meningitis. J. Immunol. 167, 4616–4626 13. Neumann, H., Misgeld, T., Matsumuro, K., and Wekerle, H. (1998) Neurotrophins inhibit major histocompatibility class II inducibility of microglia: involvement of the p75 neurotrophin receptor. Proc. Natl. Acad. Sci. USA 95, 5779–5784 14. Neumann, H., Schweigreiter, R., Yamashita, T., Rosenkranz, K., Wekerle, H., and Barde, Y. A. (2002) Tumor necrosis factor inhibits neurite outgrowth and branching of hippocampal neurons by a rho-dependent mechanism. J. Neurosci. 22, 854–862 15. Nguyen, M. D., Julien, J. P., and Rivest, S. (2002) Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat. Rev. Neurosci. 3, 216–227 16. Böttcher, T., von Mering, M., Ebert, S., Meyding-Lamade, U., Kuhnt, U., Gerber, J., and Nau, R. (2003) Differential regulation of Toll-like receptor mRNAs in experimental murine central nervous system infections. Neurosci. Lett. 344, 17–20 17. Perry, V. H., Newman, T. A., and Cunningham, C. (2003) The impact of systemic infection on the progression of neurodegenerative disease. Nat. Rev. Neurosci. 4, 103–112 18. Lehnardt, S., Massillon, L., Follett, P., Jenes, F. E., Ratan, R., Rosenber, P. A., Volpe, J. J., and Vartanian, T. (2003) Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4 dependent pathway. Proc. Natl. Acad. Sci. USA 100, 8514–8519 19. Medana, I. M., Day, N. P., Hien, T. T., Mai, N. T., Bethell, D., Phu, N. H., Farrar, J., Esiri, M. M., White, N. J., and Turner, G. D. (2002) Axonal injury in cerebral malaria. Am. J. Pathol. 160, 655–666 20. Neumann, H. (2003) Molecular mechanisms of axonal damage in inflammatory central nervous system diseases. Curr. Opin. Neurol. 16, 267–273 21. Peterson, J. W., Bo, L., Mork, S., Chang, A., and Trapp, B. D. (2001) Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann. Neurol. 50, 389–400 22. Bitsch, A., Schuchardt, J., Bunkowski, S., Kuhlmann, T., and Bruck, W. (2000) Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation. Brain 123, 1174–1183 23. Wyss-Coray, T., and Mucke, L. (2002) Inflammation in neurodegnerative disease - A double-edged sword. Neuron 35, 419–432
24. Ii, M., Sunamoto, M., Ohnishi, K., and Ichimori, Y. (1996) beta-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity. Brain Res. 720, 93–100 25. Chao, C. C., Hu, S., Molitor, T. W., Shaskan, E. G., and Peterson, P. K. (1992) Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J. Immunol. 149, 2736– 2741 26. Dawson, V. L., Brahmbhatt, H. P., Mong, J. A., and Dawson, T. M. (1994) Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neuropharmacology 33, 1425–1430 27. Bal-Price, A., and Brown, G. C. (2001) Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J. Neurosci. 21, 6480–6491 28. Kingham, P. J., Cuzner, M. L., and Pocock, J. M. (1999) Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation. J. Neurochem. 73, 538–547 29. Dawson, T. M., and Snyder, S. H. (1994) Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J. Neurosci. 14, 5147–5159 30. Chao, C. C., Hu, S., Ehrlich, L., and Peterson, P. K. (1995) Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of Nmethyl-D-aspartate receptors. Brain Behav. Immun. 9, 355–365 31. Malipiero, U., Heuss, C., Schlapbach, R., Tschopp, J., Gerber, U., and Fontana, A. (1999) Involvement of the N-methyl-D-aspartate receptor in neuronal cell death induced by cytotoxic T cell-derived secretory granules. Eur. J. Immunol. 29, 3053–3062 32. Gerber, J., Eiffert, H., Fleischer, H., Wellmer, A., Munzel, U., and Nau, R. (2001) Rifampin reduces the release of DNA from Streptococcus pneumoniae in comparison to spontaneous growth and ceftriaxone treatment. Eur. J. Clin. Microbiol. Infect. Dis. 20, 490–493 33. Nau, R., and Brück, W. (2002) Neuronal injury in bacterial meningitis: mechanisms and implications for therapy. Trends Neurosci. 25, 38–45 Received August 7, 2003; accepted October 30, 2003.
Fig. 1
Figure 1. CpG-DNA toxicity in organotypic cortico-hippocampal slices. Slices were either untreated or treated with 10 µM CpG-DNA for 72 h. Labelling with propidium iodide revealed neuronal damage. Scale bar: 200 µm.
Fig. 2
Figure 2. BDA-traced reduction of neurite length in organotypic cortico-hippocampal slices. A) Anterograde tracing of the entorhinal cortex with biotinylated dextran amine (BDA) demonstrated shortening of neurites in slices treated with CpG-DNA compared with untreated controls. The arrowheads point to neuronal somata. Scale bar: 80 µm. B) Analysis of neurite length traced with BDA. Shortening of neurites was quantified for control slices (untreated) and slices treated with CpG-DNA (CpG treated). Data are presented as mean ± SEM of three independent experiments. (Untreated vs. CpG treated: P
