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Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9) ¹

ASPAROUH I. ILIEV^*,2, ARGYRIOS K. STRINGARIS^,2, ROLAND NAU^,3 and HARALD NEUMANN^*

^* Neuroimmunology Unit, European Neuroscience Institute Göttingen, Göttingen, Germany; and
Department of Neurology, University of Göttingen, Göttingen, Germany

³Correspondence: Department of Neurology, University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. E-mail: rnau{at}gwdg.de

SPECIFIC AIMS

The extent of neuronal injury determines the clinical outcome in inflammatory brain diseases, including multiple sclerosis, viral or bacterial infection and in stroke. Unmethylated CG dinucleotide motif rich DNA is released from invading microorganisms and possibly from damaged brain cells during cytotoxic disease processes. This study was designed to determine whether activation of microglial cells by unmethylated CG dinucleotide motif rich DNA via toll-like receptor-9 (TLR9) leads to the release of neuronotoxic products and can contribute to neuronal injury.

PRINCIPAL FINDINGS

1. Unmethylated DNA induces neuronal injury in cortico-hippocampal slice cultures
Cortico-hippocampal slice cultures containing the hippocampus and the adjacent neocortex were treated with unmethylated CpG-DNA oligonucleotides, which mimic bacterial unmethylated CG dinucleotide motif rich 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 labeling of living slices with propidium iodide. The labeling indicated neuronal damage after exposure to 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, while neurite length in slices treated with 10 µM CpG-DNA for 72 h was decreased [average total neurite length 96 µm±24 µm vs. 243 µm±46 µm (mean±SEM)].

2. Microglial cells express TLR9 and produce nitric oxide and tumor necrosis factor- after stimulation with unmethylated DNA
To study the brain cell type responding to CpG-DNA challenge, TLR9 gene transcripts were assessed by RT-PCR in microglial cells isolated from postnatal hippocampal tissue and enriched to a purity of more than 96%. TLR9 gene transcripts were detected after PCR amplification in untreated microglia and microglial cell preparations treated for 24 h with 10 µM CpG-DNA. No PCR product for TLR9 was detected in cultured hippocampal neurons.

Challenge of microglia with either 1µM or 10 µM CpG-DNA for 24 h led to prominent induction of both tumor necrosis factor- (TNF-) and inducible nitric oxide synthase (iNOS) gene transcripts. No gene transcripts for TNF- and iNOS were detected in untreated microglia. Furthermore, CpG-DNA treatment for 24 h led to a strong secretion of TNF- as determined by enzyme-linked immunosorbent assay (ELISA).

3. CpG-DNA-induced damage to hippocampal neurons is mediated via microglia and starts at neurites
Treatment with 10 µM CpG-DNA for 72 h was not toxic to cultured neurons (Fig. 1 A). Unstimulated microglia had no cytotoxic effects on neurons after 72 h of co-culture (Fig. 1B ). However, strong neurotoxicity occurred after treatment of co-cultures with 10 µM CpG-DNA for 72 h (Fig. 1B ). 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. During distal degeneration of neurites, cell bodies showed no signs of damage. 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.

View larger version (55K): [in this window] [in a new window]   Figure 1. CpG-DNA induced neurotoxicity. Neurons were labeled with ß-tubulin III antibody (green) and microglia with isolectin B4 (orange). A) Neurons were either untreated or treated with CpG-DNA. Please note the absence of injury in neurons cultured alone and stimulated with CpG-DNA. Scale bar: 40 µm. B) Neurons and microglia were co-cultured and either untreated or treated with CpG-DNA. There was apparent neurite damage and a strong reduction of the number of neurites and neuronal somata in microglia-neuron co-cultures treated with CpG-DNA. Scale bar: 40 µm.

4. TNF- and nitric oxide are CpG-DNA-induced microglial effectors of damage to hippocampal neurons
Since nitric oxide (NO) and TNF- 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. 2 A). Co-cultures were then treated with the iNOS inhibitor aminoguanidine (200 µM) starting two hours prior to stimulation with 10 µM CpG-DNA for 72 h. Blockade of iNOS protected neurons from microglial CpG-DNA toxicity (Fig. 2B ). Incubation of co-cultures with TNF-R1 fusion protein (10 µg/ml) partially protected neurites from challenge with 10 µM CpG-DNA for 72 h (Fig. 2b) . Conversely, treatment of co-cultures with the glutamate (NMDA) receptor antagonist MK-801 (10 µM) did not prevent toxicity of CpG-DNA substantially (Fig. 2B ).

View larger version (30K): [in this window] [in a new window]   Figure 2. CpG-DNA-induced neurotoxicity is mainly mediated via microglial NO and TNF-. A) Density of neurites and neuronal cell bodies was analyzed after immunohistochemistry with antibodies directed against ß-tubulin III and isolectin B4 labeling of microglia. Neurons were cultured alone or co-cultured with microglia and left untreated or treated with CpG-DNA. Data are presented as percentage of untreated controls (mean±SEM of five independent experiments). B) Density of neurites and neuronal cell bodies was analyzed after immunohistochemistry with antibody against ß-tubulin III and isolectin B4 labeling of microglia. Co-cultures of microglia and neurons were treated with CpG-DNA. Neurite degeneration and neuronal cell death in response to CpG-DNA-stimulated microglia (CpG) was prevented by blocking of TNF- with recombinant human TNF receptor I-IgG1 (TNFR1-IgG) and inhibition of iNOS by aminoguanidine (Amino). The glutamate antagonist MK801 showed no significant protection. Data are presented as percentage of untreated controls (mean±SEM of five independent experiments); * P < 0.05 and ** P < 0.01 analyzed by one-way ANOVA with Bonferroni correction for repeated testing.

CONCLUSIONS AND SIGNIFICANCE

Toll-like receptors (TLRs) play an essential role in the innate recognition of "pathogen-associated molecular patterns" and trigger the protective immune response. During inflammatory reactions, TLR gene transcripts are up-regulated in the central nervous system (CNS). Stimulation of TLRs may be one of the major triggers for neurodegeneration in the CNS. Bacterial DNA has the capacity to induce sustained inflammation via stimulation of TLR9. Furthermore, self-DNA immunoglobulin complexes have been suggested to trigger autoimmunity via stimulation of TLR9. We show that CpG-DNA, which stimulates TLR9, causes neuronal and neurite injury in the presence of brain innate immune cells, but not in neurons cultured alone. In organotypic cortico-hippocampal slices, CpG-DNA induced neuronal and neurite injury in the dentate gyrus, stratum radiatum of the CA1 area and entorhinal cortex. Direct visualization of neurites by tracing with BDA demonstrated selective neurite loss in the entorhinal cortex, confirming axonal degeneration following CpG-DNA treatment. In microglial-neuronal co-cultures, time-lapse imaging 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 and autoimmune diseases of the CNS. Microglial cells ensheath neurites in inflammatory cortical lesions, and the number of activated microglia and macrophages positively correlate with the extent of axonal damage in multiple sclerosis. Neurotoxicity of microglia was observed in vitro following stimulation with amyloid-ß protein, lipopolysaccharide (LPS) and chromogranin A.

The neurotoxic mechanisms of microglia involve generation of NO, other reactive oxygen species, and TNF- and IL-1ß. We demonstrate that microglial cells express TLR9, and up-regulate gene transcripts for the enzyme NO synthase and cytokine TNF- in response to CpG-DNA treatment. Neurotoxicity of CpG-DNA was mainly mediated via release of NO and TNF- from microglia as determined by application of specific inhibitors.

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. 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. Recently, self-DNA immunoglobulin complexes have been shown to contribute to autoimmune disease by stimulation of TLR9. Therefore, it is tempting to speculate that unmethylated CG dinucleotide rich self-DNA released during autoimmune inflammation of the CNS may promote microglial stimulation and subsequent axonal damage as observed in multiple sclerosis lesions.

In conclusion, these data demonstrate for the first time that stimulation of TLR9 triggers extensive neurotoxicity, mainly directed against neurites. The study shows that neurotoxicity of unmethylated DNA is mediated via stimulation of TLR9 and microglial production of TNF- and NO (Fig. 3 ). The study supports the up-and-coming concept of stimulation of Toll-like receptors as being one of the major triggers for neurodegeneration in the central nervous system thereby linking innate immunity and neuronal injury.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0670fje

² These authors contributed equally to this work.

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