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-alpha (TNF-alpha). Blockade of NO synthase and TNF-alpha 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-alpha is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury.
Subject(s)
DNA-Binding Proteins/metabolism , Microglia/metabolism , Neurons/metabolism , Neurons/pathology , Receptors, Cell Surface/metabolism , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , CpG Islands/genetics , DNA/genetics , DNA/toxicity , DNA Methylation , Hippocampus/drug effects , Hippocampus/pathology , In Vitro Techniques , Microglia/cytology , Microglia/drug effects , Models, Biological , Neurites/drug effects , Neurites/metabolism , Neurites/pathology , Neurons/drug effects , Nitric Oxide/metabolism , Oligonucleotides/genetics , Oligonucleotides/toxicity , Toll-Like Receptor 9 , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Neuronal injury in bacterial meningitis is caused by the interplay of host inflammatory responses and direct bacterial toxicity. We investigated the mechanisms by which pneumolysin, a cytosolic pneumococcal protein, induces damage to neurons. The toxicity after exposure of human SH-SY5Y neuroblastoma cells and hippocampal organotypic cultures to pneumolysin was time- and dose-dependent. Pneumolysin led to a strong calcium influx apparently mediated by pores on the cell membrane formed by the toxin itself and not by voltage-gated calcium channels. Buffering of intracellular calcium with BAPTA-AM [1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid tetra(acetomethoxyl) ester] improved survival of neuronal cells following challenge with pneumolysin. Western blotting revealed increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) as early as 30 min after challenge with pneumolysin. SB 203580, a potent and selective inhibitor of p38 MAPK, rescued human neuronal cells from pneumolysin-induced death. Inhibition of the mitochondrial permeability transition pore using bongkrekate and caspase inhibition also improved survival following challenge with the toxin. Modulation of cell death pathways activated by pneumolysin may influence the outcome of pneumococcal meningitis.
