Poly(I:C) promotes TNFα/TNFR1-dependent oligodendrocyte death in mixed glial cultures

Background: Activation of glial cells via toll-like receptors (TLRs) and other intracellular pathogen recognition receptors promotes the release of potentially toxic acute phase reactants such as TNF alpha and nitric oxide into the extracellular space. As such, prolonged glial activation, as is thought to occur during a persistent viral infection of the CNS, may contribute to both neurodegeneration and demyelination. However, the effects of virus-induced glial activation on oligodendrocytes are not fully understood. Method: To determine the effects of glial activation on oligodendrocyte viability we treated primary glial cultures isolated from neonatal rats or mice with the RNA viral mimic poly(I:C) and in some cases other TLR ligands. TLR3 expression was determined by western blot. Cytokine levels were measured by RT-PCR, ELISA, and intracellular cytokine staining. Oligodendrocyte precursor (preOL) viability was determined by Alamar blue assays and immunocytochemistry. Result: Stimulation of mixed glial cultures with poly(I:C) resulted in microglia activation, TNF alpha production and preOL toxicity. This toxic effect of poly(I:C) was indirect as it failed to affect preOL viability in pure cultures despite the fact that preOLs express TLR3. Poly(I:C)-induced loss of preOLs was abolished in TNF alpha or TNFR1 deficient mixed glial cultures, suggesting that TNF alpha/TNFR1 signaling is required for poly(I:C) toxicity. Furthermore, although both microglia and astrocytes express functional TLR3, only microglia produced TNF alpha in culture. Consistent with these findings, other TLR agonists similarly triggered TNF alpha production and preOL toxicity in mixed glial cultures. Conclusion: Activation of microglia by poly(I:C) promotes TNF alpha/TNFR1-dependent oligodendroglial cell death. These data indicate that during an ongoing viral infection of the CNS, microglial TNF alpha may be detrimental to oligodendrocytes.