Supernatants from microglia stimulated via TLR2, TLR4, or TLR9 induce neurotoxic γδ T cells.

<p><b>(A)</b> Microglia were stimulated with Pam3CysSK4 (100 ng/ml) LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone, served as controls. Subsequently, cultures were immunostained with antibodies against CD3 (γδ T cells, green), NeuN, and neurofilament (neurons, red), magnification 100x, scale bar 50 μm. In <b>(C)</b> 1*10⁵ microglia were added to neuronal cultures concurrent with γδ T cells. <b>(D)</b> BMDMs were stimulated with Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. BMDM-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone served as controls. Subsequently, cultures were immunostained as in <b>(A)</b>. <b>(B, C, D)</b> NeuN-positive cells were quantified and expressed as relative neuronal viability. Each condition was performed in duplicate and averaged. Mean ± SEM of 3–5 individual experiments with ANOVA followed by Bonferroni multiple comparison post test, (B) <i>p</i><0.0001, (C) <i>p</i><0.0001, (D) <i>p</i><0.0001.