Treatment with 1,25(OH)₂D₃ reduced EAE-induced expression of TLR8 in the spinal cord.

<p>(<b>A</b>) Real-time RT-PCR analysis of expression of the key TLRs in the spinal cord (Prevention model, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058808#pone-0058808-g001" target="_blank">Figure 1</a> legend for 1,25(OH)₂D₃ treatment scheme). The mRNA levels of TLR3 and TLR8 were significantly increased in EAE spinal cords. 1,25(OH)₂D₃ only significantly reduced TLR8 mRNA level. Data shown are the means ± SEM (n = 4) from one of two independent experiments. (<b>B</b>) Real-time RT-PCR analysis of expression of the key TLRs in the spinal cord (Treatment model). Mice were injected with 1,25(OH)₂D₃ beginning at day 7 post-MOG injection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058808#s2" target="_blank"><i>Material and Methods</i></a>). Expression of all 4 TLRs analyzed were significantly induced by EAE and reduced by 1,25(OH)₂D₃ treatment. Data shown are the means ± SEM (n = 4) from one of two independent experiments. In both treatment and prevention models, TLR8 mRNA level showed the highest magnitude of induction by EAE and was significantly down-regulated by 1,25(OH)₂D₃ treatment. * <i>P</i><0.05; ** <i>P</i><0.01. (<b>C</b>) Representative TLR8 immunostaining of spinal cord sections. Animal treatments were described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058808#pone-0058808-g001" target="_blank">Figure 1</a> legend. Three mice were analyzed in each group. The magnification was X10 (upper panels). (<b>D</b>) FACS analysis of intracellular TLR7 and 8 expressions in the spinal cord in both models. Mononuclear cells were isolated using pooled spinal cords from 2–3 mice each group by Percoll gradients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058808#s2" target="_blank"><i>Material and Methods</i></a>). Data are representative of two independent analyses.