Figure 5.

Synergistic activation of TLR3 and TLR9 enhances microglial motility via PI3K/Akt signaling. A, Organotypic brain slices were inoculated with GL261mCherry cells, and accumulation of microglia within the tumor areas without treatment and after 120 h treatment with Poly(I:C) +CpG is shown. Microglia were labeled with GFP as MacGreen mice were used (n = 6 per group). Scale bar, 100 μm. B, Quantification of total microglial number within the tumor area of organotypic slices of untreated and Poly(I:C)-, CpG-, and Poly(I:C) CpG-treated slices determined by confocal microscopy with Imaris 3D reconstruction. C, Using the Boyden chamber assay, we determined microglia motility activity by applying 10 µg/ml Poly(I:C), 2 µM CpG, or 10 µg/ml Poly(I:C) + 2 µM CpG into both compartments, and the number of cells migrating through the membrane was quantified. Left, Cells on the membrane were labeled with Diff-Quik kit. Right, Data are quantified as cells/mm² (5 repetitions per n, n = 3 per group). D, Motility was evaluated using the agarose spot assay. Spots and fluid surrounding the spots contained either medium only or 10 µg/ml Poly(I:C), 2 µM CpG, or 10 µg/ml Poly(I:C) + 2 µM CpG. Left, Images from sample spots. Right, Quantification of migrating cells normalized to PBS control. Values are normalized to average migration in control medium (6 repetitions per n, n = 3). E, Protein lysate from cultured microglia treated with 10 µg/ml Poly(I:C), 2 µM CpG, or 10 µg/ml Poly(I:C) + 2 µM CpG for 24 h were analyzed for protein level of phosphor-Akt (p-Akt) and total Akt (t-Akt) by Western blot (n = 3). F, Quantification of p-Akt relative to t-Akt Western blot data as shown in E. G, Quantification of motility using the Boyden chamber of cells treated with 10 µg/ml Poly(I:C) + 2 μm CpG and with and without the PI3K inhibitors LY294002 and wortmannin (5 repetitions per n, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.