LCMV effects are mediated via TLR2/MyD88/Mal; CNS primary glial cells (passage 2 to 3) were challenged with LCMV and other TLR ligands for 18-24 hours in the presence of Brefeldin A at a concentration of 1 μg/ml. Cells were first stained with CD11b, which was used to distinguish microglial cells (CD11b positive subset) from astrocytes (CD11b negative subset). The intracellular expression of MCP-1 and TNF-α was determined by double staining with antibodies against mouse MCP-1 or TNF-α (Fig 2B). The expression of CD11b was used to distinguish microglial cells (CD11b⁺) from astrocytes (CD11b^-). The percentage of each subset of cells expressing either MCP-1 (A) or TNF-α (B) was gated, analyzed and shown as bar graphs. Representatives of the FACS analysis were shown in the bottom panels of Fig 2a, 2b. LCMV-infected primary microglial cells were stained with antibodies against LCMV-NP, CD11b, and TNF-α. Cells were first gated on LCMV⁺CD11b⁺ and LCMV⁺CD11b^- subsets of cells. The production of TNF-α by these two populations was separately gated (C). TLR3 and/or TLR4 KO primary mixed glial cells were included as TLR specificity controls. Values on the upper right quadrant represent the percentage of glial cells producing cytokines. One representative of at least five experiments is shown. (D) Confocal Microscopy: Cells were treated as described in the text, fixed with Paraformaldehyde, permeabilized and stained with anti-CD11b antibody (a marker for microglial cells) and anti-GFAP antibody (as a marker for astrocytes). Additionally, cells were incubated with anti-TNF-α antibodies for intracellular TNF production and Hoechst 33258 to stain nuclei.

(A) WT and MyD88 KO primary microglial cells were enriched with CD11b microbeads and challenged with LCMV. Cells were separately stained with antibodies against LCMV together with either TNF-α or MCP-1. Values on the upper right quadrant represent the percentage of microglial cells producing TNF-α; (B) Immortalized microglial cells were challenged with LCMV and other TLR ligands as described above. Cells were first stained with CD11b and then subjected to the intracellular staining of MCP-1 and TNF-α. Values on the upper left quadrant represent the percentage of microglial cells producing TNF-α; Values on the upper right quadrant represent the percentage of microglial cells producing both TNF-α and MCP-1; Values on the low right quadrant represent the percentage of microglial cells producing MCP-1. One representative of at least five experiments is shown.

Immortalized WT and TLR2 KO microglial cells were seeded into 24-well plates and stimulated with LCMV and other TLR ligands for 18-24 hours as indicated. The expression of MHC-class I (A, bottom panel), class II (A, top panel), CD40 (B, top panel), CD86 (B, bottom panel) was analyzed by FACS staining. One representative of at least three experiments is shown.

A: Primary CNS mixed glial cells were surface-stained with CD11b and intracellularly stained with anti-GFAP antibody. B: Primary CNS glial cells (passage 2) were challenged with live LCMV-Arm or UV-inactivated LCMV-Arm for 18-24 hours. As controls, cells were treated with medium, mouse recombinant TNF-α (TLR-independent), Pam2 (TLR2 ligand) and poly:IC (TLR3 ligand). The levels of MCP-1 and RANTES in the supernatants were measured by ELISA. One representative of at least five experiments is shown.

LCMV-infected primary mixed glial cells were challenged with LCMV as described in Fig 2 and stained with antibodies against LCMV-NP together with either MCP-1 (A) or TNF-α (B). Values on the upper right quadrant represent the percentage of glial cells producing cytokines.

A: Primary CNS glial cells were challenged with LCMV and various controls for 18-24 hours. IL-1R1 KO primary glial cells were included as a control. The expression of TLR2 was measured by flow cytometry. Cells were gated on CD11b negative cells. B: Immortalized WT and MyD88 KO microglial cells were seeded into 24-well plates and challenged with LCMV as well as various controls for 18-24 hours. The expression of TLR2 was determined by flow cytometry. One representative of at least three experiments is shown.