TLR7-deficient C57BL/6 mice 
were kindly provided by S. Akira (Osaka University, Osaka, Japan) and were backcrossed with Inbred Rocky Mountain White (IRW) mice for at least 10 generations 
. IRW mice and TLR7-deficient IRW mice were used for the present study. Mice were genotyped to confirm TLR7 knockout allele as previously described 
. Unc93b1 3D mice were obtained from the Mutant Mouse Regional Resource Center at University of California, Davis. All of the animal procedures were approved by and conducted in accordance with the Louisiana State University Animal Care and Use Committee guidelines or the Rocky Mountain Laboratories Animal Care and Use Committee guidelines under protocols LSU06-120 and RML2008-46.
Primary cortical cultures
Primary mixed cortical cultures were generated from the cortical tissue of 1- to 2-day-old neonatal mice. Intact brains were removed and dissected free of meninges. The midbrain and cerebellum were removed and the cortices were placed in 2% glucose/PBS and gently triturated using a 10 ml pipet. Cells were pelleted at 244 g for 5 min, and a single-cell suspension was made by triturating with a 1-ml syringe and 20 gauge needle. Cells were cultured in 25-cm2 Primaria tissue culture flasks (BD Bioscience) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units penicillin and streptomycin and maintained at 37°C, 5% CO2. When cultures reached 90 to 95% confluency in the flask, they were treated with 0.25% Trypsin, 1 mM EDTA in HBSS (0.25% Trypsin-EDTA, Gibco) for 5 minutes, harvested and replated in 12-well plates for agonist stimulation or 6-well plates for flow cytometry analysis. Immunohistochemical analysis of these cultures demonstrated that the primary cell type was astroglia, but also contained a number of microglia. Detection of neurons and other cells was rare.
Astrocyte and microglia cultures
For the generation of astrocyte cultures and for analyzing TLR7 and TLR8 expression in astrocytes and microglia, single cell suspensions were generated as described above. However, following trituration, cells were suspended in 2 ml of 70% percoll and transferred to the bottom of 30%, 0% step percoll gradient. The gradients were centrifuged at 500 g for 20 min. The microglia cell population was collected between 30% and 70% percoll layers, washed in PBS, and seeded at 5×105 in Primaria T-25 flasks containing DMEM with 10% FBS and 20% LADMAC culture supernatant (mouse bone marrow cells producing macrophage colony stimulating factor/M-CSF). The astrocyte rich cell population was collected between 0% and 30% percoll layers, washed in PBS, and seeded at 2×105 cells in Primaria T-25 flasks containing DMEM with 10% FBS. When cells reached confluency (7–10 days), flasks containing astrocyte rich cells (0/30 fraction) were orbitally shaken overnight at 250 RPM to remove any remaining microglia as well as any oligodendrocytes. Astrocytes were removed from the flasks by treating with 0.25% Trypsin-EDTA (Gibco), and microglia were removed from confluent T-25 flasks using a cell scraper for use in flow cytometric analysis. Astrocyte cultures were greater than 95% GFAP positive and microglia were greater than 95% F4/80 and Iba1 positive.
When primary cortical cultures were approximately 80% confluent, they were used for agonist stimulation. The TLR7/8 agonist CL075, a thiazoloquinoline compound also known as 3M002 (Invivogen, San Diego, CA) was prepared in endotoxin-free water, aliquoted at stock concentrations of 20 mM and was thawed only once prior to use. A 20-mer of pT-ODN (Invitrogen) was prepared in endotoxin-free water, aliquoted at stock concentrations of 2 mM and was thawed only once prior to use. Cultures were stimulated with optimal concentrations of 20 µM of CL075, and 1–5 µM of pT-ODN depending on experiment. In costimulation experiments, 1 µM was optimal in cultures generated from IRW mice, whereas 5 µM was optimal in cultures from C57BL/6 mice.
Cells were analyzed for TLR7 and TLR8 protein expression by intracellular staining. Cells were fixed for 20 min in 2% paraformaldehyde, permeabilized with 0.1% saponin in PBS (pH 7.0), and then incubated with rat anti-mouse CD16/CD32 antibodies (BD Biosciences) to block non-specific antibody binding to Fc receptors. Cells were then incubated with polyclonal rabbit anti-TLR7 antibody (Zymed), goat anti-TLR8 antibody (Capralogics), polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Dako), mouse anti-F4/80 antibody (eBioscience), rabbit anti-Iba1 antibody (Wako Inc.) or isotype control antibody overnight at 4°C. The next day, cells were incubated with the relative secondary antibody conjugated to AlexaFluor 488 (Invitrogen) in 0.1% saponin/PBS. Cells were washed twice with PBS, resuspended in 3% BSA in PBS, and analyzed on a FACSAria flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences). Data analyses were performed using FCS3 Express software (De Novo). GFAP, F4/80 and Iba1 antibodies were used to confirm cell purity.
Primary cortical cultures in 8-chamber culture slides (BD Biosciences) at 70–80% confluency were used for immunocytochemical analysis. Cells were fixed in 4% para-formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and 0.1% Sodium citrate for 30 min, and blocked using normal donkey serum blocking solution (PBS containing 2% donkey serum, 1% BSA, 0.1% cold fish skin gelatin, 0.1% Triton X-100, and 0.05% Tween 20) for 30 min. Cells were then incubated overnight at 4°C with the appropriate concentration of GFAP, Iba1 or F4/80 in the normal donkey serum blocking solution. Cells were stained using antibody concentrations stated above for flow cytometric analysis. Cells were then incubated with the relative secondary antibody conjugated to AlexaFluor 488 (Invitrogen). The slides were covered with a glass coverslip using Fluoro-Gel II with DAPI mounting solution (Electron Microscopy Sciences). Images were pseudocolored and overlaid using Olympus MicroSuite FIVE or Nikon Elements NIS Basic Research software.
Analysis of mRNA Expression by Real-time PCR
Total RNA was extracted from primary cell cultures at 48 hps using an RNA isolation kit (Zymo Research) following manufacturer's instructions. RNA was treated with DNaseI for 30 minutes and re-purified using an RNA clean-up kit (Zymo Research). cDNA was generated from RNA samples using an iScript reverse transcription kit (Bio-Rad) following manufacturer's instructions. cDNA was diluted 5 fold in RNase-free water prior to use in real-time PCR. Primers for real-time PCR analysis are shown in . All primers used for real-time PCR analysis were designed using Primer3 software 
. Primer sequences were blasted against the National Center for Biotechnology Information (NCBI) database to confirm that all primer pairs were specific for the gene of interest and that no homology to other genes was present. PCR reactions were prepared using SYBR green mix with Rox (Bio-Rad) in a 10 µl volume with approximately 10 ng of cDNA and 1.8 µM forward and reverse primers. Samples were run in triplicate on an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Analysis of dissociation curves was used to confirm the amplification of a single product for each primer pair per sample. Confirmation of a lack of DNA contamination was achieved by analyzing samples that had not undergone reverse transcription. Untranscribed controls had at least a 1,000 fold lower expression level than analyzed samples or were negative for all genes after 40 cycles. Gene expression was quantified by the cycle number at which each sample reached a fixed fluorescence threshold (CT). To control for variations in RNA amounts among samples, data were calculated as the difference in CT values (log2) between the housekeeping gene, Gapdh,
and the gene of interest for each sample (ΔCT
− CT gene of interest). Data were calculated as a percentage of Gapdh
expression for each gene of interest per sample. These data were then calculated as fold expression relative to the average of mock samples for each gene and each group.
Primers used for real-time RT-PCR analysis.
Analysis of Cytokine And Chemokine Protein Expression by Multiplex Bead Array
At 48 hps, supernatants from primary cortical cultures were collected and stored at −80°C. Just before use, supernatants were thawed to room temperature. Supernatants were analyzed for cytokine and chemokine proteins using a 20-plex multiplex bead array (BioSource) on a Luminex 100 instrument (Bio-Rad) following manufacturer's instructions. The cytokines analyzed were CCL2 (MCP-1), CCL3 (MIP-1α), CXCL9 (MIG), CXCL10 (IP-10), fibroblast growth factor (FGF), granulocyte monocyte colony stimulation factor (GM-CSF), interferon gamma (IFN-γ), Interleukin-1α (IL-1 α), IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-13, IL-17, CXCL1 (KC), TNF, and vascular endothelial growth factor (VEGF). Data were calculated as pg/ml using a standard curve from in-plate standards. CCL2 (MCP1), IFNα and IFNβ protein levels in culture supernatants were measured using cytokine-specific ELISA assays (R&D Systems, Minneapolis, MN) following manufacturer's instructions.
Agonist uptake assay
Glial cells were grown in 96-well plates until over 80% confluent. Cells were stimulated with mock, 1 µM pT-ODN, 20 µM rhodamine-labeled CL264 or 20 µM rhodamine-labeled CL264 and 1 µM pT-ODN. Cells were then incubated at 37°C and 5% CO2 for 30 min, 1 hr or 3 hr. At each time point, cells were washed three times with PBS and analyzed for rhodamine uptake. Cells were then lysed in lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris HCl, pH 7.4 and 8 mM EDTA) to release fluorescence into solution, and the fluorescence intensity was quantitated using a microplate reader (Polar Star Omega, BMG Labtech).
Cellular localization assay
For confocal microscopy analysis, glial cells were grown on Lab-Tek® II Chamber # 1.5 coverglasses and then stimulated with mock, 1 µM pT-ODN, 20 µM rhodamine-labeled CL264 or 20 µM-rhodamine labeled CL264 combined with 1 µM pT-ODN. Lyso-tracker green at 5 µM was added to each well at the same time. After 30 min incubation at 37°C and 5% CO2, cells were washed with fresh media two times and kept in 0.1 M NH4Cl in media. All images were taken using a Zeiss 510 Meta Confocal Microscope.
All of the statistical analyses were performed using Graph Pad Prism software (San Diego, CA) using the appropriate statistical test as described in the figure legends.