Table 1.
Ingenuity Pathway Analysis of disease and biomarkers pathways induced by TLR7 stimulation.
Table 2.
Comparison of fold change in mRNA levels between microarray and RT- PCRa.
Table 3.
mRNA expression following TLR9 ligand stimulationa.
Fig 1.
Venn diagram of genes with altered expression in microglia and astrocytes by TLR7 stimulation.
The number of genes significantly (A) induced or (B) down-regulated at 6 hours post stimulation (hps) in microglia (blue circles) or astrocytes (red circle) as determined by microarray analysis, with genes expressed by both cell types displayed as overlapping circles. Microglia had the largest number of genes induced or down-regulated by TLR7 stimulation, with a total of 160 genes upregulated and 60 genes down-regulated. In contrast, only 53 genes were induced in astrocytes and 5 genes down-regulated. All down-regulated genes in astrocytes were also down-regulated in microglia, while 44 of the upregulated genes in astrocytes were also upregulated in microglia. All genes identified as upregulated or down-regulated were changed at least 2-fold compared to mock controls for each cell type and were found to be significantly altered (P <0.05) compared to mock-infected controls. Data is the average of 6 mock and 6 stimulated samples for each cell type.
Fig 2.
Ranking of Genes with expression change in both microglia and astrocytes.
Genes with mRNA that were upregulated or down-regulated in both microglia and astrocytes by at least two-fold were graphed according the average increase between both microglia and astrocytes. Data are the mean fold increase relative to mock-infected samples for each cell type. n = 6 for each group including mock groups. Green bars indicate the relative increase and red bars indicate the relative decrease compared to mock over the range of all upregulated genes with a high value of 52.6 (interleukin 6) and a low value of -8.58 (Rasgrp3). Gene that are cytokines or chemokines are shaded, while genes that were chosen for further analysis by real-time PCR are shown in bold. The rank number is shown on the left side of the gene name.
Fig 3.
Genes with expression change only in microglia and not astrocytes.
Genes whose mRNA expression was upregulated or down-regulated in microglia but not astrocytes were graphed according to their average fold increase in microglia. This data is only a partial list of genes and the full set of genes is shown in S1 Fig. Data for astrocytes are also shown. Data are the mean fold increase relative to mock-infected samples for each cell type. n = 6 for each group including mock groups. Green bars indicate the relative increase over the range of all upregulated genes with a high value of 17.2 (Marco) and a low value of -8.90 (Rgs2). Gene that are cytokines or chemokines are shaded, while genes that were chosen for further analysis by real-time PCR are shown in bold. The rank number is shown on the left side of the gene name.
Fig 4.
Genes with expression change in astrocytes and not microglia.
Genes whose mRNA expression was upregulated or down-regulated in astrocytes but not microglia were graphed according to their average fold increase in astrocytes. Data are the mean fold increase relative to mock-infected samples for each cell type. n = 6 for each group including mock groups. Green bars indicate the relative increase over the range of all upregulated genes with a high value of 4.55 (Ccl2) and low value of 2.08 (Plek). Gene that are cytokines or chemokines are shaded, while genes that were chosen for further analysis by real-time PCR are shown in bold. The rank number is shown on the left side of the gene name.
Fig 5.
Real-time PCR analysis of mRNA expression in microglia and/or astrocytes following TLR7 or TLR9 stimulation.
RNA samples from mock, TLR7 or TLR9-stimulated microglia or astrocytes were analyzed at 6 hps for mRNA expression of genes that were found by microarray to be (A-D) induced in microglia only, (E-H) induced in both microglia and astrocytes, or (I-L) induced in astrocytes only. Data are shown as the fold induction relative to mock-stimulated controls for each cell type (average of 6 mock-stimulated samples). Data are the mean +/- SEM for 3–6 samples per group and are representative of two separate experiments. Statistical analysis was conducted using a one-way ANOVA to determine differences between microglia and astrocytes for each stimulation. * P<0.05, ** P<0.01, *** P<0.001.
Fig 6.
Real-time PCR analysis of mRNA expression of selected genes in brain tissue of mice with viral encephalitis.
Brain tissue from mice with signs of neurological disease following infection with MuLV or LACV was processed for RNA. Age-matched and strain-matched controls for each virus infection were processed at the same time as viral infection and are shown as controls for the respective viruses. RNA was then analyzed for expression of mRNAs of genes identified as being induced following TLR activation of microglia and/or astrocytes. Data are the mean +/- SEM of 3–6 mice per group and are shown as the fold change relative to the average mock sample for each group. Statistical analysis was completed by unpaired t test between the virus-infected brain tissue and the appropriate mock-infected control. P<0.05, ** P<0.01, *** P<0.001.