Entire microarray data sets are available as Supplementary Material. In PIC treated cul tures, IRF3 enhanced genes included IFNb, IL 29, IRF7, an inducible transcription factor which synergizes with IRF3, many ISGs, TLR7, a TLR shown to mediate antiviral and anti inflammatory Y27632 func tions in myeloid cells, and IL 10 receptor. Intrigu ingly, IL 1a and IL 1ra, as well as the IL 12 family cytokines IL 23 and IL 27 were differentially regu lated, showing increase in IL 1ra and IL 27 and decrease in IL 1a b and IL 23. These results suggest that Ad IRF3 can suppress the Th1 Th17 activation pathway and promote the Th2 pathway in microglia. Similar trends were observed in IL 1 IFNg treated microglial cultures, i.

e, downregulation Inhibitors,Modulators,Libraries of proinflammatory cytokine genes such as IL 1a, IL 1b, IL 8 and CXCL1, but upregulation of anti inflammatory genes, antiviral genes and ISGs, such as IL 1ra, IL 10, IL 10 receptor, IFNb, IFIT1, IRF7, sup pressor of cytokine signaling 1 and IL 27. The microarray data show that microglial inflamma tory and antiviral genes are differentially regulated in the presence of increased IRF3 protein expression, and that the responses are similar regardless of the stimuli used. Q PCR validation of the Ad IRF3 effects in microglial inflammatory gene expression We also employed Q PCR to examine microglial gene expression following exposure to Ad IRF3. Inhibitors,Modulators,Libraries Figure 2 shows a typical experiment in which microglial cultures derived from a single case were tested in six different conditions, uninfected, Ad GFP infected or Ad IRF3 infected, each with or without treatment with IL 1 IFNg.

Selected genes were examined based on the microarray data, and the results showed that antiviral and anti inflammatory genes such as IFNb, IFIT1 and IL 1ra were robustly upregulated by Ad IRF3, and proinflammatory genes Inhibitors,Modulators,Libraries such as IL 1b, IL 8 and TNFa were downregulated by Ad IRF3. Q PCR validation of the Ad IRF3 effects in multiple Inhibitors,Modulators,Libraries microglial cases We have analyzed data from microglial cultures derived from multiple donors in order to determine whether the Ad IRF3 effects are significant across many cases. Q PCR data were compiled from several microglial cases treated with IL 1 IFNg and grouped into significantly upregulated and downregulated genes, based on single sample t test. Ad IRF3 upregulated genes are shown in Figure 3A as ratios of gene expression in Ad IRF3 culture to Ad GFP culture in a log 10 scale.

Ad IRF3 downregulated genes are shown in Figure 3B as % inhibition, calculated by 100 ��. These results once again confirm that the two groups of genes Inhibitors,Modulators,Libraries are dif ferentially regulated by Ad IRF3 in microglia. Ad IRF3 effects kinase inhibitor EPZ-5676 on microglial cytokine protein production We next performed Luminex multiplex beads based protein analyses of IL 1 IFNg stimulated microglia to determine whether the Ad IRF3 induced mRNA changes are reflected at the protein level.