We next expanded the study to compare the responses to different stimuli in the same microglial cases, and examined the production of IL 1b, IL 1ra, IL 8 and IP 10 by individual ELISA. The results http://www.selleckchem.com/products/BIBF1120.html show that the amounts of proinflam matory cytokines such as IL 1b and IL 8 were markedly decreased by Ad IRF3, while the amounts of IL 1ra and IP 10 were increased. These results confirm that Ad IRF3 differen tially regulates microglial cytokine production, regard less of the types of stimuli applied. Ad IRF3 activates the PI3K Akt pathway in microglia In order to determine the mechanism by which Ad IRF3 mediates its effects on microglial cytokine expression, we examined cell signaling pathways altered by Ad IRF3 by western blot analysis.
Three different cases of microglial cultures were transduced with Ad IRF3 or Ad GFP for 48 h, and were subjected to western blot analysis for p Akt, p Erk, p Jnk, and Inhibitors,Modulators,Libraries total Akt. Figure 5A demonstrates Inhibitors,Modulators,Libraries a representative western blot and Figure 5B demonstrates densitometric analysis normalized to the control level from three microglial cases. The results show that the levels of p Akt increased in the presence of Ad IRF3, whereas those of p Erk or p Jnk were unchanged. Role of the PI3K Akt pathway in Ad IRF3 mediated modulation of microglial gene expression In order to determine whether pAkt contributed to Ad IRF3 Inhibitors,Modulators,Libraries mediated modulation of microglial gene expres sion, we employed a pharmacological inhibitor of PI3K, LY294002. Microglial cultures were transduced with Ad IRF3 or Ad GFP then stimulated with IL 1 IFNg in the presence or absence of LY294002, as described in the Methods.
The results were examined by microarray and also by Q PCR. In Figure 6A, gene expression ratios were expressed as % change, in which 0 represents no change, 100% represents two fold increase, and 50% represents 50% inhi bition. Inhibitors,Modulators,Libraries The results showed that the PI3K inhibitor exhibited differential effects on the expression of the two groups of genes, i. e, suppression of Ad IRF3 Inhibitors,Modulators,Libraries induced genes and increase of Ad IRF3 inhibited genes. The complete micro array data set is available as Supplemental Material. These results are validated by Q PCR. Figure 6B and 6C demonstrate Q PCR data derived from several microglial cases, shown as normalized values. They confirm that LY inhibited Ad IRF3 upregulated genes while increasing Ad IRF3 inhibited genes.
However, the effect of LY on IL 1b mRNA expression was not significant, reflecting the results obtained with microarray. Taken together, these results demonstrate that the PI3K Akt pathway significantly contributes to the differential gene regulation induced by Ad IRF3 in microglia. The role of the PI3K Akt pathway sellckchem in microglial inflammatory gene expression Because our data suggest a major role of PI3K Akt in Ad IRF3 mediated immune modulation, we next examined the effect of LY294002 on microglial cytokine gene induc tion by TLR activation or IL 1 IFNg.