Shown in Figure 7 are results from multiple microglial cases,

Shown in Figure 7 are results from multiple microglial cases, Vandetanib hypothyroidism nor malized to the values induced by LPS, PIC or IL 1 IFNg alone. They show that the PI3K Akt pathway is involved in LPS or PIC mediated induction of anti inflammatory cytokine genes, but that it had little or no effect on proinflammatory cytokine mRNA expression. Interestingly, LY294002 suppressed IL 1b protein production, Inhibitors,Modulators,Libraries although it had no significant effect on IL 1b mRNA. As noted before, human microglia responded remarkably similarly to LPS or PIC. The effects of LY294002 on cytokines induced by IL 1 IFNg were different from those observed using TLR ligands. LY294002 had no significant effects on anti inflammatory gene expression, but it had significant stimulatory effects on proinflammatory gene expression, with no change in IL 1b mRNA levels.

Inhibitors,Modulators,Libraries Since these data suggest a possible stimulus dependent role of PI3K in microglial inflammatory gene induction, we next compared PIC and IL 1 IFNg as stimuli in the same microglial case. The role of Ad IRF3 was also deter mined. Microglia were transduced with Ad IRF3 or Ad GFP and further stimulated with PIC or IL 1 IFNg in the presence Inhibitors,Modulators,Libraries or absence of LY294002. The pro duction of IFNb, Inhibitors,Modulators,Libraries IL 8 and IL 1b was determined by ELISA. Measurement of IFNb using a highly sensitive ELISA kit demonstrated that neither PIC nor IL 1 IFNg induced detectable amounts of IFNb from microglia. IFNb was produced when cells were exposed to both Ad IRF3 and immune stimuli. Furthermore, IFNb production was almost completely inhibited by LY294002.

In con trast, LY294002 had no effect Inhibitors,Modulators,Libraries on PIC induced IL 8 pro tein production, but it increased IL 8 production by IL 1 IFNg, suggesting a suppressive role of PI3K Akt in IL 1 IFNg induced IL 8 expression. Furthermore, LY294002 suppressed PIC induced IL 1b protein production, but it increased IL 1 IFNg induced IL 1b protein production. The effect of LY294002 in the presence of Ad IRF3 resembled the results obtained by microarray and Q PCR in Figure 6. For all three cytokines, PIC provided a stronger stimulus than IL 1 IFNg for microglia. Together, our experiments with LY294002 show that the PI3K Akt pathway plays a crucial role in the induc tion of key anti inflammatory and immunomodulatory genes such as IL 1ra, IL 10 and IFNb from microglia. They also show that boosting the amount of IRF3 pro tein in microglia is necessary for adequate IFNb response upon further stimulation with TLR ligands or cytokines. The PI3K Akt pathway plays dual roles in proinflammatory cytokine production from microglia, depending on the nature of the stimuli used to induce cytokines, it plays a suppressive role when cytokines are used as inducing stimuli, but shows little effects when the TLR3 4 ligands are used as stimuli.

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