IL 6 production in glia induced by treatment with LPS plus IFN was greatly reduced by downregulation of STAT3 expression by siRNA, by pharmacological inhibition with the STAT3 spe cific inhibitor JSI 124, or DAPT secretase solubility with the JAK2 inhibitor AG490, neither of which impaired cell viability. These findings indicate that STAT3 may not only propagate the IL 6 sig nal after sepsis but also contributes to inflammation induced IL 6 production in glia. IL 6 production is strongly dependent on GSK3 Since STAT3 activation requires active GSK3, systemic IL 6 production is controlled by GSK3, and GSK3 is abundantly expressed in the brain, we tested if GSK3 con trols IL 6 production by astrocytes and microglia, first with the selective inhibitor lithium using a cytokine array.

Among the 12 molecules detected to be induced by treatment with LPS plus IFN in glia, the production of Inhibitors,Modulators,Libraries TIMP 1 and VCAM 1 were independent of GSK3, whereas active GSK3 decreased the expression of CXCL2 MIP 2 and increased the expression of CXCL1 KC, IL 12p40, CCL9 MIP 1, CCL2 MCP 1, P Selectin and CCL5 RANTES. However, most promi nently, active GSK3 promoted IL 6 production by 16 fold, showing that IL 6 production by glia is strongly depend ent on GSK3. Confirmation by ELISA showed that inhibi tion of GSK3 with lithium treatment reduced the time dependent production of IL 6 induced by LPS and its potentiation by IFN in primary astrocytes. The dependency of IL 6 production in primary astrocytes on GSK3 was confirmed by the strong inhibition exerted by four other structurally diverse GSK3 inhibitors on IL 6 produced in response to LPS or to a combination of LPS plus IFN, which was not due to changes in cell viability.

Inhibition of GSK3 also strongly reduced IL 6 production in primary microglia. siRNA mediated knockdown by 75% Inhibitors,Modulators,Libraries of GSK3, but not of GSK3, greatly reduced the production of IL 6 by pri mary enriched astrocytes induced by LPS and its potentia tion by IFN, indicating Inhibitors,Modulators,Libraries that GSK3 predominantly regulates IL 6 production. GSK3 and STAT3 are sufficient to stimulate IL 6 production in glia Since both active STAT3 and active GSK3 were Inhibitors,Modulators,Libraries necessary for IL 6 production, the expression of active forms of each was introduced using adenoviruses in primary enriched astrocytes to examine their effects on IL 6 production.

These Inhibitors,Modulators,Libraries included STAT3C, in which covalent bonds between cysteines establishes the active dimer conforma tion, and constitutively active S9A GSK3 and S21A GSK3, where serines subject to inhibitory phosphoryla tion were mutated to alanines to prohibit inhibitory ser ine phosphorylation. Expression of active S21A GSK3 and S9A GSK3 together increased IL 6 production and this was increased a further 4. 5 fold with co expression of STAT3C. This demonstrates that GSK3 and STAT3 were INCB018424 both necessary, and together sufficient, to stimulate IL 6 production in glia cells.