Date on Master's Thesis/Doctoral Dissertation

5-2009

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Martin, Michael David

Author's Keywords

Interferon; Glycogen synthase kinase 3; Toll-like receptors; IL-10

Subject

Glycogen; Interferon

Abstract

It has been shown that GSK3 ß plays a critical role in the inflammation response by differentially regulating MyD88-dependent pro- and anti-inflammatory cytokines production in TLR4-stimulated innate immune cells. The work included in this dissertation demonstrates that (I) GSK3ß negatively regulates the production of the TLR4 dependent and MyD88-independent cytokine, IFN ß, by controlling the levels of total c-Jun and thereby modulating the amount of c-Jun /ATF2 complexes, and (II) that IFN ß induces IL-10 in dendritic cells by regulating GSK3 ß activity. Part I: TLR4 stimulation of macrophages has been shown to induce the production of interferon-ß (IFN-ß) via the MyD88-independent pathway. Here we demonstrate that glycogen synthase kinase 3-ß (GSK3-ß) plays a fundamental role in this process. Suppression of GSK3-ß activity by pharmacological inhibition, siRNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-ß mutant, augmented IFN-ß production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-ß mutant severely attenuated IFN-ß production. GSK3-ß was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. siRNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-ß inhibition to augment IFN-ß production by TLR4 stimulated macrophages. Inhibition of GSK3 in vivo resulted in potently augmenting the systemic levels of IFN-ß in mice that were administered LPS. These findings identified a novel regulatory pathway controlling IFN-ß production by TLR4-stimulated innate immune cells. Part II: IFN-ß is known to induce the production of IL-10 by innate immune cells, yet the underlying cellular mechanisms responsible for this effect are unknown. Here, we demonstrate that the constitutively active serine/threonine kinase, GSK3-ß, controls the IFN-ß-mediated production of IL-10 by human dendritic cells. Stimulation of cells with IFN-ß induced the activation of the phosphatidylinositol 3-kinase (PI3K) pathway and blockade of PI3K activity inhibited the ability of IFN-ß to induce IL-10 production. Assessment of downstream kinases within the PI3K pathway demonstrated that IFN-ß induced the phosphorylation and subsequent suppression of GSK3ß activity. Direct inhibition of GSK3 activity via pharmacological inhibition, siRNA-mediated knockdown of GSK3-ß, or ectopic expression of a kinase dead GSK3-ß potently augmented the levels of IL-10 produced by type I IFN-stimulated dendritic cells, whereas no affect on the production of pro-inflammatory cytokines was observed. In contrast, ectopic expression of a constitutively active GSK3-ß mutant severely attenuated the levels of IL-10 produced by IFN-ß-stimulated cells. Analysis of transcription factors involved in the regulation of IL-10 showed that IFN-ß increased the nuclear levels of phospho-CREB and this effect was dependent upon the ability of IFN-ß to induce PI3K activity and inactivate GSK3-ß. These findings identify the cellular mechanism by which IFN-ß induces IL-10 production by innate immune cells.

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