Inducible IL-23p19 expression in human microglia via p38 MAPK and NF-κB signal pathways

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Abstract

Activated microglia can release a variety of proinflammatory cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). IL-23, a novel proinflammatory cytokine, is required for the induction of experimental autoimmune encephalomyelitis. Previously we demonstrated that IL-23 is expressed in MS lesions and that microglia are one cellular source of IL-23 in MS patients. In the present study we investigated the inducible expression and regulation of p19, a key subunit of IL-23, in human microglia. We demonstrated the inducible expression of IL-23p19 by lipopolysaccharide-stimulated microglial cells. Using signaling pathway-specific inhibitors, we showed that blocking p38 MAP kinase or NF-κB signaling pathway significantly reduced p19 expression in microglia. The regulatory role of p38 MAP kinase in p19 expression was further confirmed by decreased expression in microglia transduced with dominant-negative p38. We concluded that the p38 MAP kinase and NF-κB signaling pathways play an important role in regulation of IL-23p19 expression on human microglia, and are thus potential therapeutic targets in the treatment of MS.

Introduction

IL-23, a heterodimer cytokine in the IL-12 family, is composed of IL-12p40 and p19 subunits (Oppmann et al., 2000). Cytokines IL-12 and IL-23 are both produced by monocytes, but they promote two distinct T-cell subsets. In contrast to the role of IL-12 in inducing IFN-γ-producing Th1 cells, IL-23 drives the expansion of a novel T-cell population, Th17, characterized by the secretion of IL-17A, IL-17F, IL-6 and tumor necrosis factor (TNF) (Park et al., 2005, Veldhoen et al., 2006, Mangan et al., 2006, Zhou et al., 2007). Importantly the IL-23-driven Th17 cells are linked to autoimmune inflammation and mediated tissue destruction in chronic inflammatory disease such as multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), which serves as the animal model of MS, IL-23-driven TIL17 cells can transfer demyelinating disease into the brain, indicating that IL-23 is a highly potent inducer of CNS immune pathology (Langrish et al., 2005). In addition, therapeutic treatment with anti-IL-23p19 antibodies in the relapsing and remitting EAE model reduced the serum level of proinflammatory cytokines and prevented disease relapse by inhibiting epitope spreading initiated inside the brain (Chen et al., 2006). Thus, IL-23 plays a critical role in the development of neuroinflammatory disease.

Microglia are cells of the monocyte/macrophage lineage that reside in brain parenchyma in two major states: nonactivated (ramified, or resting) and activated (amoeboid). Microglia have been proposed to play a role in host defense and tissue repair in the CNS. However, activation of microglia and consequent release of proinflammatory and/or cytotoxic factors, such as IL-1β, TNF-α, nitric oxide and reactive oxygen species, are believed to contribute to the pathophysiology of several neurodegenerative diseases, including MS and Alzheimer's disease. In our previous studies, we identified human microglia, represented by CD11b+CD68+ as one of the major cellular sources of IL-23 in MS brain sections, and IL-23p19 production inside the brain correlated with MS disease severity (Li et al., 2007). Thus, therapeutic targets for precise and specific fine tuning of IL-23 responses including IL-23 production and its signaling will inhibit the inflammatory immune response and consequently benefit MS patients.

Aimed at therapeutic reduction of IL-23, we further investigated the intrinsic signaling pathways that mediate IL-23 production in microglia. Here we report the inducible expression of IL-23p19 in human microglia stimulated by LPS, and the involvement of p38 mitogen activated protein (MAP) kinase and NF-κB signal pathways in the regulation of IL-23p19 expression using pharmacological inhibitors and genetic manipulation.

Section snippets

Microglia cell cultures and activation

Because of the limited availability of naive microglia from human brain, microglial cell lines are a necessary alternative to human microglia in in vitro studies and have provided significant information for the biology of human microglia in vivo (Nagai et al., 2005). Thus, in the present study we used commercially available human microglial cell lines to elucidate the regulation and inhibition of IL-23p19 production. The studies on human microglia were performed in accordance with the

Inducible production of IL-23 in activated microglia

We have previously demonstrated that IL-23p19 expression colocalized in CD11b+CD68+ cells in MS patients' brain tissue sections and verified that macrophages/microglia are one of the cellular sources of IL-23 in the pathogenesis of MS (Li et al., 2007). In order to study the regulation of IL-23p19 in microglia, highly enriched microglial cells were generated after several weeks' culture in vitro. Stained with FITC-conjugated RCA-1, the purity of microglial cells was validated by flow cytometry

Discussion

In the present study, we demonstrate that IL-23 is induced in cultured human microglia in vitro and that the intrinsic signaling of p38 MAPK and NF-κB is involved in the regulation of IL-23 expression. We further confirm that phosphorylated Threonine 180 and Tyrosine 182 of p38, which cause activation of the p38 signaling pathway, is necessary for IL-23p19 production in human microglia.

In the pathogenesis of MS and EAE, it is believed that auto-aggressive myelin-reactive T lymphocytes migrate

Acknowledgments

This study was supported by the NIH, the National Multiple Sclerosis Society, and the Groff Foundation. We thank Katherine Regan for editorial assistance.

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