Abstract
When activated through toll-like receptors (TLRs), macrophages generate IL-33, an IL-1 family member that induces innate immune responses through ST2 signaling. LPS, a TLR4 ligand, induces macrophages to generate prostaglandin E2 (PGE2) through inducible COX-2 and microsomal PGE2 synthase 1 (mPGES-1) (1). We demonstrate that IL-33 production by bone marrow-derived murine macrophages (bmMFs) requires the generation of endogenous PGE2 and the intrinsic expression of EP2 receptors to amplify NF-κB-dependent, LPS-induced IL-33 expression via exchange protein activated by cAMP (EPAC). Compared with WT cells, bmMFs lacking either mPGES-1 or EP2 receptors displayed reduced LPS-induced IL-33 levels. A selective EP2 agonist and, to a lesser extent, EP4 receptor agonist potentiated LPS-induced IL-33 generation from both mPGES-1-null and WT bmMFs, whereas EP1 and EP3 receptor agonists were inactive. The effects of PGE2 depended on cAMP, were mimicked by an EPAC-selective agonist, and were attenuated by EPAC-selective antagonism and knockdown. LPS-induced p38 MAPK and NF-κB activations were necessary for both IL-33 production and PGE2 generation, and exogenous PGE2 partly reversed the suppression of IL-33 production caused by p38 MAPK and NF-κB inhibition. Mice lacking mPGES-1 showed lower IL-33 levels and attenuated lung inflammation in response to repetitive Alternaria inhalation challenges. Cumulatively, our data demonstrate that endogenous PGE2, EP2 receptors, and EPAC are prerequisites for maximal LPS-induced IL-33 expression and that exogenous PGE2 can amplify IL-33 production via EP2 and EP4 receptors. The ubiquitous induction of mPGES-1-dependent PGE2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders.
Highlights
IL-33 is a recently discovered member of the IL-1 cytokine family that binds to toll-like receptors (TLRs)/IL1R2 superfamily receptor ST2
Exogenous prostaglandin E2 (PGE2) can potentiate IL-33 expression by dendritic bone marrow-derived murine macrophage; EPAC, exchange protein activated by cAMP; WB, Western blotting; qPCR, quantitative PCR; CREB, cAMP-response element-binding protein; ILC2, group 2 innate lymphoid cell; recombinant murine MCSF (rmMCSF), recombinant murine macrophage colony-stimulating factor; 6-bnz-cAMP, N6-benzoyladenosine-3Ј, 5Ј-cyclic monophosphate; 8-pCPT2-O-Me-cAMP AM, 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-3Ј, 5Ј-cyclic monophosphate; FPA 124, dichloro[(2Z)-2-[(4-oxo-4H-1-benzopyran-3-yl)methylene]hydrazinecarbothioamide; 10-DEBC, 10-[4Ј-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride; APC, allophycocyanin
Stimulation with LPS dose-dependently induced the release of PGE2 by WT cells, which was significantly reduced in cells lacking microsomal PGE2 synthase 1 (mPGES-1) (Fig. 1A)
Summary
To determine the role of endogenous PGE2 in LPS-mediated IL-33 production, we stimulated bmMFs with LPS at concentrations of 0.5 and 1.0 g/ml. Stimulation of WT and mPGES-1 KO bmMFs with the EP2 receptor-selective agonist AE1-259-01 (1.0 M) in combination with LPS significantly enhanced IL-33 production but failed to do so in EP2 KO cells (Fig. 1E). To determine whether cAMP was involved in potentiation of IL-33 production, we stimulated bmMFs with LPS in the absence or presence of the pharmacologic adenyl cyclase activator forskolin (50.0 M) or a cell-permeable cAMP analog, 8-bromo-cAMP (100.0 M). Both agents significantly potentiated LPS-induced IL-33 expression in mPGES-1 KO and EP2 KO cells and potentiated the response of WT cells (Fig. 3A). ILC2 expansion, and IL-33 expression were identified in EP2 receptor knock-out mice (n ϭ 1 experiment, data not shown)
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