Endogenous prostaglandin E2 amplifies IL-33 production by macrophages through an E prostanoid (EP)2/EP4-cAMP-EPAC-dependent pathway

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.

Keywords: EPAC; IL-33; PGE2; bone marrow macrophages; cAMP; cytokine; innate immunity; mPGES-1; macrophage; prostaglandin.

Figure 1.

Endogenous PGE₂ is required for IL-33 production in response to LPS and it signals through EP₂ and EP₄. A, PGE₂ levels in cell supernatants from LPS-stimulated WT and mPGES-1 KO bmMFs after 8 h. B, IL-33 protein percentage production levels in corresponding cell lysates by ELISA. C, potentiation of IL-33 production by exogenous PGE₂ at concentrations in mPGES-1 KO and WT cells. D, exogenous PGE₂ induction to LPS-stimulated to EP₂ KO bmMFs. E, enhanced IL-33 production by the EP₂ receptor-selective agonist AE1-259-01 and the selective EP₄ receptor agonist AE-329. F, WB showing LPS-induced IL-33 protein and COX-2 levels in WT, mPGES-1 KO, and EP₂ KO cells in response to EP₂ and EP₄ agonists. G, IL-33 production in the presence of the EP₁ receptor-selective agonist D004 and the EP₃ receptor agonist AE248. Data are presented as mean ± S.E. of at least three independent experiments. Statistical significance was determined using unpaired t test and one-sample t test (comparing fixed 100%). p < 0.05 was considered statistically significant. *, p < 0.05; **, p < 0.005.

Figure 2.

LPS induces coordinate expression of PGE₂ pathway constituents needed to amplify IL-33 expression. A–E, we stimulated bmMFs with LPS (1.0 μg/ml) and collected RNA every 2 h until 8 h and monitored (A) IL-33, (B) EP₂ receptor, (C) EP₄ receptor, (D) COX-2, and (E) mPGES-1 mRNA levels. Data are presented as mean ± S.E. of three independent experiments. Statistical significance was determined using unpaired t test. p < 0.05 was considered statistically significant. *, p < 0.05.

Figure 3.

cAMP mediates potentiation of LPS-induced IL-33 production. A, IL-33 production by LPS-stimulated (1.0 μg/ml) bmMFs in the presence of 50.0 μm forskolin and 100 μm 8-bromo-cAMP (8-Br-cAMP). Data are presented as mean ± S.E. of three independent experiments. Statistical significance was determined using one-sample t test (comparing fixed 100%). p < 0.05 was considered statistically significant. *, p < 0.05. B, WB showing LPS-induced IL-33 protein levels in WT cells further enhanced by addition of PGE₂, 8-Br-cAMP, EP₂ and EP₄ agonists. C, Western blots showing the effects of PGE₂ and 8-Br-cAMP on IL-33 and COX-2 expression in cells from the indicated genotypes.

Figure 4.

PGE₂-enhanced IL-33 production involves EPAC but not PKA or PI3K. A, IL-33 production by LPS-stimulated bmMFs (1.0 μg/ml) in the presence of selective cAMP analogs for PKA and EPAC. B, levels of IL-33 production by LPS-stimulated bmMFs with or without PGE₂ in the presence of the PKA inhibitor H89 (10.0 μm). C, effect of ESI09 (10.0 μm) on IL-33 production by LPS-stimulated bmMFs. D, effects of H89 and ESI09 alone and in combination on LPS-induced IL-33 production by WT bmMFs. E, effects of H89 and ESI09 on LPS-induced IL-6 production by the same samples as in D. F, effects of ESI09 on PGE₂-enhanced IL-33 production by bmMFs. G, effects of ESI09 on potentiation of IL-33 production by WT bmMFs stimulated with AE1-259-01 but not with AE-329. H, effect of wortmannin (1.0 μm) on LPS-induced IL-33 production and its potentiation by PGE₂. Data are presented as mean ± S.E. of at least three independent experiments. Statistical significance was determined using one-sample t test (comparing fixed 100%). p < 0.05 was considered statistically significant. *, p < 0.05; **, p < 0.005.

Figure 5.

Gene silencing confirms that PGE₂ enhances IL-33 production in part through EPAC. A and B, effect of EPAC knockdown on 8-pCPT-2-O-Me-cAMP-AM-enhanced (A) or PGE₂-enhanced (B) IL-33 production. C and D, mRNA levels of EPAC1 (C) and EPAC2 (D) in the presence of the respective siRNAs in PGE₂/8-pCPT-2-O-Me-cAMP-AM-stimulated bmMFs. Data are presented as mean ± S.E. of three independent experiments. Statistical significance was determined using unpaired t test. p < 0.05 was considered statistically significant. *, p < 0.05.

Figure 6.

PGE₂-mediated potentiation of LPS-stimulated IL-33 production is independent of p38 MAPK and NF-κB activation. A, effect of the p38 MAPK inhibitors SKF 86002 and SB706504 on IL-33 production by LPS (1.0 μg/ml) with or without PGE₂. B, protein lysates from WT, mPGES-1 KO, and EP₂ KO bmMFs 30 min after LPS stimulation were analyzed by WB, showing levels of phospho-p38 MAPK in WT, mPGES-1 KO, and EP₂ KO bmMFs. C, IL-33 production by bmMFs in the presence of NF-κB inhibitor. D, NF-κB activation levels upon LPS stimulation in WT bmMFs in the presence of other selective agonists. E, WB analysis showing NF-κB activation by IκB phosphorylation in WT, mPGES-1 KO, and EP₂ KO cells upon LPS stimulation and PGE₂ addition. F, IL-33 levels with the ERK1/2 inhibitor FR180204 (1.0 μm) in the absence or presence of PGE₂. G, effect of the JNK signaling inhibitor SP 600125 (100.0 μm) on IL-33 levels. Data are presented as mean ± S.E. of at least three independent experiments. Statistical significance was determined using one-sample t test (comparing fixed 100%). p < 0.05 was considered statistically significant. *, p < 0.05; **, p < 0.005.

Figure 7.

Endogenous PGE₂ is necessary for inducible IL-33 expression and type 2 immunopathology in response to Alternaria. WT and mPGES-1 KO mice were treated with four intranasal doses of Alternaria (12 μg) over 9 days. On day 10, mice from the indicated groups were euthanized for the studies. A–C, total lung cell numbers (A), total lung eosinophils (B), and lung ILC2 numbers (C) in WT and mPGES-1 KO mice in PBS and Alternaria-challenged mice. D, qPCR quantification of the IL-33 mRNA transcript in lung single-cell suspensions in WT and mPGES-1 KO Alternaria-challenged mice (IL-33 transcripts were below detection levels in PBS-challenged mice). Data are presented as mean ± S.D. of at least three independent experiments. Statistical significance was determined using an unpaired t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Figure 8.

Schematic of the tentative pathway involved in IL-33 augmentation by PGE₂. Shown is a schematic of IL-33 augmentation by LPS-stimulated bmMFs in response to PGE₂ acting through EPAC but not PKA.