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Impact of mPGES-1 on the resolution of zymosan-induced peritonitis in mice

ABSTRACT: Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mf) by microsomal PGE2-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE2-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mf during the resolution process. mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mf upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mf, but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE2 contributes to resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.

ORGANISM(S): Mus musculus

PROVIDER: GSE164364 | GEO | 2021/02/10

REPOSITORIES: GEO

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Project description:Prostaglandin E2 (PGE2) is involved in several inflammatory conditions including periodontitis. The aim of this study was to investigate the global gene expression profile of tumor necrosis factor alpha (TNFalpha) stimulated human gingival fibroblasts, focusing on signal pathways related to PGE2 production and the new PGE2-synthesizing enzymes, prostaglandin E synthases (PGES). The expression of microsomal prostaglandin E synthase-1 (mPGES-1) as well as the upstream cyclooxygenase-2 (COX-2) was up-regulated by TNFalpha, accompanied by increased PGE2 production. In contrast, the expression of microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFalpha. Using microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, we identified differentially expressed genes in response to TNFalpha treatment. Enrichment analysis of microarray data identified two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappaB (NF-kappaB). We used specific inhibitors and phosphorylation analysis to confirm their role in PGE2 regulation. Both JNK and NF-kappaB inhibitors reduced the TNFalpha-stimulated up-regulation of mPGES-1 and COX-2 as well as subsequent PGE2 production. The novel finding that TNFalpha-stimulated mPGES-1 is regulated by JNK suggests this kinase as a potential future target for treatment strategies in inflammatory disorders, including periodontitis. Keywords: Time course, gene expression, factorial design. Three human gingival fibroblast cell lines were established from gingival biopsies obtained from 3 healthy patients, 3 to 12 years of age, with no clinical signs of periodontal disease. Cells were incubated with or without TNF-alpha (20 ng/ml) for 1, 3 or 6h. After incubation for 1, 3 or 6 h in the two conditions, the cells were immediately frozen in liquid nitrogen and then stored at -70°C for subsequent isolation of total RNA. The experimental design of the microarray study was set up as a time-course factorial design, to best observe the TNF-alpha induced gene expression changes over time. C++ program (G.F. Glonek, P.J. Solomon, Factorial and time course designs for cDNA microarray experiments, Biostatistics 5 (2004) 89-111) was used to determine the exact layout of the design in order to estimate the interaction effect between treatment and time, i.e. genes that are differentially expressed over time, with optimal statistical efficiency. Cyanine 5 and cyanine 3 were used for labeling. For each cell line 12 hybridizations were performed in a time-course factorial design. In total, 36 hybridizations were performed.

Project description:Inhibitors of microsomal prostaglandin E synthase-1 (mPges-1) are in the early phase of clinical development. Deletion of mPges-1 in mice confers analgesia, restrains atherogenesis and fails to accelerate thrombogenesis, while suppressing prostaglandin (PG) E2, but increasing biosynthesis of prostacyclin (PGI2). In hyperlipidemic mice, this last effect represents the dominant mechanism by which mPges-1 deletion restrains thrombogenesis, while suppression of PGE2 accounts for its anti-atherogenic effect. However, the impact of mPges-1 depletion on blood pressure (BP) in this setting remains unknown. To address how differential effects on PGE2 and PGI2 might modulate salt-evoked BP responses in the absence of mPges-1, we generated mice lacking the I prostanoid (Ipr) receptor or mPges-1 on a hyperlipidemic background caused by deletion of the low density lipoprotein receptor (Ldlr). Here, mPges-1 depletion significantly increased the BP response to salt loading in male Ldlr-/- mice, whereas, despite the direct vasodilator properties of PGI2, Ipr deletion suppressed it. Furthermore, combined deletion of the Ipr abrogated the exaggerated BP response in male mPges-1-/- mice. Suppression of PGE2 biosynthesis was enough to explain the exaggerated BP response to salt loading by either mPges-1/Ldlr depletion or by an MPGES-1 inhibitor in mice expressing human mPGES-1. However, the lack of a hypertensive response to salt in Ipr-deficient mice was attributable to reactive activation of the atrial natriuretic peptide (ANP) pathway. Interestingly, these unexpected BP phenotypes were not observed in female mice fed a high salt diet. This is attributable to the protective effect of estrogen in Ldlr-/- mice and in Ipr-/- /Ldlr-/- mice. Thus, estrogen compensates for a deficiency in PGI2 to maintain BP homeostasis in response to high salt in hyperlipidemic female mice. In males, by contrast, augmented formation of ANP plays a similar compensatory role, restraining hypertension and oxidant stress in the setting of Ipr depletion. In male mice, depletion of Lactobacilius in the gut microbiome coincided with a reduction in their fecal product, indole-3-lactic acid and plasma levels of indoxyl sulfate/ tryptophan. These metabolites act via the Aryl Hydrocarbon Receptor to restrain inflammation and oxidative stress and their depletion may augment salt induced hypertension in males. Hyperlipidemic males on a high salt diet might be at risk of a hypertensive response to mPGES-1 inhibitors.

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Impact of mPGES-1 on the resolution of zymosan-induced peritonitis in mice

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