The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Exhibits Anti-Inflammatory Activity in LPS-Stimulated Organotypic Hippocampal Cultures.

Ewa Trojan,Kinga Tylek,Enza Lacivita,Władysław Lasoń,Marcello Leopoldo,Agnieszka Basta-Kaim,Monika Leśkiewicz,Lars-Ove Brandenburg

doi:10.3390/cells10061524

Abstract

Accumulating evidence indicates a pivotal role for chronic inflammatory processes in the pathogenesis of neurodegenerative and psychiatric disorders. G protein-coupled formyl peptide receptor 2 (FPR2) mediates pro-inflammatory or anti-/pro-resolving effects upon stimulation with biased agonists. We aimed to evaluate the effects of a new FPR2 ureidopropanamide agonist, compound MR-39, on neuroinflammatory processes in organotypic hippocampal cultures (OHCs) derived from control (WT) and knockout FPR2−/− mice (KO) exposed to bacterial endotoxin (lipopolysaccharide; LPS). Higher LPS-induced cytokine expression and basal release were observed in KO FPR2 cultures than in WT cultures, suggesting that a lack of FPR2 enhances the OHCs response to inflammatory stimuli. Pretreatment with MR-39 abolished some of the LPS-induced changes in the expression of genes related to the M1/M2 phenotypes (including Il-1β, Il-6, Arg1, Il-4, Cd74, Fizz and Cx3cr1) and TNF-α, IL-1β and IL-4 release in tissue derived from WT but not KO mice. Receptor specificity was confirmed by adding the FPR2 antagonist WRW4, which abolished the abovementioned effects of MR-39. Further biochemical data showed an increase in the phospho-p65/total p65 ratio after LPS stimulation in hippocampal tissues from both WT and KO mice, and MR-39 only reversed this effect on WT OHCs. LPS also increased TRAF6 levels, which are critical for the TLR4-mediated NF-κB pro-inflammatory responses. MR-39 attenuated the LPS-evoked increase in the levels of the NLRP3 and caspase-1 proteins in WT but not KO hippocampal cultures. Since NLRP3 may be involved in the pyroptosis, a lytic type of programmed cell death in which the main role is played by Gasdermin D (GSDMD), we examined the effects of LPS and/or MR-39 on the GSDMD protein level. LPS only increased GSDMD production in the WT tissues, and this effect was ameliorated by MR-39. Collectively, this study indicates that the new FPR2 agonist efficiently abrogates LPS-induced neuroinflammation in an ex vivo model, as evidenced by a decrease in pro-inflammatory cytokine expression and release as well as the downregulation of NLRP3 inflammasome-related pathways.

Highlights

Neuroinflammation is a complex multicellular process that plays an important role in the onset and progression of several neurodegenerative and psychiatric disorders [1,2].Acute inflammation is considered a physiological response to defend the host against pathogens and to maintain homeostasis
1β), Tnf-a (Tumor necrosis factor α), Il-6 (Interleukin 6), Cd40 (Cluster of differentiation 40), CD68 (Cluster of differentiation 68), Il-23a (Interleukin 23a), Igf-1 (Insulin-like growth factor 1), Arg-1 (Arginase 1), Il-4 (Interleukin 4), Il-10 (Interleukin 10), Il-13 (Interleukin 13), Cd74 (Cluster of differentiation 74), Ym1 (Chitinase 3-like-3 Chi3 l3/Ym1), Fizz (Found in inflammatory zone/resistinlike molecule), Il-27 (Interleukin 27), Cx3cl1 (Fractalkine) and Cx3cr1 (Cx3c chemokine receptor 1) and the FastStart Universal Probe Master (Rox) kit (Roche, Basel, Switzerland) using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) as we described previously [36,38]
The results presented in this study were derived from three independent knockout FPR2−/− mice (KO) formyl peptide receptor 2 (FPR2)−/− or wild type (WT) organotypic hippocampal cultures (OHCs), and the “n” for each culture was 2–5

Summary

Introduction

Neuroinflammation is a complex multicellular process that plays an important role in the onset and progression of several neurodegenerative and psychiatric disorders [1,2].Acute inflammation is considered a physiological response to defend the host against pathogens and to maintain homeostasis. FPR2 ( referred to in the literature as FPRL1 or ALX/FPR2) is a G protein-coupled receptor (GPCR) that binds LXA4 and 15-epi-LXA4 with high affinity This receptor is expressed on various types of immune cells and in the brain on some neurons, astroglia and microglia [12]. The ability of FPR2 to mediate opposing effects (pro-inflammatory vs pro-resolving) is mechanistically related to receptor dimerization, which is induced in a ligand-specific manner [18,19]. Chen et al [22] showed a decrease in the inflammatory response in FPR2-deficient (FPR2−/−) mice. In a mouse model of pneumococcal meningitis, FPR2−/− mice showed a significantly increased glial cell density, whereas immune responses, including the expression of anti-inflammatory cytokines, were decreased [24]

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