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

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.

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Abstract 069: Intrarenal Arteries Sense Mitochondrial N-formyl Peptides via Formyl Peptide Receptor in Wistar and Spontaneously Hypertensive Rats (shr)

Hypertension ◽

10.1161/hyp.66.suppl_1.069 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Camilla F Wenceslau ◽

Cameron G McCarthy ◽

Safia Ogbi ◽

Paul M O'Connor ◽

R. Clinton Webb

Keyword(s):

Protein Expression ◽

Ex Vivo ◽

Kidney Injury ◽

Formyl Peptide Receptor ◽

Peptide Receptor ◽

Immune System Activation ◽

Formyl Peptide ◽

Wistar Kyoto ◽

Formyl Peptides

It is well established that chronic immune system activation contributes to hypertension and kidney injury. Mitochondria carry hallmarks of their bacterial ancestry and thus have emerged as a significant source of inflammatogenic damage-associated molecular patterns (DAMPs). One of these hallmarks is that it still uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Recently, we have observed that mitochondrial DAMPs are elevated in the circulation of SHR, and that mitochondrial N-formyl peptides (F-MIT) infusion in rats induces systemic inflammation and vascular dysfunction via formyl peptide receptor (FPR) activation. However, we do not know if FPR plays a role in kidney injury and hypertension. We hypothesized that F-MIT activate FPR and lead to intrarenal dysfunction in 12 week old male Wistar and SHR (n=4-8). Wistar rats were treated with F-MIT (0.02 mg/kg) or non-formylated peptide (control) for 6 h and intrarenal arteries (diameter >100 μm) were isolated. To exclude systemic effects of F-MIT, intrarenal arteries were also isolated from control rats and treated ex vivo with 100 nM F-MIT or non-formylated peptide. F-MIT treatment in vivo increased intrarenal arteries FPR protein expression (2.3-fold vs. control) and decreased β-arrestin 2 (protein that internalizes FPR upon activation) and phosphorylation of endothelial nitric oxide synthase (4-fold vs. control). These results were reproduced in isolated arteries incubated with F-MIT or control for 6 h ex vivo. Similarly, in intrarenal arteries from untreated SHR, we found that FPR protein expression was higher (1.5-fold vs. Wistar Kyoto, WKY) and β-arrestin 2 protein expression was decreased (2-fold vs. WKY). Interestingly, although treatment with hydrochlorothiazide (10-55 mg/kg/day) and reserpine (0.6-4.5 mg/kg/day) for 7 weeks attenuated the increase in blood pressure in SHR, anti-hypertensive therapy did not change FPR and β-arrestin 2 protein expression. Additionally, it was observed that the co-localization of FPR and β-arrestin 2 was decreased in intrarenal arteries from SHR. Overall, these data suggest that intrarenal arteries sense F-MIT. Also, FPR activation parameters following F-MIT treatment of normotensive rats are similar to those observed in SHR.

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The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Improves Ex Vivo and In Vivo Amyloid Beta (1–42)-Induced Neuroinflammation in Mouse Models of Alzheimer’s Disease

Molecular Neurobiology ◽

10.1007/s12035-021-02543-2 ◽

2021 ◽

Author(s):

Ewa Trojan ◽

Kinga Tylek ◽

Nicole Schröder ◽

Iris Kahl ◽

Lars-Ove Brandenburg ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Inflammatory Cytokines ◽

Ex Vivo ◽

Neuronal Loss ◽

Formyl Peptide Receptor ◽

Peptide Receptor ◽

Formyl Peptide ◽

Anti Inflammatory

Abstract The major histopathological hallmarks of Alzheimer’s disease (AD) include β-amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. Aβ 1–42 (Aβ1-42) has been shown to induce neurotoxicity and secretion of proinflammatory mediators that potentiate neurotoxicity. Proinflammatory and neurotoxic activities of Aβ1-42 were shown to be mediated by interactions with several cell surface receptors, including the chemotactic G protein-coupled N-formyl peptide receptor 2 (FPR2). The present study investigated the impact of a new FPR2 agonist, MR-39, on the neuroinflammatory response in ex vivo and in vivo models of AD. To address this question, organotypic hippocampal cultures from wild-type (WT) and FPR2-deficient mice (knockout, KO, FPR2−/−) were treated with fibrillary Aβ1-42, and the effect of the new FPR2 agonist MR-39 on the release of pro- and anti-inflammatory cytokines was assessed. Similarly, APP/PS1 double-transgenic AD mice were treated for 20 weeks with MR-39, and immunohistological staining was performed to assess neuronal loss, gliosis, and Aβ load in the hippocampus and cortex. The data indicated that MR-39 was able to reduce the Aβ1-42-induced release of proinflammatory cytokines and to improve the release of anti-inflammatory cytokines in mouse hippocampal organotypic cultures. The observed effect was apparently related to the inhibition of the MyD88/TRAF6/NFкB signaling pathway and a decrease in NLRP3 inflammasome activation. Administration of MR-39 to APP/PS1 mice improved neuronal survival and decreased microglial cell density and plaque load.These results suggest that FPR2 may be a promising target for alleviating the inflammatory process associated with AD and that MR-39 may be a useful therapeutic agent for AD.

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