The Crosstalk between N-Formyl Peptide Receptors and uPAR in Systemic Sclerosis: Molecular Mechanisms, Pathogenetic Role and Therapeutic Opportunities.

Systemic Sclerosis (SSc) is a heterogeneous autoimmune disease characterized by widespread vasculopathy, the presence of autoantibodies and the progressive fibrosis of skin and visceral organs. There are still many questions about its pathogenesis, particularly related to the complex regulation of the fibrotic process, and to the factors that trigger its onset. Our recent studies supported a key role of N-formyl peptide receptors (FPRs) and their crosstalk with uPAR in the fibrotic phase of the disease. Here, we found that dermal fibroblasts acquire a proliferative phenotype after the activation of FPRs and their interaction with uPAR, leading to both Rac1 and ERK activation, c-Myc phosphorylation and Cyclin D1 upregulation which drive cell cycle progression. The comparison between normal and SSc fibroblasts reveals that SSc fibroblasts exhibit a higher proliferative rate than healthy control, suggesting that an altered fibroblast proliferation could contribute to the initiation and progression of the fibrotic process. Finally, a synthetic compound targeting the FPRs/uPAR interaction significantly inhibits SSc fibroblast proliferation, paving the way for the development of new targeted therapies in fibrotic diseases.

FPR1: A critical gatekeeper of the heart and brain.

G protein-coupled receptors (GPCRs) are currently the most widely focused drug targets in the clinic, exerting their biological functions by binding to chemicals and activating a series of intracellular signaling pathways. Formyl-peptide receptor 1 (FPR1) has a typical seven-transmembrane structure of GPCRs and can be stimulated by a large number of endogenous or exogenous ligands with different chemical properties, the first of which was identified as formyl-methionine-leucyl-phenylalanine (fMLF). Through receptor-ligand interactions, FPR1 is involved in inflammatory response, immune cell recruitment, and cellular signaling regulation in key cell types, including neutrophils, neural stem cells (NSCs), and microglia. This review outlines the critical roles of FPR1 in a variety of heart and brain diseases, including myocardial infarction (MI), ischemia/reperfusion (I/R) injury, neurodegenerative diseases, and neurological tumors, with particular emphasis on the milestones of FPR1 agonists and antagonists. Therefore, an in-depth study of FPR1 contributes to the research of innovative biomarkers, therapeutic targets for heart and brain diseases, and clinical applications.

Knockout of formyl peptide receptor 1 reduces osteogenesis and bone healing.

AIMS: Formyl peptide receptor 1 (FPR1), from a G-protein coupled receptor family, was previously well-characterized in immune cells. But the function of FPR1 in osteogenesis and fracture healing was rarely reported. This study, using the FPR1 knockout (KO) mouse, is one of the first studies that try to investigate FPR1 function to osteogenic differentiation of bone marrow-derived stem cells (BMSCs) in vitro and bone fracture healing in vivo. MATERIALS AND METHODS: Primary BMSCs were isolated from both FPR1 KO and wild type (WT) mice. Cloned mouse BMSCs (D1 cells) were used to examine role of FoxO1 in FPR1 regulation of osteogenesis. A closed, transverse fracture at the femoral midshaft was created to compare bone healing between KO and WT mice. Biomechanical and structural properties of femur were compared between healthy WT and KO mice. KEY FINDINGS: FPR1 expression increased significantly during osteogenesis of both primary and cloned BMSCs. Compared to BMSCs from FPR1 KO mice, WT BMSCs displayed considerably higher levels of osteogenic markers as well as mineralization. Osteogenesis by D1 cells was inhibited by either an FPR1 antagonist cFLFLF or a specific inhibitor of FoxO1, AS1842856. In addition, the femur from WT mice had better biomechanical properties than FPR1 KO mice. Furthermore, bone healing in WT mice was remarkably improved compared to FPR1 KO mice analyzed by X-ray and micro-CT. SIGNIFICANCE: These findings indicated that FPR1 played a vital role in osteogenic differentiation and regenerative capacity of fractured bone, probably through the activation of FoxO1 related signaling pathways.

FPR1 Antagonist (BOC-MLF) Inhibits Amniotic Epithelial-mesenchymal Transition.

OBJECTIVE: Premature rupture of membranes (PROM) is a common pregnancy disorder that is closely associated with structural weakening of fetal membranes. Studies have found that formyl peptide receptor 1 (FPR1) activates inflammatory pathways and amniotic epithelialmesenchymal transition (EMT), stimulates collagen degradation, and leads to membrane weakening and membrane rupture. The purpose of this study was to investigate the anti-inflammatory and EMT inhibitory effects of FPR1 antagonist (BOC-MLF) to provide a basis for clinical prevention of PROM. METHODS: The relationship between PROM, FPR1, and EMT was analyzed in human fetal membrane tissue and plasma samples using Western blotting, PCR, Masson staining, and ELISA assays. Lipopolysaccharide (LPS) was used to establish a fetal membrane inflammation model in pregnant rats, and BOC-MLF was used to treat the LPS rat model. We detected interleukin (IL)-6 in blood from the rat hearts to determine whether the inflammatory model was successful and whether the anti-inflammatory treatment was effective. We used electron microscopy to analyze the structure and collagen expression of rat fetal membrane. RESULTS: Western blotting, PCR and Masson staining indicated that the expression of FPR1 was significantly increased, the expression of collagen was decreased, and EMT appeared in PROM. The rat model indicated that LPS caused the collapse of fetal membrane epithelial cells, increased intercellular gaps, and decreased collagen. BOC-MLF promoted an increase in fetal membrane collagen, inhibited EMT, and reduced the weakening of fetal membranes. CONCLUSION: The expression of FPR1 in the fetal membrane of PROM was significantly increased, and EMT of the amniotic membrane was obvious. BOC-MLF can treat inflammation and inhibit amniotic EMT.

Larixol is not an inhibitor of Gαi containing G proteins and lacks effect on signaling mediated by human neutrophil expressed formyl peptide receptors.

Neutrophils express several G protein-coupled receptors (GPCRs) connected to intracellular Gαi or Gαq containing G proteins for down-stream signaling. To dampen GPCR mediated inflammatory processes, several inhibitors targeting the receptors and/or their down-stream signals, have been developed. Potent and selective inhibitors for Gαq containing G proteins are available, but potent and specific inhibitors of Gαi containing G proteins are lacking. Recently, Larixol, a compound extracted from the root of Euphorbia formosana, was shown to abolish human neutrophil functions induced by N-formyl-methionyl-leucyl-phenylalanine (fMLF), an agonist recognized by formyl peptide receptor 1 (FPR1) which couple to Gαi containing G proteins. The inhibitory effect was suggested to be due to interference with/inhibition of signals transmitted by ßγ complexes of the Gαi containing G proteins coupled to FPR1. In this study, we applied Larixol, obtained from two different commercial sources, to determine the receptor- and G protein- selectivity of this compound in human neutrophils. However, our data show that Larixol not only lacks inhibitory effect on neutrophil responses mediated through FPR1, but also on responses mediated through FPR2, a Gαi coupled GPCR closely related to FPR1. Furthermore, Larixol did not display any features as a selective inhibitor of neutrophil responses mediated through the Gαq coupled GPCRs for platelet activating factor and ATP. Hence, our results imply that the inhibitory effects described for the root extract of Euphorbia formosana are not mediated by Larixol and that the search for a selective inhibitor of G protein dependent signals generated by Gαi coupled neutrophil GPCRs must continue.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Microglia Depletion Attenuates the Pro-Resolving Activity of the Formyl Peptide Receptor 2 Agonist AMS21 Related to Inhibition of Inflammasome NLRP3 Signalling Pathway: A Study of Organotypic Hippocampal Cultures.

Microglial cells have been demonstrated to be significant resident immune cells that maintain homeostasis under physiological conditions. However, prolonged or excessive microglial activation leads to disturbances in the resolution of inflammation (RoI). Formyl peptide receptor 2 (FPR2) is a crucial player in the RoI, interacting with various ligands to induce distinct conformational changes and, consequently, diverse biological effects. Due to the poor pharmacokinetic properties of endogenous FPR2 ligands, the aim of our study was to evaluate the pro-resolving effects of a new ureidopropanamide agonist, compound AMS21, in hippocampal organotypic cultures (OHCs) stimulated with lipopolysaccharide (LPS). Moreover, to assess whether AMS21 exerts its action via FPR2 specifically located on microglial cells, we conducted a set of experiments in OHCs depleted of microglial cells using clodronate. We demonstrated that the protective and anti-inflammatory activity of AMS21 manifested as decreased levels of lactate dehydrogenase (LDH), nitric oxide (NO), and proinflammatory cytokines IL-1ß and IL-6 release evoked by LPS in OHCs. Moreover, in LPS-stimulated OHCs, AMS21 treatment downregulated NLRP3 inflammasome-related factors (CASP1, NLRP3, PYCARD) and this effect was mediated through FPR2 because it was blocked by the FPR2 antagonist WRW4 pre-treatment. Importantly this beneficial effect of AMS21 was only observed in the presence of microglial FPR2, and absent in OHCs depleted with microglial cells using clodronate. Our results strongly suggest that the compound AMS21 exerts, at nanomolar doses, protective and anti-inflammatory properties and an FPR2 receptor located specifically on microglial cells mediates the anti-inflammatory response of AMS21. Therefore, microglial FPR2 represents a promising target for the enhancement of RoI.

Development of potent isoflavone-based formyl peptide receptor 1 (FPR1) antagonists and their effects in gastric cancer cell models.

Formyl peptide receptor-1 (FPR1) is a G protein-coupled chemoattractant receptor that plays a crucial role in the trafficking of leukocytes into the sites of bacterial infection and inflammation. Recently, FPR1 was shown to be expressed in different types of tumor cells and could play a significant role in tumor growth and invasiveness. Starting from the previously reported FPR1 antagonist 4, we have designed a new series of 4H-chromen-2-one derivatives that exhibited a substantial increase in FPR1 antagonist potency. Docking studies identified the key interactions for antagonist activity. The most potent compounds in this series (24a and 25b) were selected to study the effects of the pharmacological blockade of FPR1 in NCl-N87 and AGS gastric cancer cells. Both compounds potently inhibited cell growth through a combined effect on cell proliferation and apoptosis and reduced cell migration, while inducing an increase in angiogenesis, thus suggesting that FPR1 could play a dual role as oncogene and onco-suppressor.

Dynamic Evolution of Bacterial Ligand Recognition by Formyl Peptide Receptors.

The detection of invasive pathogens is critical for host immune defense. Cell surface receptors play a key role in the recognition of diverse microbe-associated molecules, triggering leukocyte recruitment, phagocytosis, release of antimicrobial compounds, and cytokine production. The intense evolutionary forces acting on innate immune receptor genes have contributed to their rapid diversification across plants and animals. However, the functional consequences of immune receptor divergence are often unclear. Formyl peptide receptors (FPRs) comprise a family of animal G protein-coupled receptors which are activated in response to a variety of ligands including formylated bacterial peptides, pathogen virulence factors, and host-derived antimicrobial peptides. FPR activation in turn promotes inflammatory signaling and leukocyte migration to sites of infection. Here we investigate patterns of gene loss, diversification, and ligand recognition among FPRs in primates and carnivores. We find that FPR1, which plays a critical role in innate immune defense in humans, has been lost in New World primates. Amino acid variation in FPR1 and FPR2 among primates and carnivores is consistent with a history of repeated positive selection acting on extracellular domains involved in ligand recognition. To assess the consequences of FPR divergence on bacterial ligand interactions, we measured binding between primate FPRs and the FPR agonist Staphylococcus aureus enterotoxin B, as well as S. aureus FLIPr-like, an FPR inhibitor. We found that few rapidly evolving sites in primate FPRs are sufficient to modulate recognition of bacterial proteins, demonstrating how natural selection may serve to tune FPR activation in response to diverse microbial ligands.

Transcriptome sequencing and experiments reveal the effect of formyl peptide receptor 2 on liver homeostasis.

BACKGROUND: Formyl peptide receptor 2 (Fpr2) is an important receptor in host resistance to bacterial infections. In previous studies, we found that the liver of Fpr2-/- mice is the most severely damaged target organ in bloodstream infections, although the reason for this is unclear. AIM: To investigate the role of Fpr2 in liver homeostasis and host resistance to bacterial infections. METHODS: Transcriptome sequencing was performed on the livers of Fpr2-/- and wild-type (WT) mice. Differentially expressed genes (DEGs) were identified in the Fpr2-/- and WT mice, and the biological functions of DEGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) en-richment analysis. Quantitative real time-polymerase chain reaction (qRT-PCR) and western blot (WB) analyses were used to further validate the expression levels of differential genes. Cell counting kit-8 assay was employed to investigate cell survival. The cell cycle detection kit was used to measure the distribution of cell cycles. The Luminex assay was used to analyze cytokine levels in the liver. The serum biochemical indices and the number of neutrophils in the liver were measured, and hepatic histopathological analysis was performed. RESULTS: Compared with the WT group, 445 DEGs, including 325 upregulated genes and 120 downregulated genes, were identified in the liver of Fpr2-/- mice. The enrichment analysis using GO and KEGG showed that these DEGs were mainly related to cell cycle. The qRT-PCR analysis confirmed that several key genes (CycA, CycB1, Cdc20, Cdc25c, and Cdk1) involved in the cell cycle had significant changes. The WB analysis confirmed a decrease in the expression of CDK1 protein. WRW4 (an antagonist of Fpr2) could inhibit the proliferation of HepG2 cells in a concentration dependent manner, with an increase in the number of cells in the G0/G1 phase, and a decrease in the number of cells in the S phase. Serum alanine aminotransferase levels increased in Fpr2-/- mice. The Luminex assay measurements showed that interleukin (IL)-10 and chemokine (C-X-C motif) ligand (CXCL)-1 levels were significantly reduced in the liver of Fpr2-/- mice. There was no difference in the number of neutrophils, serum C-reactive protein levels, and liver pathology between WT and Fpr2-/- mice. CONCLUSION: Fpr2 participates in the regulation of cell cycle and cell proliferation, and affects the expression of IL-10 and CXCL-1, thus playing an important protective role in maintaining liver homeostasis.

Anti-Inflammatory and Pro-Resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function.

Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

Unleashing the power of formyl peptide receptor 2 in cardiovascular disease.

N-formyl peptide receptors (FPRs) are seven-transmembrane, G protein-coupled receptors with a wide distribution in immune and non-immune cells, recognizing N-formyl peptides from bacterial and mitochondrial origin and several endogenous signals. Three FPRs have been identified in humans: FPR1, FPR2, and FPR3. Most FPR ligands can activate a pro-inflammatory response, while a limited group of FPR agonists can elicit anti-inflammatory and homeostatic responses. Annexin A1 (AnxA1), a glucocorticoid-induced protein, its N-terminal peptide Ac2-26, and lipoxin A4 (LXA4), a lipoxygenase-derived eicosanoid mediator, exert significant immunomodulatory effects by interacting with FPR2 and/or FPR1. The ability of FPRs to recognize both ligands with pro-inflammatory or inflammation-resolving properties places them in a crucial position in the balance between activation against harmful events and maintaince of tissue integrity. A new field of investigation focused on the role of FPRs in the setting of heart injury. FPRs are expressed on cardiac macrophages, which are the predominant immune cells in the myocardium and play a key role in heart diseases. Several endogenous (AnxA1, LXA4) and synthetic compounds (compound 43, BMS-986235) reduced infarct size and promoted the resolution of inflammation via the activation of FPR2 on cardiac macrophages. Further studies should evaluate FPR2 role in other cardiovascular disorders.

Hepatocyte-secreted FAM3D ameliorates hepatic steatosis by activating FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide; however, the underlying mechanisms remain poorly understood. FAM3D is a member of the FAM3 family; however, its role in hepatic glycolipid metabolism remains unknown. Serum FAM3D levels are positively correlated with fasting blood glucose levels in patients with diabetes. Hepatocytes express and secrete FAM3D, and its expression is increased in steatotic human and mouse livers. Hepatic FAM3D overexpression ameliorated hyperglycemia and steatosis in obese mice, whereas FAM3D-deficient mice exhibited exaggerated hyperglycemia and steatosis after high-fat diet (HFD)-feeding. In cultured hepatocytes, FAM3D overexpression or recombinant FAM3D protein (rFAM3D) treatment reduced gluconeogenesis and lipid deposition, which were blocked by anti-FAM3D antibodies or inhibition of its receptor, formyl peptide receptor 1 (FPR1). FPR1 overexpression suppressed gluconeogenesis and reduced lipid deposition in wild hepatocytes but not in FAM3D-deficient hepatocytes. The addition of rFAM3D restored FPR1's inhibitory effects on gluconeogenesis and lipid deposition in FAM3D-deficient hepatocytes. Hepatic FPR1 overexpression ameliorated hyperglycemia and steatosis in obese mice. RNA sequencing and DNA pull-down revealed that the FAM3D-FPR1 axis upregulated the expression of heterogeneous nuclear ribonucleoprotein U (hnRNP U), which recruits the glucocorticoid receptor (GR) to the promoter region of the short-chain acyl-CoA dehydrogenase (SCAD) gene, promoting its transcription to enhance lipid oxidation. Moreover, FAM3D-FPR1 axis also activates calmodulin-Akt pathway to suppress gluconeogenesis in hepatocytes. In conclusion, hepatocyte-secreted FAM3D activated the FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation in hepatocytes. Under obesity conditions, increased hepatic FAM3D expression is a compensatory mechanism against dysregulated glucose and lipid metabolism.

ALX/FPR2 Activation by Stereoisomers of D1 Resolvins Elucidating with Molecular Dynamics Simulation.

Chronic inflammation contributes to several diseases, but its resolution is driven by specialized pro-resolving mediators (SPM) such as resolvin D1 (RvD1) and its epimer aspirin-triggered resolvin D1 (AT-RvD1), both biosynthesized from ω-3 fatty docosahexaenoic acid (DHA). RvD1 and AT-RvD1 have anti-inflammatory and pro-resolution potentials, and their effects could be mediated by formyl peptide receptor type 2 receptor ALX/FPR2, a G-protein-coupled receptor (GPCR). In this work, we performed 44 µs of molecular dynamics simulations with two complexes: FPR2@AT-RvD1 and FPR2@RvD1. Our results show the following: (i) in the AT-RvD1 simulations, the ALX/FPR2 receptor remained in the active state in 62% of the frames, while in the RVD1 simulations, the receptor remained in the active state in 74% of the frames; (ii) two residues, R201 and R205, of ALX/FPR2 appear, establishing interactions with both resolvins in all simulations (22 in total); (iii) RvD1 hydrogen bonds with R201 and R205 presented higher frequency than AT-RvD1; and (iv) residues R201 and R205 are the two receptor hotspots, demonstrated by the binding free calculations. Such results show that the ALX/FPR2 receptor remained in the active state for longer in the FPR2@RvD1 simulations than in the FPR2@AT-RvD1 simulations.

Developmental and homeostatic signaling transmitted by the G-protein coupled receptor FPR2.

Formyl peptide receptor 2 (FPR2) and its mouse counterpart Fpr2 are the members of the G protein-coupled receptor (GPCR) family. FPR2 is the only member of the FPRs that interacts with ligands from different sources. FPR2 is expressed in myeloid cells as well as epithelial cells, endothelial cells, neurons, and hepatocytes. During the past years, some unusual properties of FPR2 have attracted intense attention because FPR2 appears to possess dual functions by activating or inhibiting intracellular signal pathways based on the nature, concentration of the ligands, and the temporal and spatial settings of the microenvironment in vivo, the cell types it interacts with. Therefore, FPR2 controls an abundant array of developmental and homeostatic signaling cascades, in addition to its "classical" capacity to mediate the migration of hematopoietic and non-hematopoietic cells including malignant cells. In this review, we summarize recent development in FPR2 research, particularly in its role in diseases, therefore helping to establish FPR2 as a potential target for therapeutic intervention.

AZ2158 is a more potent formyl peptide receptor 1 inhibitor than the commonly used peptide antagonists in abolishing neutrophil chemotaxis.

Formyl peptide receptor 1 (FPR1), a G protein-coupled receptor expressed in phagocytes, recognizes short N-formylated peptides originating from proteins synthesized by bacteria and mitochondria. Such FPR1 agonists are important regulators of neutrophil functions and by that, determinants of inflammatory reactions. As FPR1 is implicated in promoting both pro-inflammatory and pro-resolving responses associated with inflammatory diseases, characterization of ligands that potently and selectively modulate FPR1 induced functions might be of high relevance. Accordingly, a number of FPR1 specific antagonists have been identified and shown to inhibit agonist binding or receptor down-stream signaling as well as neutrophil functions such as granule secretion and NADPH oxidase activity. The inhibitory effect on neutrophil chemotaxis induced by FPR1 agonists has generally not been part of basic antagonist characterization. In this study we show that the inhibitory effects on neutrophil chemotaxis of established FPR1 antagonists (i.e., cyclosporin H, BOC1 and BOC2) are limited. Our data demonstrate that the recently described small molecule AZ2158 is a potent and selective FPR1 antagonist in human neutrophils. In contrast to the already established FPR1 antagonists, AZ2158 also potently inhibits chemotaxis. Whereas the cyclosporin H inhibition was agonist selective, AZ2158 inhibited the FPR1 response induced by both a balanced and a biased FPR1 agonist equally well. In accordance with the species specificity described for many FPR1 ligands, AZ2158 was not recognized by the mouse orthologue of FPR1. Our data demonstrate that AZ2158 may serve as an excellent tool compound for further mechanistic studies of human FPR1 mediated activities.

Targeting formyl peptide receptor 1 with anteiso-C13-surfactin for neutrophil-dominant acute respiratory distress syndrome.

BACKGROUND AND PURPOSE: Acute respiratory distress syndrome (ARDS) is a catastrophic pulmonary inflammatory dysfunction with a high mortality rate. An overwhelming immune response by neutrophils is a key feature in infective or sterile ARDS. The formyl peptide receptor 1 (FPR1) is a crucial damage-sensing receptor for inflammatory reactions in the initiation and progression of neutrophil-mediated ARDS. However, effective targets for controlling dysregulated neutrophilic inflammatory injuries in ARDS are limited. EXPERIMENTAL APPROACH: Human neutrophils were used to explore the anti-inflammatory effects of cyclic lipopeptide anteiso-C13-surfactin (IA-1) from marine Bacillus amyloliquefaciens. The lipopolysaccharide-induced model of ARDS in mice was used to determine the therapeutic potential of IA-1 in ARDS. Lung tissues were harvested for histology analyses. KEY RESULTS: The lipopeptide IA-1 inhibited immune responses of neutrophils, including respiratory burst, degranulation, and expression of adhesion molecules. IA-1 inhibited the binding of N-formyl peptides to FPR1 in human neutrophils and in hFPR1-transfected HEK293 cells. We identified IA-1 as a competitive FPR1 antagonist, thus diminishing the downstream signalling pathways involving calcium, mitogen-activated protein kinases and Akt. Furthermore, IA-1 ameliorated the inflammatory damage to lung tissue, by decreasing neutrophil infiltration, reducing elastase release and oxidative stress in endotoxemic mice. CONCLUSION AND IMPLICATIONS: The lipopeptide IA-1 could serve as a therapeutic option for ARDS by inhibiting FPR1-mediated neutrophilic injury.

HCH6-1, an antagonist of formyl peptide receptor-1, exerts anti-neuroinflammatory and neuroprotective effects in cellular and animal models of Parkinson's disease.

Microglial activation-induced neuroinflammation contributes to onset and progression of sporadic and hereditary Parkinson's disease (PD). Activated microglia secrete pro-inflammatory and neurotoxic IL-1ß, IL-6 and TNF-α, which subsequently promote neurodegeneration. Formyl peptide receptor-1 (FPR1) of CNS microglia functions as pattern recognition receptor and is activated by N-formylated peptides, leading to microglial activation, induction of inflammatory responses and resulting neurotoxicity. In this study, it was hypothesized that FPR1 activation of microglia causes loss of dopaminergic neurons by activating inflammasome and upregulating IL-1ß, IL-6 or TNF-α and that FPR1 antagonist HCH6-1 exerts neuroprotective effect on dopaminergic neurons. FPR1 agonist fMLF induced activation of microglia cells by causing activation of NLRP3 inflammasome and upregulation and secretion of IL-1ß, IL-6 or TNF-α. Conditioned medium (CM) of fMLF-treated microglia cells, which contains neurotoxic IL-1ß, IL-6 and TNF-α, caused apoptotic death of differentiated SH-SY5Y dopaminergic neurons by inducing mitochondrial oxidative stress and activating pro-apoptotic signaling. FPR1 antagonist HCH6-1 prevented fMLF-induced activation of inflammasome and upregulation of pro-inflammatory cytokines in microglia cells. HCH6-1 co-treatment reversed CM of fMLF-treated microglia-induced apoptotic death of dopaminergic neurons. FPR1 antagonist HCH6-1 inhibited rotenone-induced upregulation of microglial marker Iba-1 protein level, cell death of dopaminergic neurons and motor impairment in zebrafish. HCH6-1 ameliorated rotenone-induced microglial activation, upregulation of FPR1 mRNA, activation of NLRP3 inflammasome, cell death of SN dopaminergic neurons and PD motor deficit in mice. Our results suggest that FPR1 antagonist HCH6-1 possesses anti-neuroinflammatory and neuroprotective effects on dopaminergic neurons by inhibiting microglial activation and upregulation of inflammasome activity and pro-inflammatory cytokines.

Formyl peptide receptor 2 is an emerging modulator of inflammation in the liver.

Formyl peptide receptors (FPRs), which are seven-membrane G-protein coupled receptors, recognize chemotactic signals to protect hosts from pathogenic infections and mediate inflammatory responses in the body. There are three isoforms of FPRs in humans-FPR1, FPR2, and FPR3-and they bind to N-formyl peptides, except FPR3, and to various endogenous agonists. Among FPR family members, FPR2 has a lower affinity for N-formyl peptides than FPR1 and binds with a wide range of endogenous or exogenous agonists. Thus, FPR2 is considered the most ambiguous member. Accumulating evidence has shown that FPR2 is involved in the host's defense against bacterial infection and inflammation in liver diseases, such as nonalcoholic fatty liver disease, liver fibrosis, and liver cancer, suggesting the pathophysiological relevance of FPR2 to the liver. However, FPR2 has been shown to promote or suppress inflammation, depending on the type of FPR2-expressing cell and FPR2-bound ligands in the liver. Therefore, it is important to understand FPR2's function per se and to elucidate the mechanism underlying immunomodulation initiated by ligand-activated FPR2 before suggesting FPR2 as a novel therapeutic agent for liver diseases. In this review, up-to-date knowledge of FPR2, with general information on the FPR family, is provided. We shed light on the dual action of FPR2 in the liver and discuss the hepatoprotective roles of FPR2 itself and FPR2 agonists in mediating anti-inflammatory responses.

The G-Protein-Coupled Formyl Peptide Receptor 2 Promotes Endothelial-Mesenchymal Transition in Diabetic Retinopathy.

INTRODUCTION: In proliferative diabetic retinopathy (PDR), retinal neovascularization is the essential pathogenic process that is linked to endothelial-to-mesenchymal transition (EndoMT) induced by high glucose (HG). This pathophysiological process may be regulated by a G-protein-coupled chemoattractant receptor FPR2 (mouse Fpr2), involved in inflammatory cell migration and proliferation. In the current study, we investigated the role of Fpr2 in regulating EndoMT and the underlying mechanisms during diabetic retinopathy progression. METHODS: FPR2 agonist or inhibitor was added to human microvascular endothelial cells (HMECs) exposed to normal glucose or HG. Morphologic, phenotypic, and functional changes of HMECs as well as the formation of microvasculature related to EndoMT were assessed. EndoMT biomarkers were detected in the retinal tissues of diabetic mice and fibrovascular epiretinal membranes (FVMs) from patients with PDR. RESULTS: HG upregulated FPR2 in HMECs, which triggered morphological changes, and the cells acquired mesenchymal phenotype, with enhanced cell migration, viability, and angiogenic process shown by tube formation and aortic ring sprouting. Inhibition of FPR2 attenuated HG-induced EndoMT and endothelial cell migration to form vessel-like tube structures. RNA sequence and protein analysis further revealed that inhibition of FPR2 decreased the expression of genes associated with EndoMT. ERK1/2 and P38 signaling pathway was activated in HMECs, promoting neovascularization in HG-induced EndoMT of HMECs. In vivo, increased expression of mesenchymal markers was detected in the retina of diabetic mice and FVMs from patients with PDR. FPR2 deficiency was associated with diminished EndoMT-related phenotypic changes in the retina of diabetic mice. CONCLUSIONS: FPR2 is actively involved in the progression of EndoMT that may contribute to the pathogenesis of PDR. Thus, FPR2 may be a potential therapeutic target for PDR.