Renoprotective effect of isoliquiritigenin on cisplatin-induced acute kidney injury through inhibition of FPR2 in macrophage.

Acute kidney injury (AKI) is a serious complication in critically ill patients. Accumulating evidences indicated that macrophages play an important pro-inflammatory role in AKI and isoliquiritigenin (ISL) can inhibit macrophagic inflammation, but its role in AKI and the underlying mechanism are unknown. The present study aims to investigate the renoprotective effect of ISL on AKI and the role of Formyl peptide receptors 2 (FPR2) in this process. In this study, cisplatin-induced AKI model and lipopolysaccharide-induced macrophage inflammatory model were employed to perform the in vivo and in vitro experiments. The results showed that ISL strongly relieved kidney injury and inhibited renal inflammation in vivo and suppress macrophagic inflammatory response in vitro. Importantly, it was found that FPR2 was significantly upregulated compared to the control group in AKI and LPS-induced macrophage, whereas it was strongly suppressed by ISL. Interestingly, overexpression of FPR2 with transfection of pcDNA3.1-FPR2 effectively reversed the anti-inflammatory effect of ISL in macrophage, suggesting that FPR2 may be the potential target for ISL to prevent inflammation and improve kidney injury of AKI. Take together, these findings indicated that ISL improved cisplantin-induced kidney injury by inhibiting FPR2 involved macrophagic inflammation, which may provide a potential therapeutic option for AKI.

Suppression of FPR2 expression inhibits inflammation in preeclampsia by improving the biological functions of trophoblast via NF-κB pathway.

PURPOSE: The dysfunction of trophoblast during inflammation plays an important role in PE. Formyl peptide receptor 2 (FPR2) plays crucial roles in the development of inflammation-associated disease. This present study aimed to explore the effect of FPR2 on a trophoblast cellular model of preeclampsia. METHODS: The expression of FPR2 in placenta was detected by immunohistochemical staining and western blotting. Transfection of siRNA was used to knockdown FPR2 in HTR-8/SVneo cells. Inflammatory cytokines were detected by ELISA. CCK8, Transwell, wound healing, FACS and tube formation assays were performed to observe the abilities of cell proliferation, migration, invasion, apoptosis and angiogenesis. Western blotting was implemented to clarify that NF-κB signaling pathway was downstream of FPR2. RESULTS: The expression levels of FPR2 were higher in placental tissues of patients with PE. Knockdown of FPR2 expression by siFPR2 or inhibition of its activity by WRW4 decreased the release of proinflammatory cytokines in HTR8/SVneo cells treated with LPS. Knockdown of FPR2 expression or inhibition of its activity further reversed the LPS-induced attenuation of the proliferation, migration, invasion and angiogenesis and increase in apoptosis in HTR8/SVneo cells. Moreover, the NF-κB signaling pathway was activated in both placental tissues of patients with PE and LPS-treated HTR8/SVneo cells. However, the activation was attenuated when FPR2 was knocked down or inhibited. CONCLUSION: Suppression of FPR2 expression alleviated the effects of inflammation induced by LPS on trophoblasts via the NF-κB signaling pathway, which provided a novel and potential strategy for the treatment of PE.

FPR2-based anti-inflammatory and anti-lipogenesis activities of novel meroterpenoid dimers from Ganoderma.

Four pairs of novel meroterpenoid dimers, (±)-applandimeric acids A-D (1-4) with an unprecedented spiro[furo[3,2-b]benzofuran-3,2'-indene] core were isolated from the fruiting bodies of Ganoderma applanatum. Their planar structures were unambiguously determined via extensive spectroscopic analysis. Their relative and absolute configurations were confirmed through calculated internuclear distance, coupling constant, 13C NMR with DP4 + analysis and electronic circular dichroism (ECD). Furthermore, the molecular docking-based method was used to evaluate their interaction with formyl peptide receptor 2 (FPR2) associated with inflammation. Interestingly, (±)-applandimeric acid D (4) can bond with FPR2 by some key hydrogen bonds. Furthermore, an in vitro bioassay verified that 4 can inhibit the expression of FPR2 with IC50 value of 7.93 µM. In addition, compared to the positive control LiCl (20 mM), 4 showed comparable anti-lipogenesis activity at the concentration of 20 µM. Meanwhile, 4 can suppress the protein levels of peroxisome proliferators-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-ß (C/EBP-ß), adipocyte fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS) through activating AMP-activated protein kinase (AMPK) signaling pathway. Thus, our findings indicate that compound 4 could be a lead compound to treat obesity and obesity-related diseases by inhibiting lipid accumulation in adipocyte and alleviating inflammation.

Formyl-Peptide Receptor Activation Enhances Phagocytosis of Community-Acquired Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Formyl-peptide receptors (FPRs) are important pattern recognition receptors that sense specific bacterial peptides. Formyl-peptide receptors are highly expressed on neutrophils and monocytes, and their activation promotes the migration of phagocytes to sites of infection. It is currently unknown whether FPRs may also influence subsequent processes such as bacterial phagocytosis and killing. Staphylococcus aureus, especially highly pathogenic community-acquired methicillin-resistant S aureus strains, release high amounts of FPR2 ligands, the phenol-soluble modulins. METHODS: We demonstrate that FPR activation leads to upregulation of complement receptors 1 and 3 as well as FCγ receptor I on neutrophils and, consequently, increased opsonic phagocytosis of S aureus and other pathogens. RESULTS: Increased phagocytosis promotes killing of S aureus and interleukin-8 release by neutrophils. CONCLUSIONS: We show here for the first time that FPRs govern opsonic phagocytosis. Manipulation of FPR2 activation could open new therapeutic opportunities against bacterial pathogens.

Human cathelicidin antimicrobial peptide LL-37 promotes lymphangiogenesis in lymphatic endothelial cells through the ERK and Akt signaling pathways.

LL-37, the only member of the cathelicidin family of cationic antimicrobial peptides in humans has been shown to exhibit a wide variety of biological actions in addition to its antimicrobial activity. However, the lymphangiogenic effect of LL-37 has not been elucidated yet. In this study, we examined the effects of LL-37 on lymphangiogenesis and evaluated the underlying molecular mechanisms. LL-37 treatment significantly increased the migration and tube-like formation of human dermal lymphatic microvascular endothelial cells (HDLECs) and promoted the expression of lymphangiogenic factor in HDLECs. Treatment with LL-37 increased phosphorylation of ERK and Akt proteins in HDLECs, and pretreatment with ERK and Akt inhibitors significantly blocked the LL-37-induced HDLEC migration and tube-like formation. Furthermore, to investigate the involvement of formyl peptide receptor-like 1 (FPRL1) signaling in LL-37-induced lymphangiogenesis, HDLECs were treated with an FPRL1 antagonist. Pretreatment with the FPRL1 antagonist inhibited LL-37-induced phosphorylation of ERK and Akt proteins and attenuated LL-37-induced HDLEC migration and tube-like formation. These data indicated that LL-37 induces lymphangiogenesis in lymphatic endothelial cells via FPRL1, and the activation of the ERK and Akt-dependent signaling pathways.

The Lipoxin Receptor/FPR2 Agonist BML-111 Protects Mouse Skin Against Ultraviolet B Radiation.

Excessive exposure to UV, especially UVB, is the most important risk factor for skin cancer and premature skin aging. The identification of the specialized pro-resolving lipid mediators (SPMs) challenged the preexisting paradigm of how inflammation ends. Rather than a passive process, the resolution of inflammation relies on the active production of SPMs, such as Lipoxins (Lx), Maresins, protectins, and Resolvins. LXA4 is an SPM that exerts its action through ALX/FPR2 receptor. Stable ALX/FPR2 agonists are required because SPMs can be quickly metabolized within tissues near the site of formation. BML-111 is a commercially available synthetic ALX/FPR2 receptor agonist with analgesic, antioxidant, and anti-inflammatory properties. Based on that, we aimed to determine the effect of BML-111 in a model of UVB-induced skin inflammation in hairless mice. We demonstrated that BML-111 ameliorates the signs of UVB-induced skin inflammation by reducing neutrophil recruitment and mast cell activation. Reduction of these cells by BML-111 led to lower number of sunburn cells formation, decrease in epidermal thickness, collagen degradation, cytokine production (TNF-α, IL-1ß, IL-6, TGF, and IL-10), and oxidative stress (observed by an increase in total antioxidant capacity and Nrf2 signaling pathway), indicating that BML-111 might be a promising drug to treat skin disorders.

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus.

The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

Resolvin D1 Programs Inflammation Resolution by Increasing TGF-ß Expression Induced by Dying Cell Clearance in Experimental Autoimmune Neuritis.

UNLABELLED: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies (AIDP), an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. EAN and AIDP are characterized by self-limitation with spontaneous recovery; however, endogenous pathways that regulate inflammation resolution in EAN and AIDP remain elusive. A pathway of endogenous mediators, especially resolvins and clearance of apoptotic cells, may be involved. Here, we determined that resolvin D1 (RvD1), its synthetic enzyme, and its receptor were greatly increased in PNS during the recovery stage of EAN. Both endogenous and exogenous RvD1 increased regulatory T (Treg) cell and anti-inflammatory macrophage counts in PNS, enhanced inflammation resolution, and promoted disease recovery in EAN rats. Moreover, RvD1 upregulated the transforming growth factor-ß (TGF-ß) level and pharmacologic inhibition of TGF-ß signaling suppressed RvD1-induced Treg cell counts, but not anti-inflammatory macrophage counts, and RvD1-improved inflammation resolution and disease recovery in EAN rats. Mechanistically, the RvD1-enhanced macrophage phagocytosis of apoptotic T cells leading to reduced apoptotic T-cell accumulation in PNS induced TGF-ß production and caused Treg cells to promote inflammation resolution and disease recovery in EAN. Therefore, these data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest its potential as a therapeutic target in human neuropathies. SIGNIFICANCE STATEMENT: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies, an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. Here, we demonstrated that resolvin D1 (RvD1) promoted macrophage phagocytosis of apoptotic T cells in PNS, thereby upregulating transforming growth factor-ß by macrophages, increased local Treg cell counts, and finally promoted inflammation resolution and disease recovery in EAN. These data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis.

Human Mesenchymal Stem (Stromal) Cells Promote the Resolution of Acute Lung Injury in Part through Lipoxin A4.

Previous studies demonstrated that bone marrow-derived mesenchymal stem (stromal) cells (MSCs) reduce the severity of acute lung injury in animal models and in an ex vivo perfused human lung model. However, the mechanisms by which MSCs reduce lung injury are not well understood. In the present study, we tested the hypothesis that human MSCs promote the resolution of acute lung injury in part through the effects of a specialized proresolving mediator lipoxin A4 (LXA4). Human alveolar epithelial type II cells and MSCs expressed biosynthetic enzymes and receptors for LXA4. Coculture of human MSCs with alveolar epithelial type II cells in the presence of cytomix significantly increased the production of LXA4 by 117%. The adoptive transfer of MSCs after the onset of LPS-induced acute lung injury (ALI) in mice led to improved survival (48 h), and blocking the LXA4 receptor with WRW4, a LXA4 receptor antagonist, significantly reversed the protective effect of MSCs on both survival and the accumulation of pulmonary edema. LXA4 alone improved survival in mice, and it also significantly decreased the production of TNF-α and MIP-2 in bronchoalveolar lavage fluid. In summary, these experiments demonstrated two novel findings: human MSCs promote the resolution of lung injury in mice in part through the proresolving lipid mediator LXA4, and LXA4 itself should be considered as a therapeutic for acute respiratory distress syndrome.

Non-peptide ligand binding to the formyl peptide receptor FPR2--A comparison to peptide ligand binding modes.

Ligands of the FPR2 receptor initiate many signaling pathways including activation of phospholipase C, protein kinase C, the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/protein kinase B pathway. The possible actions include also calcium flux, superoxide generation, as well as migration and proliferation of monocytes. FPR2 activation may induce a pro- and anti-inflammatory effect depending on the ligand type. It is also found that this receptor is involved in tumor growth. Most of currently known FPR2 ligands are agonists since they were designed based on N-formyl peptides, which are natural agonists of formyl receptors. Since the non-peptide drugs are indispensable for effective treatment strategies, we performed a docking study of such ligands employing a generated dual template homology model of the FPR2 receptor. The study revealed different binding modes of particular classes of these drugs. Based on the obtained docking poses we proposed a detailed location of three hydrophobic pockets in orthosteric binding site of FPR2. Our model emphasizes the importance of aromatic stacking, especially with regard to residues His102(3.29) and Phe257(6.51), for binding of FPR2 ligands. We also identified other residues important for non-peptide ligand binding in the binding site of FPR2.

The leukocyte chemotactic receptor FPR2, but not the closely related FPR1, is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop.

Lipidated peptides (pepducins) can activate certain G-protein coupled receptors (GPCRs) through a unique allosteric modulation mechanism involving cytosolic receptor domains. Pepducins with the amino acid sequence of the third intracellular loop of the neutrophil formyl peptide receptors (FPRs) as a common denominator were N-terminally conjugated with palmitic acid. F2Pal16, containing the 16 amino acids present in the third intracellular loop of FPR2, induced superoxide production in human neutrophils and the activity was sensitive to FPR2 antagonists. Cells over-expressing FPR2 were similarly responsive and responded with a transient increase in cytosolic calcium. No such effects were observed with the corresponding FPR1 pepducin. The peptide alone, lacking palmitic acid, did not activate neutrophils. A ten amino acid long pepducin F2Pal10, that was a more potent neutrophil activator than F2Pal16, was used for amino acid substitution studies. The sequences of FPR1 and FPR2 in the third intracellular loop differ by only two amino acids, and a pepducin with the FPR2-specific K231 replaced by the FPR1-specific Q231 lost all activity. The active F2Pal10 pepducin also triggered a response in cells expressing a mutated FPR2 with the third intracellular loop identical to that of FPR1. The data presented suggest that the same signaling pathways are activated when the signaling cascade is initiated by a classical receptor agonist (outside-in signaling) and when signaling starts on the cytosolic side of the membrane by a pepducin (inside-in signaling). A fundamental difference is also disclosed between the two neutrophil FPRs regarding their sensitivities to third intracellular loop pepducins.

A membrane-tethering pepducin that inhibits formyl peptide receptor 2-induced signaling.

Since formyl peptide receptor 2 (FPR2) plays a key role in the regulation of innate immune response and inflammation, it has been a hot topic to develop molecules which inhibit FPR2-induced cellular responses. In this study, we investigated the effect of an FPR2-derived pepducin in human neutrophils and human umbilical vein endothelial cells (HUVECs). The pepducin (F2pal-12) selectively inhibited FPR2 agonists (MMK-1 and serum amyloid A)-stimulated neutrophil chemotaxis. MMK-1-stimulated superoxide anion production was also inhibited by F2pal-12. HUVECs also express FPR2; FPR2 agonists-stimulated HUVECs migration and tube formation were also selectively inhibited by F2pal-12 but not by scrambled control pepducin. Since FPR2 mediates inflammatory response by inducing chemotactic migration of inflammatory cells, F2pal-12 can be used as a useful material to modulate FPR2-mediated inflammatory responses.

Immuno-chemotherapeutic platinum(IV) prodrugs of cisplatin as multimodal anticancer agents.

There is growing consensus that the clinical therapeutic efficacy of some chemotherapeutic agents depends on their off-target immune-modulating effects. Pt anticancer drugs have previously been identified to be potent immunomodulators of both the innate and the adaptive immune system. Nevertheless, there has been little development in the rational design of Pt-based chemotherapeutic agents to exploit their immune-activating capabilities. The FPR1/2 formyl peptide receptors are highly expressed in immune cells, as well as in many metastatic cancers. Herein, we report a rationally designed multimodal Pt(IV) prodrug containing a FPR1/2-targeting peptide that combines chemotherapy with immunotherapy to achieve therapeutic synergy and demonstrate the feasibility of this approach.

BML-111 attenuates hemorrhagic shock-induced acute lung injury through inhibiting activation of mitogen-activated protein kinase pathway in rats.

BACKGROUND: Hemorrhagic shock activates cellular stress signals and can lead to systemic inflammatory response, organ injury, and death. Mitogen-activated protein kinase (MAPK) acts as a sensor of tissue injury in models of ischemia-reperfusion injury. Lipoxins are endogenous lipid mediators with potent anti-inflammatory and pro-resolving actions. We hypothesized that BML-111 (a lipoxin A4-receptor agonist) attenuates hemorrhagic shock-induced acute lung injury (ALI) through inhibiting activation of the MAPK pathway. METHODS: We randomized Sprague-Dawley rats into four groups: sham, hemorrhagic shock-resuscitation (HS), HS plus BML-111 (BML-111), and HS plus BML-111 and BOC-2 (BOC-2). Two hours after resuscitation, we collected samples of lung. We obtained bronchoalveolar lavage fluid for neutrophil count. We performed optical microscopy to examine pathologic changes in lungs. Wet/dry ratios, myeloperoxidase expression, interleukin (IL)-1ß and IL-6 levels in lung were measured. We evaluated MAPK activation and the DNA binding activity of activator protein-1 in lung. RESULTS: Treatment with BML-111 reduced the lung damage and wet/dry ratio, neutrophil count in bronchoalveolar lavage fluid, expression of myeloperoxidase, and production of IL-1ß and IL-6 in lung. Phosphorylation of MAPK was also decreased by BML-111 in lung. Furthermore, the DNA binding activity of activator protein-1 was blocked by BML-111. An antagonist of the lipoxin A4-receptor, BOC-2, reversed the protective effect of BML-111 on ALI induced by hemorrhagic shock. CONCLUSIONS: This study indicates that BML-111 attenuated hemorrhagic shock-induced ALI via the MAPK/activator protein-1 signaling pathway. Therefore, BML-111 may have therapeutic potential for hemorrhagic shock-induced ALI.

Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2.

The role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast tumor cell lines. In MCF-7 cells, ANXA1-targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well-characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin A(4) (LXA(4)), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MCF-7 and MDA-MB-231 cells that was attenuated by incubation with FPR2 antagonists WRW(4) (1 µM) or Boc2 (100 nM) or by siRNA against FPR2. FCS-induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNA against FPR2 and, to a lesser extent, FPR1. LXA(4) increased phosphorylation of Akt, p70(S6K) but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA(4) were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohistochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen-activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.

Lipoxin A4 is a novel estrogen receptor modulator.

Inflammation is intimately linked with naturally occurring remodeling events in the endometrium. Lipoxins comprise a group of short-lived, nonclassic eicosanoids possessing potent anti-inflammatory and proresolution properties. In the present study, we investigated the role of lipoxin A(4) (LXA(4)) in the endometrium and demonstrated that 15-LOX-2, an enzyme necessary for LX biosynthesis, is expressed in this tissue. Our results establish that LXA(4) possesses robust estrogenic activity through its capacity to alter ERE transcriptional activity, as well as expression of estrogen-regulated genes, alkaline phosphatase activity, and proliferation in human endometrial epithelial cells. Interestingly, LXA(4) also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. This estrogenic activity was directly mediated through estrogen receptors (ERs). Subsequent investigations determined that the actions of LXA(4) are exclusively mediated through ERα and closely mimic those of the potent estrogen 17ß-estradiol (E2). In binding assays, LXA(4) competed with E2 for ER binding, with an IC(50) of 46 nM. Furthermore, LXA(4) exhibited estrogenic activity in vivo, increasing uterine wet weight and modulating E2-regulated gene expression. These findings reveal a previously unappreciated facet of LXA(4) bioactions, implicating this lipid mediator in novel immunoendocrine crosstalk mechanisms.

A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.

The members of the formyl peptide receptor (FPR) family are involved in the sensing of chemoattractant substances, including bacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractant receptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptide receptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks the FPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr and identical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis induced by the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar range of FLIPr, its antagonism of the FPR is approximately 100-fold more potent than that of FLIPr and comparable to that of CHIPS. The second N-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, the deletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with that of CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmed that FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus, FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulate these chemoattractant receptors.

Involvement of Phospholipase D 1 and 2 in the subcellular localization and activity of formyl-peptide-receptors in the human colonic cell line HT29.

Epithelial cells of the alimentary tract play a central role in the mucosal host defence against pathogens and in the recognition of agonists that interact with mucosal surfaces. In particular, the formyl peptide receptor (FPR) family and their three human subtypes: FPR, formyl-peptide-receptor-like-1 (FPRL1) and FPRL2, are involved in the host defence against pathogens that mediate epithelial responses thus upregulating inflammation. To elucidate the mechanisms by which FPR function, we examined the influence of phospholipase D (PLD) 1 and 2 on the activity and signal transduction of human enterocytes cell line HT29. PLD is a key enzyme involved in secretion, endocytosis and receptor signalling. We inhibited PLD1 and 2 by small interference RNA (siRNA) and determined the activity of formyl peptide receptors using Western blotting and cAMP level measurements. We then analyzed the distribution of formyl peptide receptors FPR, FPRL1 and FPRL2 compared to a control. In this study, we demonstrated that the depletion of PLD1 and 2 resulted in a marked reduction of formyl peptide receptor activity due to inhibited extracellular-signal regulated kinases 1/2 (ERK1/2), phosphorylation and cAMP level reduction. In addition, we observed an intracellular accumulation of FPR, FPRL1 and FPRL2 as a result of receptor recycling inhibition using fluorescence microscopy. The constitutive internalization rate was unaffected. Our results support the importance of PLD1 and 2 in formyl peptide receptor function and the role of endocytosis, receptor recycling and reactivation for receptor activity.

Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.

Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) < or = 4 microM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 270 +/- 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving to be an efficient means to screen for selective receptor ligand probes.

FPRL1-mediated induction of superoxide in LL-37-stimulated IMR90 human fibroblast.

Molecular mechanisms underlying the generation of reactive oxygen species in LL-37-stimulated cells are poorly understood. Previously, we demonstrated that in human fibroblasts the exposure to WKYMVm induced p47(phox) phosphorylation and translocation and, in turn, NADPH oxidase activation. These effects were mediated by the activation of the Formyl-peptide receptor-like 1 (FPRL1) and the downstream signaling involved ERKs phosphorylation and PKCalpha- and PKCdelta-activation. Since LL-37 uses FPRL1 as a receptor to mediate its action on several cell types, we investigated in LL-37-stimulated IMR90 cells molecular mechanisms involved in NADPH-dependent superoxide generation. The exposure to LL-37, which is expressed in fibroblasts, induced ERKs activation, p47(phox) phosphorylation and translocation as well as NADPH oxidase activation. These effects were prevented by pertussis toxin, PD098059 and WRWWWW, a FPRL1-selective antagonist. Furthermore, the stimulation with LL-37 of HEK293 cells, transfected to stably express FPRL1, induced a rapid activation of ERKs and p47(phox) phosphorylation.