Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Interferon-ß regulates proresolving lipids to promote the resolution of acute airway inflammation.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-ß (IFN-ß) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli-evoked lung injury, and suppresses production of IFN-ß and the proresolving lipid mediators 15-epi-LXA4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-ß delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-ß accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-ß-mediated resolution. These findings point to a pivotal role of IFN-ß in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-ß-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

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.

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.

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.

The anti-inflammatory effect of ε-viniferin by specifically targeting formyl peptide receptor 1 on human neutrophils.

The uncontrol respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of ε-viniferin, a lignan from the root of Vitis thunbergii var. thunbergii, inhibits N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP) induced respiratory burst by antagonizing formyl peptide receptor 1 in human neutrophils. Briefly, ε-viniferin specifically inhibited fMLP (0.1 µM: formyl peptide receptor 1 agonist or 1 µM: formyl peptide receptor 1, 2 agonist)-induced superoxide anion production in a concentration-dependent manner (IC50 = 2.30 ± 0.96 or 9.80 ± 0.21 µM, respectively) without affecting this induced by formyl peptide receptor 2 agonist (WKYMVM). ε-viniferin inhibited fMLP (0.1 µM)-induced phosphorylation of ERK, Akt, Src or intracellular calcium mobilization without affecting these caused by WKYMVM. The synergistic suppression of fMLP (1 µM)-induced superoxide anion production was observed only in the combination of ε-viniferin and formyl peptide receptor 2 antagonist (WRW4) but not in combination of ε-viniferin and formyl peptide receptor 1 antagonist (cyclosporine H). ε-viniferin inhibited FITC-fMLP binding to formyl peptide receptors. Moreover, the synergistic suppression of FITC-fMLP binding was observation only in the combination of ε-viniferin and WRW4 but not in other combinations. ATPγS induced superoxide anion production through formyl peptide receptor 1 in fMLP desensitized neutrophils and this effect was inhibited by ε-viniferin. The concentration-response curve of fMLP-induced superoxide anion was not parallel shifted by ε-viniferin. Furthermore, the inhibiting effect of ε-viniferin on fMLP-induced superoxide anion production was reversible. These results suggest that ε-viniferin is an antagonist of formyl peptide receptor 1 in a reversible and non-competitive manner.

Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo.

Psoriasis is a long-lasting inflammatory skin disease lacking proper cure. Dysregulated activation of neutrophils is a major pathogenic factor in psoriasis. Formyl peptide receptor 1 (FPR1) triggers neutrophil activation in response to bacteria- or mitochondria-derived N-formyl peptides, but its significance in neutrophilic psoriasis remains unknown. In this study, we discovered two derivatives of ursolic acid, 3ß-hydroxyurs-12,18-dien-28-oic acid (randialic acid B, RAB) and 3ß-hydroxyurs-12,19-dien-28-oic acid (tomentosolic acid, TA), as FPR1 inhibitors in human neutrophils with ability to suppress psoriatic symptoms in mice. Both RAB and TA, triterpenoids of traditional medicinal plant Ilex kaushue, selectively inhibited reactive oxygen species production, elastase release, and CD11b expression in human neutrophils activated by FPR1, but not non-FPR1 agonists. Importantly, RAB and TA inhibited the binding of N-formyl peptide to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells, indicating FPR1 antagonism. Moreover, in assays induced by various concentrations of FPR1 agonist, both RAB and TA acted competitively for its binding to the FPR1 receptor. The FPR1-downstream signaling such as Ca2+ mobilisation and activation of Akt and MAPKs was also competitively inhibited. In addition, imiquimod-induced psoriasis-like symptoms, including epidermal hyperplasia, desquamation with scaling, neutrophil skin infiltration, and transepidermal water loss were significantly reduced by both RAB and TA. The results illustrate a possible role of human neutrophils FPR1 receptor in psoriasis-like inflammation. Accordingly, triterpenoids RAB and TA represent novel FPR1 antagonists and exhibit therapeutic potential for treating neutrophilic inflammatory skin diseases.

Formyl peptide receptor 2 (FPR2) antagonism is a potential target for the prevention of Brucella abortus 544 infection.

Here, we explore the potential role of formyl peptide receptor 2 (FPR2) during Brucella abortus infection. FPR2 manipulation affected B. abortus internalization but not its growth within macrophages. During the activation of FPR2 induced by its agonist AGP-8694, a high level of Brucella uptake was accompanied by an increase in ERK phosphorylation, while intracellular survival at 24 h postincubation was observed to be associated with slightly reduced nitrite accumulation but augmented superoxide anion production. Attenuated secretion of IL-6 and IL-10 were observed 48 h postincubation in the bone marrow-derived macrophages (BMDMs) treated with the FPR2 antagonist WRW4. An opposite pattern of bacterial uptake was observed upon treatment with the FPR2 antagonist, but no significant changes in the activation of MAPKs or the production of nitrite or superoxide anion were observed. Interestingly, AGP-8694 treatment of mice did not lead to differences in spleen or liver weight but slightly enhanced bacterial proliferation was observed in the spleen. Although the weights of the spleen or liver did not differ, WRW4 treatment led to reduced bacterial proliferation in the spleen. Furthermore, FPR2 antagonist treatment was associated with high serum levels of the proinflammatory cytokines IL-12, TNF-α, IFN-γ and MCP-1, while the production of TNF-α was inhibited in AGP-8694-treated mice. IL-6 and IL-10 levels were slightly increased in AGP-8694-treated mice at 24 h postinfection. Our findings demonstrated the contribution of FPR2 via manipulating this receptor using its reported agonist AGP-8694 and antagonist WRW4 in both in vitro and in vivo systems. Although activation of the receptor did not consistently induced Brucella infection, FPR2 inhibition may be a promising strategy to treat brucellosis in animals which encourages further investigation.

Electroacupuncture decreases inflammatory pain through a pro-resolving mechanism involving the peripheral annexin A1-formyl peptide receptor 2/ALX-opioid receptor pathway.

The pro-resolving mechanism is a recently described endogenous process that controls inflammation. The present study evaluated components of this mechanism, including annexin 1 (ANXA1) and the formyl peptide receptor 2/ALX (FPR2/ALX) receptor, in the antihyperalgesic effect induced by electroacupuncture (EA) in an animal model of persistent peripheral inflammation. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2-10 Hz, ST36-SP6) or subcutaneous BML-111 injection (FPR2/ALX agonist) for 5 consecutive days. In a separate set of experiments, on the first and fifth days after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or naloxone (non-selective opioid receptor antagonist) before EA or BML-111 injection. Paw protein levels of FPR2/ALX and ANXA1 were evaluated on the second day after CFA injection by western blotting technique. EA and BML-111 reduced mechanical hyperalgesia. I.pl. naloxone or WRW4 prevented the antihyperalgesic effect induced by either EA or BML-111. EA increased ANXA1 but did not alter FPR2/ALX receptor levels in the paw. Furthermore, i.pl. pretreatment with WRW4 prevented the increase of ANXA1 levels induced by EA. This work demonstrates that the EA antihyperalgesic effect on inflammatory pain involves the ANXA1/FPR2/ALX pro-resolution pathway. This effect appears to be triggered by the activation of FPR2/ALX receptors and crosstalk communication with the opioid system.

Resolvin D1 Attenuates Innate Immune Reactions in Experimental Subarachnoid Hemorrhage Rat Model.

Excessive inflammation is a major cause contributing to early brain injury (EBI) and is associated with negative or catastrophic outcomes of subarachnoid hemorrhage (SAH). Resolvin D1 (RvD1) exerts strong anti-inflammatory and pro-resolving effects on either acute or chronic inflammation of various origin. Henceforth, we hypothesized that RvD1 potentially attenuates excessive inflammation in EBI following SAH. Therefore, we generated a filament perforation SAH model and administered 3 different doses (0.3, 0.6, and 1.2 nmol) of RvD1 after experimental SAH. Neurological scores, brain edema, and blood-brain barrier integrity were evaluated; besides, neutrophil infiltration, neuronal deaths, and microglial pro-inflammatory polarization were observed using histopathology or immunofluorescence staining, western blots, and qPCR. After confirming the effectiveness of RvD1 in SAH, we administered the FPR2-specific antagonist Trp-Arg-Trp-Trp-Trp-Trp-NH2 (WRW4) 30 min before SAH establishment to observe whether this compound could abolish the anti-inflammatory effect of RvD1. Altogether, our results showed that RvD1 exerted a strong anti-inflammatory effect and markedly reduced neutrophil infiltration and microglial pro-inflammatory activation, leading to remarkable improvements in neurological function and brain tissue restoration. After addition of WRW4, the anti-inflammatory effects of RvD1 were abolished. These results indicated that RvD1 could exert a good anti-inflammatory effect and alleviate EBI, which suggested that RvD1 might be a novel therapeutic alternative for SAH-induced injury.

N-Formylated Peptide Induces Increased Expression of Both Formyl Peptide Receptor 2 (Fpr2) and Toll-Like Receptor 9 (TLR9) in Schwannoma Cells-An In Vitro Model for Early Inflammatory Profiling of Schwann Cells.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCß. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.

Enterobactin induces the chemokine, interleukin-8, from intestinal epithelia by chelating intracellular iron.

Iron is an indispensable nutrient for both mammals and microbes. Bacteria synthesize siderophores to sequester host iron, whereas lipocalin 2 (Lcn2) is the host defense protein that prevent this iron thievery. Enterobactin (Ent) is a catecholate-type siderophore that has one of the strongest known affinities for iron. Intestinal epithelial cells (IECs) are adjacent to large microbial population and are in contact with microbial products, including Ent. We undertook this study to investigate whether a single stimulus of Ent could affect IEC functions. Using three human IEC cell-lines with differential basal levels of Lcn2 (i.e. HT29 < DLD-1 < Caco-2/BBe), we demonstrated that iron-free Ent could induce a dose-dependent secretion of the pro-inflammatory chemokine, interleukin 8 (IL-8), in HT29 and DLD-1 IECs, but not in Caco-2/BBe. Ent-induced IL-8 secretion was dependent on chelation of the labile iron pool and on the levels of intracellular Lcn2. Accordingly, IL-8 secretion by Ent-treated HT29 cells could be substantially inhibited by either saturating Ent with iron or by adding exogenous Lcn2 to the cells. IL-8 production by Ent could be further potentiated when co-stimulated with other microbial products (i.e. flagellin, lipopolysaccharide). Water-soluble microbial siderophores did not induce IL-8 production, which signifies that IECs are specifically responding to the lipid-soluble Ent. Intriguingly, formyl peptide receptor (FPR) antagonists (i.e. Boc2, cyclosporine H) abrogated Ent-induced IL-8, implicating that such IEC response could be, in part, dependent on FPR. Taken together, these results demonstrate that IECs sense Ent as a danger signal, where its recognition results in IL-8 secretion.

Application of small molecule FPR1 antagonists in the treatment of cancers.

The formylpeptide receptor-1 (FPR1) is a member of the chemotactic GPCR-7TM formyl peptide receptor family, whose principle function is in trafficking of various leukocytes into sites of bacterial infection and inflammation. More recently, FPR1 has been shown to be expressed in different types of cancer and in this context, plays a significant role in their expansion, resistance and recurrence. ICT12035 is a selective and potent (30 nM in calcium mobilisation assay) small molecule FPR1 antagonist. Here, we demonstrate the efficacy of ICT12035, in a number of 2D and 3D proliferation and invasion in vitro assays and an in vivo model. Our results demonstrate that targeting FPR1 by a selective small molecule antagonist, such as ICT12035, can provide a new avenue for the treatment of cancers.

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.

Inhibition of formyl peptide receptors improves the outcome in a mouse model of Alzheimer disease.

BACKGROUND: An important hallmark of Alzheimer's disease (AD) is the increase of Aß1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia cell function represents an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aß1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model. METHODS: To address this question, APP/PS1 double-transgenic AD mice were treated for 20 weeks with either the pro-inflammatory FPR agonist fMLF, the FPR1/2 antagonist Boc2 or the anti-inflammatory FPR2 agonist Ac2-26. Spatial learning and memory were evaluated using a Morris water maze test. Immunohistological staining, gene expression studies, and flow cytometry analyses were performed to study neuronal loss, gliosis, and Aß-load in the hippocampus and cortex, respectively. RESULTS: FPR antagonism by Boc2-treatment significantly improved spatial memory performance, reduced neuronal pathology, induced the expression of homeostatic growth factors, and ameliorated microglia, but not astrocyte, reactivity. Furthermore, the elevated levels of amyloid plaques in the hippocampus were reduced by Boc2-treatment, presumably by an induction of amyloid degradation. CONCLUSIONS: We suggest that the modulation of FPR signaling cascades might be considered as a promising therapeutic approach for alleviating the cognitive deficits associated with early AD. Additional studies are now needed to address the downstream effectors as well as the safety profile of Boc2.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

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.

Targeting the Formyl Peptide Receptor type 1 to prevent the adhesion of ovarian cancer cells onto mesothelium and subsequent invasion.

BACKGROUND: The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84-95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available. METHODS: Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson's Chi-square (χ2) test. RESULTS: Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84-95 Abs, or to the RI-3 peptide, blocking the uPAR84-95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1. CONCLUSIONS: Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84-95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.

A New Formyl Peptide Receptor-1 Antagonist Conjugated Fullerene Nanoparticle for Targeted Treatment of Degenerative Disc Diseases.

Intervertebral disc degeneration associated back pain is the most common cause of disability worldwide; however, no safe and effective treatments have been available. Here, we report a new functionalized nanofullerene conjugated with a peptide that binds specifically to a formyl peptide receptor-1 (FPR-1) expressed on activated macrophages. The new nanoparticle (aka FT-C60) was synthesized by conjugating carboxyl-C60 with the primary amine group of the peptide with a fluorescence dye for easy detection. The new nanoparticle was characterized by X-ray photoelectron spectroscopy, mass spectroscopy, and gel electrophoresis. It possessed effective radical (hydroxyl and superoxide anions) scavenging capabilities in electron paramagnetic resonance spectroscopy. In cultured cells, the nanoparticle FT-C60 demonstrated preferential binding to FPR-1 on activated macrophages and significantly attenuated mRNA expressions of proinflammatory factors including interleukin-6, interleukin-1, tumor necrosis factor-alpha, and cyclooxygenase-2. In vivo animal studies exhibited that a single intravenous injection of FT-C60 effectively alleviated pain in an established mouse model of radiculopathy for up to post-operation day (POD) 12. Ex vivo near-infrared fluorescence imaging of the mouse spine confirmed the targeting property of FT-C60 toward the injured disc on POD 14. Quantitative analysis of histological staining on spine sections showed that nanoparticle FT-C60 dramatically reduced inflammation at the local injury site compared to injury only on POD 7. In summary, we developed a novel targeted nanoparticle for treatment of lumbar radiculopathy by systemic delivery. This is a first-of-its-kind study for developing a novel class of targeted and systemic nanoparticle therapeutics to treat degenerative disc diseases.

FPR1 is the plague receptor on host immune cells.

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.