The Lipidated Peptidomimetic Lau-((S)-Aoc)-(Lys-βNphe)6-NH2 Is a Novel Formyl Peptide Receptor 2 Agonist That Activates Both Human and Mouse Neutrophil NADPH Oxidase
2016; Elsevier BV; Volume: 291; Issue: 38 Linguagem: Inglês
10.1074/jbc.m116.736850
ISSN1083-351X
AutoresAndré Holdfeldt, Sarah Line Skovbakke, Malene Winther, Michael Gabl, Christina Nielsen, Iris Perez-Gassol, Camilla Josephine Larsen, Ji Ming Wang, Anna Karlsson, Cláes Dahlgren, Huamei Forsman, Henrik Franzyk,
Tópico(s)Immune Response and Inflammation
ResumoNeutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stable α-peptide/β-peptoid hybrid Lau-((S)-Aoc)-(Lys-βNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat ((Lys-βNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases. Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stable α-peptide/β-peptoid hybrid Lau-((S)-Aoc)-(Lys-βNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat ((Lys-βNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases. The molecular basis for directional neutrophil migration toward inflammatory sites is their ability to sense "danger signals" produced by microorganisms (i.e. pathogen-associated molecular patterns) and damaged host cells or tissues (i.e. damage-associated molecular patterns) (1Kolaczkowska E. Kubes P. Neutrophil recruitment and function in health and inflammation.Nat. Rev. Immunol. 2013; 13: 159-175Crossref PubMed Scopus (3142) Google Scholar). Human neutrophils express two members of the formyl peptide receptor family (FPR1 3The abbreviations used are: FPR, formyl peptide receptor; βNphe, N-phenylmethyl-β-alanine; Aoc, 2-aminooctanoic acid; GPCR, G-protein-coupled receptor; KRG, Krebs-Ringer phosphate buffer supplemented with glucose; Lat A, latrunculin A; Lau, lauroyl; PAF, platelet-activating factor; PAFR, PAF receptor; PMA, phorbol 12-myristate 13-acetate; fMLF, formylmethionylleucylphenylalanine; PBP10, RhB-QRLFQVKGRR; F2M2, Lau-((S)-Aoc)-(Lys-βNphe)6-NH2. 3The abbreviations used are: FPR, formyl peptide receptor; βNphe, N-phenylmethyl-β-alanine; Aoc, 2-aminooctanoic acid; GPCR, G-protein-coupled receptor; KRG, Krebs-Ringer phosphate buffer supplemented with glucose; Lat A, latrunculin A; Lau, lauroyl; PAF, platelet-activating factor; PAFR, PAF receptor; PMA, phorbol 12-myristate 13-acetate; fMLF, formylmethionylleucylphenylalanine; PBP10, RhB-QRLFQVKGRR; F2M2, Lau-((S)-Aoc)-(Lys-βNphe)6-NH2. and FPR2), belonging to the family of G-protein-coupled receptors (GPCRs), which recognize pathogen-associated molecular patterns in the form of peptides with an N-terminal formyl-methionine residue, originating from bacterial (and mitochondrial) protein synthesis (2Dahlgren C. Gabl M. Holdfeldt A. Winther M. Forsman H. Basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria.Biochem. Pharmacol. 2016; (10.1016/j.bcp.2016.04.014)Crossref Scopus (100) Google Scholar, 3Ye R.D. Boulay F. Wang J.M. Dahlgren C. Gerard C. Parmentier M. Serhan C.N. Murphy P.M. International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family.Pharmacol. Rev. 2009; 61: 119-161Crossref PubMed Scopus (590) Google Scholar). 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Although the two human neutrophil FPRs, FPR1 and FPR2, display significant sequence similarity, they have distinct, albeit partially overlapping, ligand recognition profiles (2Dahlgren C. Gabl M. Holdfeldt A. Winther M. Forsman H. Basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria.Biochem. Pharmacol. 2016; (10.1016/j.bcp.2016.04.014)Crossref Scopus (100) Google Scholar, 6Rabiet M.J. Huet E. Boulay F. Human mitochondria-derived N-formylated peptides are novel agonists equally active on FPR and FPRL1, while Listeria monocytogenes-derived peptides preferentially activate FPR.Eur. J. Immunol. 2005; 35: 2486-2495Crossref PubMed Scopus (158) Google Scholar, 7Kretschmer D. Gleske A.K. Rautenberg M. Wang R. Köberle M. Bohn E. Schöneberg T. Rabiet M.J. Boulay F. Klebanoff S.J. van Kessel K.A. van Strijp J.A. Otto M. Peschel A. Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus.Cell Host Microbe. 2010; 7: 463-473Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The mouse genome contains at least eight mouse Fprs, among which Fpr1 and Fpr2 are regarded as the orthologues of the receptors expressed in human neutrophils (8He H.Q. Liao D. Wang Z.G. Wang Z.L. Zhou H.C. Wang M.W. Ye R.D. Functional characterization of three mouse formyl peptide receptors.Mol. Pharmacol. 2013; 83: 389-398Crossref PubMed Scopus (50) Google Scholar, 9Onnheim K. Bylund J. Boulay F. Dahlgren C. Forsman H. Tumour necrosis factor (TNF)-α primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization.Immunology. 2008; 125: 591-600Crossref PubMed Scopus (32) Google Scholar). Studies using mice deficient in Fpr1 or Fpr2 have demonstrated roles of these receptors not only in host defense but also in immune regulation and in the resolution of inflammation (10Gao J.L. Lee E.J. Murphy P.M. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor.J. Exp. Med. 1999; 189: 657-662Crossref PubMed Scopus (241) Google Scholar, 11Chen K. Le Y. Liu Y. Gong W. Ying G. Huang J. Yoshimura T. Tessarollo L. Wang J.M. A critical role for the G protein-coupled receptor mFPR2 in airway inflammation and immune responses.J. Immunol. 2010; 184: 3331-3335Crossref PubMed Scopus (104) Google Scholar12Dufton N. Hannon R. Brancaleone V. Dalli J. Patel H.B. Gray M. D'Acquisto F. Buckingham J.C. Perretti M. Flower R.J. Anti-inflammatory role of the murine formyl-peptide receptor 2: ligand-specific effects on leukocyte responses and experimental inflammation.J. Immunol. 2010; 184: 2611-2619Crossref PubMed Scopus (246) Google Scholar). The therapeutic potential of targeting these receptors in inflammatory/infectious conditions, such as atherosclerosis, cancer, neurodegenerative diseases, and sepsis (see a recent review (13Li L. Chen K. Xiang Y. Yoshimura T. Su S. Zhu J. Bian X.W. Wang J.M. New development in studies of formyl-peptide receptors: critical roles in host defense.J. Leukoc. Biol. 2016; 99: 425-435Crossref PubMed Scopus (51) Google Scholar)), justifies the search for receptor-specific ligands that can function as agonists, antagonists, or allosteric modulators. However, a direct translation of knowledge between humans and mice is not possible, due to the fact that there are many species-associated differences in both agonist and antagonist recognition profiles by the receptors. This is clearly illustrated by the fact that fMLF, the potent prototype FPR1 agonist, is a very weak agonist for Fpr1 (14He R. Tan L. Browning D.D. Wang J.M. Ye R.D. The synthetic peptide Trp-Lys-Tyr-Met-Val-d-Met is a potent chemotactic agonist for mouse formyl peptide receptor.J. Immunol. 2000; 165: 4598-4605Crossref PubMed Scopus (69) Google Scholar). Similarly, the potent FPR2 agonist WKYMVM (Fig. 1A) is a weak agonist for Fpr2 (9Onnheim K. Bylund J. Boulay F. Dahlgren C. Forsman H. Tumour necrosis factor (TNF)-α primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization.Immunology. 2008; 125: 591-600Crossref PubMed Scopus (32) Google Scholar, 15Bylund J. Samuelsson M. Collins L.V. Karlsson A. NADPH-oxidase activation in murine neutrophils via formyl peptide receptors.Exp. Cell Res. 2003; 282: 70-77Crossref PubMed Scopus (53) Google Scholar). To our knowledge, the short formyl peptides formyl-MIVIL and formyl-MIFL from Listeria monocytogenes and Staphylococcus aureus, respectively, are the most potent Fpr1 agonists currently identified (16Southgate E.L. He R.L. Gao J.L. Murphy P.M. Nanamori M. Ye R.D. Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils.J. Immunol. 2008; 181: 1429-1437Crossref PubMed Scopus (84) Google Scholar), whereas potent Fpr2-selective ligands remain to be identified. By contrast, several FPR2-selective ligands have successfully been described and characterized. Potent and stable modulators targeting both human and mouse receptors (especially FPR2 and Fpr2) thus remain to be identified. Such compounds would facilitate Fpr-based studies in mouse models of disease, important for understanding the precise roles of FPRs in health and disease, and for the validation of potential drug lead candidates. A major obstacle for therapeutic use of peptides is their inherent susceptibility to degradation by endogenous proteases, resulting in low bioavailability. Thus, investigation of proteolytically stable peptidomimetics, partly composed of unnatural residues, appears advantageous. Stable peptidomimetics belonging to the class of α-peptide/β-peptoid hybrids, displaying a design with alternating cationic α-amino acids and lipophilic peptoid residues, have been shown to imitate the biological effects of natural antimicrobial peptides (e.g. antimicrobial activity, antibiofilm activity, and immunomodulatory activity) (17Jahnsen R.D. Haney E.F. Franzyk H. Hancock R.E. Characterization of a proteolytically stable multifunctional host defense peptidomimetic.Chem. Biol. 2013; 20: 1286-1295Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar18Jahnsen R.D. Frimodt-Møller N. Franzyk H. 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Chem. 2015; 58: 801-813Crossref PubMed Scopus (18) Google Scholar). We have previously identified a novel class of FPR2 antagonists by screening an array of peptidomimetics comprising several subclasses of α-peptide/β-peptoid hybrids (see Fig. 1B for generic structures), using GPCR-dependent neutrophil production of superoxide as a read-out (22Skovbakke S.L. Heegaard P.M. Larsen C.J. Franzyk H. Forsman H. Dahlgren C. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.Biochem. Pharmacol. 2015; 93: 182-195Crossref PubMed Scopus (23) Google Scholar). Among these compounds, the most promising FPR2-interacting peptidomimetic, palmitoyl-(Lys-βNSpe)6-NH2 (F2M1 in Fig. 2), was found to inhibit neutrophil function, selectively through FPR2, with a potency comparable with that of the hitherto most potent FPR2-selective inhibitor known, namely the gelsolin-derived peptide PBP10 (RhB-QRLFQVKGRR) functionalized with an N-terminal rhodamine B moiety (23Forsman H. Andréasson E. Karlsson J. Boulay F. Rabiet M.J. Dahlgren C. Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending from a PIP2-binding domain of gelsolin.J. Immunol. 2012; 189: 629-637Crossref PubMed Scopus (37) Google Scholar, 24Fu H. Björkman L. Janmey P. Karlsson A. Karlsson J. Movitz C. Dahlgren C. The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide.BMC Cell Biol. 2004; 5: 50Crossref PubMed Scopus (44) Google Scholar). In the present work, we have identified a novel FPR2-interacting peptidomimetic, F2M2, (Lau-((S)-Aoc)-(Lys-βNphe)6-NH2; Fig. 2) with NADPH oxidase-activating effect, and which also activates mouse neutrophils through Fpr2. Further analysis of a number of structural variants of F2M2 revealed that the N-acyl 2-aminooctanoic acid residue and the peptidomimetic backbone of the molecule both were required for the agonistic activity. This novel class of FPR2-activating peptidomimetic ligands may serve as valuable tools for further delineation of the ligand recognition and signaling by FPR2 as well as for exploration of the therapeutic potential of targeting FPR2 in disease. In an earlier study, an array of peptidomimetics representing different structural subclasses was screened, using the neutrophil NADPH oxidase assay as a read-out system (25Dahlgren C. Karlsson A. Bylund J. Measurement of respiratory burst products generated by professional phagocytes.Methods Mol. Biol. 2007; 412: 349-363Crossref PubMed Scopus (75) Google Scholar, 26Bylund J. Björnsdottir H. Sundqvist M. Karlsson A. Dahlgren C. Measurement of respiratory burst products, released or retained, during activation of professional phagocytes.Methods Mol. Biol. 2014; 1124: 321-338Crossref PubMed Scopus (63) Google Scholar). A potent FPR2-specific antagonist was identified (22Skovbakke S.L. Heegaard P.M. Larsen C.J. Franzyk H. Forsman H. Dahlgren C. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.Biochem. Pharmacol. 2015; 93: 182-195Crossref PubMed Scopus (23) Google Scholar), and in addition, compounds displaying other lipid headgroups were found to partially inhibit the activity induced by known FPR2-specific agonists. This finding was not further explored, but we now show that the peptidomimetic F2M2 (i.e. Lau-((S)-Aoc)-(Lys-βNphe)6-NH2) in fact activates human neutrophils to produce/release superoxide anions (Fig. 3). The activity induced was concentration-dependent, with an EC50 of ∼150 nm with a maximal response reached at a concentration of ∼500 nm (Fig. 3B). Dose response of the FPR2-specific peptide agonist WKYMVM was included for comparison (Fig. 3C). The onset and overall kinetics of the F2M2-induced response, reaching the peak in <1 min and subsiding in <5 min (Fig. 3A), resembles that induced by other typical GPCR agonists. Based on the structural similarity to the earlier described FPR2 antagonist, F2M2 appeared to represent a novel FPR agonist. To establish the involvement of FPRs in the neutrophil-activating effect of F2M2, two different approaches were used: (i) reciprocal receptor desensitization induced by receptor-specific agonists, and (ii) inhibitory effects of receptor-specific antagonists. Reciprocal receptor desensitization is a process by which a receptor upon stimulation with a specific agonist becomes non-responsive (desensitized) to a second dose, not only of the same agonist but also of other agonists that bind to the receptor involved. Neutrophils triggered by F2M2 were desensitized, not only to a new dose of F2M2 but also to the FPR2-specific peptide agonists PSMα2 and WKYMVM (shown for WKYMVM in Fig. 4A). In contrast, F2M2-stimulated human neutrophils were fully responsive or even primed in response to FPR1 agonists (data not shown). The desensitization effect was reciprocal (i.e. independent of the order in which the agonists were applied) (Fig. 4A). These data suggest that F2M2 activates neutrophils selectively through FPR2 without interference by FPR1. The receptor preference of F2M2 was investigated also through the use of well-characterized selective FPR antagonists. The two FPR2-specific inhibitors PBP10 and F2M1 (22Skovbakke S.L. Heegaard P.M. Larsen C.J. Franzyk H. Forsman H. Dahlgren C. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.Biochem. Pharmacol. 2015; 93: 182-195Crossref PubMed Scopus (23) Google Scholar, 23Forsman H. Andréasson E. Karlsson J. Boulay F. Rabiet M.J. Dahlgren C. Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending from a PIP2-binding domain of gelsolin.J. Immunol. 2012; 189: 629-637Crossref PubMed Scopus (37) Google Scholar) both completely abolished the response induced by F2M2 (Fig. 4B). In contrast, no inhibitory effect was obtained with the FPR1-specific antagonist CsH (27Stenfeldt A.L. Karlsson J. Wennerås C. Bylund J. Fu H. Dahlgren C. Cyclosporin H, Boc-MLF and Boc-FLFLF are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor.Inflammation. 2007; 30: 224-229Crossref PubMed Scopus (98) Google Scholar) (Fig. 4B). Taken together, these data strongly suggest that F2M2 is a novel FPR2 agonist that triggers an activation of the neutrophil NADPH oxidase in human cells. Unequivocal confirmation of the inferred FPR2 specificity of F2M2 was obtained in studies of its ability to activate non-differentiated HL60 cells stably expressing FPR2, measured as an increase in cytosolic Ca2+ concentration. F2M2 proved capable of evoking an intracellular Ca2+ response in FPR2-expressing cells, with a maximal response at 250 nm and with an EC50 value of 50–100 nm (Fig. 5A). No response was induced in FPR1-overexpressing HL60 cells (that readily responded to the FPR1-specific agonist fMLF; Fig. 5B). To gain a deeper insight into the structure-function relationship for FPR2 activation by F2M2, we designed and synthesized an array of variants (Fig. 2 and Table 1). Different structural features were varied independently: (i) the length of the N-terminal fatty acid (i.e. F2M3–F2M6); (ii) the length of the linear alkyl side chain in the hydrophobic amino acid (i.e. F2M7–F2M9); (iii) the stereochemistry of the 2-aminooctanoic acid (Aoc) (i.e. F2M10); (iv) the backbone (α-peptide analogue F2M11); and (v) the hydrophobicity of the repetitive peptidomimetic part (i.e. F2M12–F2M14). The neutrophil-activating effects of the structural variants were investigated by their ability to trigger the production of superoxide anions, whereas their preference for FPR2 was evaluated in FPR1- and FPR2-overexpressing HL60 cells.TABLE 1Structure-activity relationships of lipidated peptidomimetics for stimulation of human FPR2Ca2+ response in HL60 cells overexpressingCompoundaAs indicated in Fig. 1.StructureNADPH-oxidase response EC50 valuebEC50 values are calculated based on data from three independent experiments. 95% confidence intervals (asymmetrical) are shown in parentheses.FPR2FPR1nmF2M2Lau-((S)-Aoc)-(Lys-βNphe)6-NH2167 (142–197)+++−Variation of N-terminal fatty acid lengthF2M3Oct-((S)-Aoc)-(Lys-βNphe)6-NH2−−−F2M4Dec-((S)-Aoc)-(Lys-βNphe)6-NH2≪+−F2M5Myr-((S)-Aoc)-(Lys-βNphe)6-NH2176 (153–202)++−F2M6Pam-((S)-Aoc)-(Lys-βNphe)6-NH2173 (151–198)++−Incomplete lipid headgroupF2M15Ac-((S)-Aoc-(S)-Aoc)-(Lys-βNphe)6-NH2−−−F2M16Ac-((S)-Aoc)-(Lys-βNphe)6-NH2−NT−F2M17Lau-(Lys-βNphe)6-NH2AntagonistcMeasured as inhibition of the response induced by WKYMVM.NT−F2M18Ac-(Lys-βNphe)6-NH2−NT−Variation of hydrophobic amino acidF2M7Lau-((S)-Nle)-(Lys-βNphe)6-NH2Antagonist−−F2M8Lau-((S)-OGly)-(Lys-βNphe)6-NH2Antagonist−−F2M9Lau-((S)-Dod)-(Lys-βNphe)6-NH2Antagonist−−Alternative stereochemistry of AocF2M10Lau-((R)-Aoc)-(Lys-βNphe)6-NH2≪++−Comparison with peptide backbone structureF2M11Lau-((S)-Aoc)-(Lys-Phe)6-NH2−−−Variation of β-peptoid hydrophobicityF2M12Lau-((S)-Aoc)-(Lys-βNF3phe)6-NH2−−−F2M13Lau-((S)-Aoc)-(Lys-βNFphe)6-NH275 (66–85)+++−F2M14Lau-((S)-Aoc)-(Lys-βNMephe)6-NH289 (78–102)+++−a As indicated in Fig. 1.b EC50 values are calculated based on data from three independent experiments. 95% confidence intervals (asymmetrical) are shown in parentheses.c Measured as inhibition of the response induced by WKYMVM. Open table in a new tab The fatty acid moiety linked to the N-terminal hydrophobic amino acid proved to be critical for the agonistic effect of F2M2 because F2M15, F2M16, and F2M18 (all lacking the fatty acid) were devoid of activity. The octanoyl analogue (F2M3), displaying an N-terminal fatty acid that was four carbons shorter than F2M2, was likewise devoid of agonistic effect in the tested concentration range (up to 2 μm). Decanoyl, myristoyl, and palmitoyl analogues (i.e. F2M4–F2M6), displaying alkyl chains that were two carbon atoms shorter or two and four carbon atoms longer than that present in the parent peptidomimetic F2M2, were active but with a reduced potency (Table 1). Shortening of the fatty acid resulted in considerable loss of activity, consistent with the lack of activity of F2M15, being the 2-acetamido-substituted analogue of F2M3. To test the importance of the hydrophobic amino acid, F2M17, which entirely lacked the Aoc unit, was evaluated for comparison. This lack of an additional alkyl side chain besides the fatty acid resulted in loss of all agonistic activity in the tested concentration range (up to 2 μm), and instead F2M17 acted as an FPR2 antagonist, reducing the response induced by the agonist WKYMVM (Table 1). To examine whether the length of the amino acid alkyl side chain was optimal in F2M2, three analogues were synthesized: F2M7 (displaying (S)-norleucine) with a two-carbon shorter side chain, F2M8 (analogue with (S)-octylglycine) with two additional carbon atoms in its side chain, and F2M9 (with (S)-2-aminododecanoic acid) with a four-carbon longer side chain (Fig. 2). All of these analogues lacked agonistic activity but maintained an interaction with FPR2, albeit as weak antagonists, underlining the importance of the exact structure of the lipid headgroup to confer agonistic activity. Noticeably, the analogue F2M10 with altered stereochemistry (i.e. the Aoc unit in F2M2 was changed from (S)-form to (R)-form), exhibited significantly reduced neutrophil activation as compared with that of F2M2 (Table 1). The influence of the peptidomimetic backbone on the agonistic activity of F2M2 was assessed by testing the α-peptide analogue (i.e. F2M11). The peptide was entirely inert in the activation assays, indicating that the α-peptide/β-peptoid peptidomimetic backbone is crucial for the ability of this novel agonist type to activate FPR2 (Table 1). Next, we examined the effects of increased hydrophobicity of the peptoid residues via introduction of fluorine or methyl substituents on the benzyl side chain of βNphe (F2M12–F2M14; Fig. 2). These more hydrophobic analogues displayed higher potencies in activating neutrophils as compared to the parental compound F2M2 (Fig. 6A and Table 1), suggesting a highly lipophilic binding site. Interestingly, peptidomimetics F2M13 and F2M14 inhibited/desensitized the response induced by FPR2 agonists but also by the FPR1 agonist fMLF as well as by phorbol 12-myristate 13-acetate (PMA), a PKC activator that bypasses membrane receptors (Fig. 6B), indicating that increased hydrophobicity leads to reduced FPR2 selectivity. In addition, increasing hydrophobicity appeared to confer cytotoxic properties to the heavily fluorinated analogue F2M12, which activated neutrophils to produce superoxide at low concentrations (≤250 nm) but rendered the cells non-responsive at higher concentrations (up to 1 μm) and abrogated the responsiveness to a second stimulation with PMA (data not shown). All agonistic variants of F2M2, described above, displayed FPR2 selectivity, because the responses induced in human neutrophils were inhibited by the FPR2-specific inhibitor PBP10 but were unaffected by the FPR1-specific inhibitor CsH (data not shown). In addition, all peptidomimetic agonist variants triggered calcium transients in FPR2-overexpressing cells, but not in FPR1-overexpressing cells (Table 1). Taken together, F2M2 was the most potent FPR2-selective neutrophil-activating peptidomimetic of the variants tested. In accordance with this, the interaction of F2M2 with primary human neutrophils was investigated further to shed more light on how FPR2 regulation is mediated by this novel type of agonist. FPRs are known to couple to pertussis toxin-sensitive Gi-protein for signaling. To characterize the effect of pertussis toxin on the F2M2 response, neutrophils were treated with pertussis toxin at different time points before stimulation with F2M2. Also, the FPR2 agonist WKYMVM and the receptor-independent PMA were included as positive and negative controls, respectively, at each time point. Pertussis toxin treatment for 2 h abolished both the WKYMVM- and F2M2-induced superoxide production (Fig. 7A), showing that a pertussis toxin-sensitive G-protein is involved in signaling downstream from FPR2 upon F2M2 binding. The cell response to PMA was unaffected by pertussis toxin (Fig. 7A). To study the binding mode of F2M2 on FPR2, we quantified the binding of Cy5-WKYMVM to neutrophils in the presence or absence of F2M2 by flow cytometry. Binding of Cy5-WKYMVM was inhibited by excess (100-fold) non-labeled WKYMVM (Fig. 7B) but not by excess (500-fold) F2M2 (Fig. 7B). These data suggest that F2M2 and WKYMVM most likely interact with different binding sites. Neutrophil production/release of superoxide anions in response to other FPR agonist differs in magnitude, depending on the state of the cells, being either naive (low responders) or primed (high responders). Priming of neutrophils can be achieved with a non-activating cytokine, such as TNF-α. Disruption of the neutrophil cytoskeleton with an inhibitor of actin polymerization, such as latrunculin A, also primes the response to FPR agonists (9Onnheim K. Bylund J. Boulay F. Dahlgren C. Forsman H. Tumour necrosis factor (TNF)-α primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization.Immunology. 2008; 125: 591-600Crossref PubMed Scopus (32) Google Scholar, 28Fu H. Bylund J. Karlsson A. Pellmé S. Dahlgren C. The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8.Immunology. 2004; 112: 201-210Crossref PubMed Scopus (56) Google Scholar, 29Almkvist J. Dahlgren C. Leffler H. Karlsson A. Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1.J. Immunol. 2002; 168: 4034-4041Crossref PubMed Scopus (78) Google Scholar). In accordance with this, neutrophils pretreated with TNF-α at 37 °C for 20 min produced increased amounts of supero
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