Function and regulation of GPR84 in human neutrophils.

Human neutrophils are components of the innate immune system and are the most abundant white blood cells in the circulation. They are professional phagocytes and express several G protein-coupled receptors (GPCRs), which are essential for proper neutrophil functions. So far, the two formyl peptide receptors, FPR1 and FPR2, have been the most extensively studied group of neutrophil GPCRs, but recently, a new group, the free fatty acid (FFA) receptors, has attracted growing attention. Neutrophils express two FFA receptors, GPR84 and FFA2, which sense medium- and short-chain fatty acids respectively, and display similar activation profiles. The exact pathophysiological role of GPR84 is not yet fully understood, but it is generally regarded as a pro-inflammatory receptor that mediates neutrophil activation. In this review, we summarize current knowledge of how GPR84 affects human neutrophil functions and discuss the regulatory mechanisms that control these responses, focusing on the similarities and differences in comparison to the two FPRs and FFA2. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc.

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

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

The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

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

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal

An increase in the cytosolic concentration of free calcium ions activates the neutrophil NADPH-oxidase provided that the free fatty acid receptor 2 has been allosterically modulated.

Signals generated by free fatty acid receptor 2 (FFA2R) can activate the neutrophil NADPH-oxidase without involvement of any orthosteric FFA2R agonist. The initiating signals may be generated by P2Y2R, the receptor for ATP. An FFA2R specific allosteric modulator (PAM; Cmp58) was required for this response and used to investigate the mechanism by which signals generated by ATP/P2Y2R activate an FFA2R dependent process. The P2Y2R induced signal that together with the modulated FFA2R activates neutrophils, was generated downstream of the Gαq containing G protein coupled to P2Y2R. A rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the important signal. The hypothesis gained support from the finding that the modulator transferred the neutrophils to a Ca2+sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the neutrophils provided that an allosteric modulator was bound to FFA2R. The activity of the superoxide generating NADPH-oxidase induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the FFA2R agonist propionate or by the signal generated by ATP/P2Y2R. The non-responding state of FFA2R was, however, revoked by a cross-activating allosteric FFA2R modulator. The [Ca2+]i mediated activation of neutrophils with their FFA2Rs allosterically modulated, represent a unique regulatory receptor crosstalk mechanism by which the activation potency of a G protein coupled receptor is controlled by a receptor-crosstalk signaling system operating from the cytosolic side of the plasma membrane.

The allosterically modulated FFAR2 is transactivated by signals generated by other neutrophil GPCRs.

Positive allosteric modulators for free fatty acid receptor 2 (FFAR2/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFAR2 was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFAR2 from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFAR2 in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFAR2s, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFAR2 activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFAR2 activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFAR2 activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFAR2 modulator.

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

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

The ketone body acetoacetate activates human neutrophils through FFAR2.

Neutrophils express many surface receptors that sense environmental changes. One such sensor is FFAR2 (free fatty acid receptor 2), a receptor that detects gut microbiota-derived short-chain fatty acids. As such, FFAR2 has been regarded as a molecular link between metabolism and inflammation. Our recent studies on FFAR2, using its endogenous agonist propionate in combination with allosteric modulators, have identified several novel aspects of FFAR2 regulation. A recent study has also identified the ketone body acetoacetate as an endogenous ligand for mouse FFAR2. Whether human FFAR2 also recognizes acetoacetate and how this recognition modulates human neutrophil functions has not been investigated. In this study, we found that acetoacetate can induce a decrease of cAMP and translocation of ß-arrestin in cells overexpressing FFAR2. In addition, we show that similar to propionate, FFAR2-specific allosteric modulators enhance acetoacetate-induced transient rise in cytosolic calcium, production of reactive oxygen species, and cell migration in human neutrophils. In summary, we demonstrate that human neutrophils recognize the ketone body acetoacetate through FFAR2. Thus, our data further highlight the key role of FFAR2 in inflammation and metabolism.

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition, activation/modulation, signaling and receptor regulated functions.

Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor-proteins span the plasma membrane in expressing cells seven times and the down-stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization, and reactivation mechanisms as well as communication (receptor transactivation/cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.

Phenol-soluble modulin α and ß display divergent roles in mice with staphylococcal septic arthritis.

Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMß which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmß). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMß has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMß strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMß play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMß protects arthritis development.

Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators-What is the Evidence so far?

Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.

GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by ß-arrestin recruiting GPR84 agonists.

In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84- but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by ß-arrestin recruitment data. The ROS production induced by a non ß-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this ß-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with ß-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.

Structural Determinants in the Staphylococcus aureus-Derived Phenol-Soluble Modulin α2 Peptide Required for Neutrophil Formyl Peptide Receptor Activation.

Highly pathogenic Staphylococcus aureus strains produce phenol-soluble modulins (PSMs), which are N-formylated peptides. Nanomolar concentrations of PSMα2 are recognized by formyl peptide receptor 2 (FPR2), but unlike the prototypic FPR2 agonist WKYMVM, PSMα2 is a biased signaling agonist. The truncated N-terminal PSMα2 variant, consisting of the five N-terminal residues, is no longer recognized by FPR2, showing that the C-terminal part of PSMα2 confers FPR2 selectivity, whereas the N-terminal part may interact with the FPR1 binding site. In the current study, a combined pharmacological and genetic approach involving primary human neutrophils and engineered FPR knock-in and knockout cells was used to gain molecular insights into FPR1 and FPR2 recognition of formyl peptides as well as the receptor downstream signaling induced by these peptides. In comparison with the full-length PSMα2, we show that the peptide in which the N-terminal part of PSMα2 was replaced by fMet-Ile-Phe-Leu (an FPR1-selective peptide agonist) potently activates both FPRs for production of superoxide anions and ß-arrestin recruitment. A shortened analog of PSMα2 (PSMα21-12), lacking the nine C-terminal residues, activated both FPR1 and FPR2 to produce reactive oxygen species, whereas ß-arrestin recruitment was only mediated through FPR1. However, a single amino acid replacement (Gly-2 to Ile-2) in PSMα21-12 was sufficient to alter FPR2 signaling to include ß-arrestin recruitment, highlighting a key role of Gly-2 in conferring FPR2-biased signaling. In conclusion, we provide structural insights into FPR1 and FPR2 recognition as well as the signaling induced by interaction with formyl peptides derived from PSMα2, originating from S. aureus bacteria.

Allosteric receptor modulation uncovers an FFA2R antagonist as a positive orthosteric modulator/agonist in disguise.

A novel receptor crosstalk activation mechanism, through which signals generated by the agonist-occupied P2Y2R (the neutrophil receptor for ATP) activate allosterically modulated free fatty acid 2 receptor (FFA2R) without the involvement of any FFA2R agonist, was used to determine the inhibitor profiles of two earlier-described, FFA2R-specific antagonists, CATPB and GLPG0974. These antagonists have been shown to have somewhat different receptor-interaction characteristics at the molecular/functional level, although both are recognized by the orthosteric site in FFA2R. The antagonists inhibited neutrophil activation induced by ATP, an activation occurred only in the presence of either of the two positive allosteric FFA2R modulators (PAMs) AZ1729 and Cmp58. No neutrophil activation was induced by either AZ1729 or Cmp58 alone, whereas together they acted as co-agonistic PAMs and activated the superoxide-generating NADPH-oxidase in neutrophils. This response was inhibited by CATPB but not by GLPG0974. In contrast, GLPG0974 acted as a positive modulator, increasing the potency, albeit not the efficacy, of the co-agonistic PAMs. GLPG0974 also altered signaling downstream of FFA2R when activated by the co-agonistic PAMs. In the presence of GLPG0974, the response of neutrophils induced by the co-agonistic PAMs included an increase in the cytosolic concentration of free calcium ions (Ca2+), and this effect was reciprocal in that GLPG0974 triggered an increase in intracellular Ca2+, demonstrating that GLPG0974 acted as an FFA2R agonist. In summary, by studying the effects of the FFA2R ligand GLPG0974 on neutrophil activation induced by the co-agonists AZ1729 + Cmp58, we show that GLPG0974 is not only an FFA2R antagonist, but also displays agonistic and positive FFA2R-modulating functions that affect NADPH-oxidase activity and alter the receptor-downstream signaling induced by the co-agonistic PAMs.

Multiple ligand recognition sites in free fatty acid receptor 2 (FFA2R) direct distinct neutrophil activation patterns.

The allosteric modulating free fatty acid receptor 2 ligands Cmp58 and AZ1729, increased the activity induced by orthosteric receptor agonists mediating a rise in intracellular calcium ions and activation of the neutrophil NADPH-oxidase. Together, the two modulators triggered an orthosteric-agonist-independent activation of the oxidase without any rise in the concentration of intracellular calcium ions. In this study, structurally diverse compounds presumed to be ligands for free fatty acid receptor 2 were used to gain additional insights into receptor-modulation/signaling. We identified two molecules that activate neutrophils on their own and we classified one as allosteric agonist and the other as orthosteric agonist. Ten compounds were classified as allosteric FFA2R modulators. Of these, one activated neutrophils when combined with AZ1729; the nine remaining compounds activated neutrophils solely when combined with Cmp58. The activation signals were primarily biased when stimulated by two allosteric modulators interacting with different binding sites, such that two complementary modulators together triggered an activation of the NADPH-oxidase but no increase in the intracellular concentration of calcium ions. No neutrophil activation was induced when allosteric receptor modulators suggested to be recognized by the same binding site were combined, results in agreement with our proposed model for activation, in which the receptor has two different sites that selectively bind allosteric modulators. The down-stream signaling mediated by cross-sensitizing allosteric receptor modulators, occurring independent of any orthosteric agonist, represent a new mechanism for activation of the neutrophil NADPH oxidase.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex.

The formyl peptide receptors FPR1 and FPR2 are abundantly expressed by neutrophils, in which they regulate proinflammatory tissue recruitment of inflammatory cells, the production of reactive oxygen species (ROS), and resolution of inflammatory reactions. The unique dual functionality of the FPRs makes them attractive targets to develop FPR-based therapeutics as novel anti-inflammatory treatments. The small compound RE-04-001 has earlier been identified as an inducer of ROS in differentiated HL60 cells but the precise target and the mechanism of action of the compound was has until now not been elucidated. In this study, we reveal that RE-04-001 specifically targets and activates FPR1, and the concentrations needed to activate the neutrophil NADPH-oxidase was very low (EC50 ∼1 nM). RE-04-001 was also found to be a neutrophil chemoattractant, but when compared to the prototype FPR1 agonist N-formyl-Met-Leu-Phe (fMLF), the concentrations required were comparably high, suggesting that signaling downstream of the RE-04-001-activated-FPR1 is functionally selective. In addition, the RE-04-001-induced response was strongly biased toward the PLC-PIP2 -Ca2+ pathway and ERK1/2 activation but away from ß-arrestin recruitment. Compared to the peptide agonist fMLF, RE-04-001 is more resistant to inactivation by the MPO-H2 O2 -halide system. In summary, this study describes RE-04-001 as a novel small molecule agonist specific for FPR1, which displays a biased signaling profile that leads to a functional selective activating of human neutrophils. RE-04-001 is, therefore, a useful tool, not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1-specific drug therapeutics.

Data showing effects of a PI3K-δ inhibitor on neutrophil superoxide production during FPR2 activation and reactivation.

Reactive oxygen species (ROS) generated by the NADPH oxidase are crucial for antimicrobial host defense and also play a role in the regulation of inflammatory processes. Signals generated by formyl peptide receptor 2 (FPR2) activate the neutrophil ROS generating NADPH oxidase; such signals are mediated when the receptors bind an activating agonist, as well as when agonist desensitized FPR2 are reactivated by the receptor for platelet-activating factor (PAF). We present data on the effects of Idelalisib, a specific inhibitor for the PI3Kδ isoform, on ROS production during FPR2 activation and reactivation by PAF, respectively. Neutrophils were isolated from peripheral blood of healthy adults obtained from the blood bank at Sahlgrenska University Hospital and ROS release was measured using isoluminol-amplified chemiluminescence.

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis.

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to ß-arrestin recruitment. ß-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of ß-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent ß-arrestin/AP2 interaction and ß-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/ß-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of ß-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in ß-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of ß-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of ß-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/ß-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.

The PAR4-derived pepducin P4Pal10 lacks effect on neutrophil GPCRs that couple to Gαq for signaling but distinctly modulates function of the Gαi-coupled FPR2 and FFAR2.

A novel mechanism of action was described for the protease-activated receptor 4 (PAR4)-derived pepducin (P4Pal10), when it was shown to exhibit inhibitory efficacy towards G protein coupling to multiple Gαq-coupled receptors (Carr, R., 3rd et al., Mol. Pharmacol. 2016(89) 94). We could confirm that P4Pal10, similar to an earlier-characterized Gαq inhibitor (YM-254890), inhibited platelet aggregation induced by agonists for the Gαq-coupled receptors PAR1 and PAR4. Next, we applied P4Pal10 as a tool compound and investigated its modulatory effect on several Gαq- and Gαi-coupled GPCRs expressed by human neutrophils. P4Pal10 had, however, no inhibitory effects on signaling downstream of the Gαq-coupled receptors for ATP (P2Y2R) and PAF (PAFR). Instead, P4Pal10 inhibited signaling downstream the Gαi-coupled FPR2. The inhibition was not due to a direct effect on Gαi as the closely related FPR1 was unaffected. In addition, we found that the pepducin activated allosterically modulated short chain fatty acid receptor (FFAR2), a Gαi/Gαq coupled GPCR that is functionally expressed in neutrophils. Taken together, we show that pepducins are unique tool-compounds for mechanistic studies of GPCR signaling and modulation in neutrophils. The data presented add also lipopeptides into the known ligand recognition lists for the two pattern recognition receptors FPR2 and FFAR2, receptors that primarily sense formylated peptides and short free fatty acids, respectively, inflammatory mediators of microbial origin.