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Maturation of the secondary antibody repertoire requires class-switch recombination (CSR), which switches IgM to other immunoglobulins (Igs), and somatic hypermutation, which promotes the production of high-affinity antibodies. Following immune response or infection within the body, activation of T cell-dependent and T cell-independent antigens triggers the activation of activation-induced cytidine deaminase, initiating the CSR process. CSR has the capacity to modify the functional properties of antibodies, thereby contributing to the adaptive immune response in the organism. Ig CSR defects, characterized by an abnormal relative frequency of Ig isotypes, represent a rare form of primary immunodeficiency. Elucidating the molecular basis of Ig diversification is essential for a better understanding of diseases related to Ig CSR defects and could provide clues for clinical diagnosis and therapeutic approaches. Here, we review the most recent insights on the diversification of five Ig isotypes and choose several classic diseases, including hyper-IgM syndrome, Waldenström macroglobulinemia, hyper-IgD syndrome, selective IgA deficiency, hyper-IgE syndrome, multiple myeloma, and Burkitt lymphoma, to illustrate the mechanism of Ig CSR deficiency. The investigation into the underlying mechanism of Ig CSR holds significant potential for the advancement of increasingly precise diagnostic and therapeutic approaches.
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In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most-abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger-signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the NADPH oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including TNFα and GM-CSF. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions.
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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.
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Neutrófilos , Transducción de Señal , Humanos , Receptores de Formil Péptido , Fagocitos , Receptores Acoplados a Proteínas GRESUMEN
This review gives an overview of the protective role of CD8+ T cells in SARS-CoV-2 infection. The cross-reactive responses intermediated by CD8+ T cells in unexposed cohorts are described. Additionally, the relevance of resident CD8+ T cells in the upper and lower airway during infection and CD8+ T-cell responses following vaccination are discussed, including recent worrisome breakthrough infections and variants of concerns (VOCs). Lastly, we explain the correlation between CD8+ T cells and COVID-19 severity. This review aids in a deeper comprehension of the association between CD8+ T cells and SARS-CoV-2 and broadens a vision for future exploration.
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COVID-19 , Humanos , SARS-CoV-2 , Linfocitos T CD8-positivos , Reacciones Cruzadas , VacunaciónRESUMEN
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.
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Neutrófilos , Receptores de Formil Péptido , Humanos , Receptores de Formil Péptido/metabolismo , Transducción de Señal , Proteínas de Unión al GTP/metabolismoRESUMEN
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
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To increase the imaging resolution and detection capability, the field strength of static magnetic fields (SMFs) in magnetic resonance imaging (MRI) has significantly increased in the past few decades. However, research on the side effects of high magnetic field is still very inadequate and the effects of SMF above 1 T (Tesla) on B cells have never been reported. Here, we show that 33.0 T ultra-high SMF exposure causes immunosuppression and disrupts B cell differentiation and signaling. 33.0 T SMF treatment resulted in disturbance of B cell peripheral differentiation and antibody secretion and reduced the expression of IgM on B cell membrane, and these might be intensity dependent. In addition, mice exposed to 33.0 T SMF showed inhibition on early activation of B cells, including B cell spreading, B cell receptor clustering and signalosome recruitment, and depression of both positive and negative molecules in the proximal BCR signaling, as well as impaired actin reorganization. Sequencing and gene enrichment analysis showed that SMF stimulation also affects splenic B cells' transcriptome and metabolic pathways. Therefore, in the clinical application of MRI, we should consider the influence of SMF on the immune system and choose the optimal intensity for treatment.
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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.
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Calcio , Neutrófilos , Neutrófilos/metabolismo , Calcio/metabolismo , Ácidos Grasos no Esterificados/metabolismo , NADP/metabolismo , Ionomicina/metabolismo , Iones/metabolismo , Adenosina Trifosfato/metabolismo , Oxidorreductasas , NADPH Oxidasas/metabolismo , Superóxidos/metabolismoRESUMEN
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.
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Neutrófilos , Receptores de Formil Péptido , Humanos , Animales , Ratones , Receptores de Formil Péptido/metabolismo , Quimiotaxis , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
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.
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Neutrófilos , Propionatos , Neutrófilos/metabolismo , Propionatos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Adenosina Trifosfato/metabolismo , Regulación AlostéricaRESUMEN
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.
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Propionatos , Receptores Acoplados a Proteínas G , Humanos , Ratones , Animales , Receptores Acoplados a Proteínas G/metabolismo , Propionatos/farmacología , Neutrófilos/metabolismo , Acetoacetatos/farmacología , Acetoacetatos/metabolismo , Cuerpos Cetónicos/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismoRESUMEN
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.
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Neutrófilos , Receptores Acoplados a Proteínas G , Humanos , Ligandos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/farmacología , Regulación AlostéricaRESUMEN
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.
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Artritis Infecciosa , Toxinas Bacterianas , Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Artritis Infecciosa/metabolismo , Toxinas Bacterianas/metabolismo , Ratones , NADP/metabolismo , Oxidorreductasas/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidadRESUMEN
CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.
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ADP-Ribosil Ciclasa 1/inmunología , Linfocitos B , Glicoproteínas de Membrana/inmunología , Receptores de Antígenos de Linfocitos B , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD19/metabolismo , Humanos , Inmunoglobulina M , Activación de Linfocitos , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
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.
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Receptores de Formil Péptido , Staphylococcus aureus , Toxinas Bacterianas , Humanos , Neutrófilos/metabolismo , Péptidos/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/química , Staphylococcus aureus/metabolismoRESUMEN
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.
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Quinasa 2 del Receptor Acoplado a Proteína-G , NADPH Oxidasas , Neutrófilos , Receptores Acoplados a Proteínas G , beta-Arrestinas , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , NADP/farmacología , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/agonistas , beta-Arrestinas/metabolismoRESUMEN
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.
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Ácidos Grasos no Esterificados , Receptores de Superficie Celular , Regulación Alostérica , Calcio/farmacología , Neutrófilos , Transducción de SeñalRESUMEN
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.
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Guanidinas/farmacología , Isoquinolinas/farmacología , Neutrófilos/fisiología , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/metabolismo , Calcio/metabolismo , Descubrimiento de Drogas , Regulación de la Expresión Génica/efectos de los fármacos , Guanidinas/química , Humanos , Isoquinolinas/química , Ligandos , Estructura Molecular , NADPH Oxidasas , Relación Estructura-ActividadRESUMEN
Neutrophils express the two formyl peptide receptors (FPR1 and FPR2) and the medium-chain fatty acid receptor GPR84. The FPRs are known to define a hierarchy among neutrophil G protein-coupled receptors (GPCRs), that is, the activated FPRs can either suppress or amplify GPCR responses. In this study, we investigated the position of GPR84 in the FPR-defined hierarchy regarding the activation of neutrophil nicotine adenine dinucleotide phosphate (NADPH) oxidase, an enzyme system designed to generate reactive oxygen species (ROS), which are important regulators in cell signaling and immune regulation. When resting neutrophils were activated by GPR84 agonists, a modest ROS release was induced. However, vast amounts of ROS were induced by these GPR84 agonists in FPR2-desensitized neutrophils, and the response was inhibited not only by a GPR84-specific antagonist but also by an FPR2-specific antagonist. This suggests that the amplified GPR84 agonist response is achieved through a reactivation of desensitized FPR2s. In addition, the GPR84-mediated FPR2 reactivation was independent of ß-arrestin recruitment and sensitive to a protein phosphatase inhibitor. In contrast to FPR2-desensitized cells, FPR1 desensitization primarily resulted in a suppressed GPR84 agonist-induced ROS response, indicating a receptor hierarchical desensitization of GPR84 by FPR1-generated signals. In summary, our data show that the two FPRs in human neutrophils control the NADPH oxidase activity with concomitant ROS production by communicating with GPR84 through different mechanisms. While FPR1 desensitizes GPR84 and by that suppresses the release of ROS induced by GPR84 agonists, amplified ROS release is achieved by GPR84 agonists through reactivation of the desensitized FPR2.
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NADPH Oxidasas , Receptores de Formil Péptido , Receptores de Lipoxina , Adenina , Humanos , Activación Neutrófila , Neutrófilos , Fosfatos , Receptores Acoplados a Proteínas GRESUMEN
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 â¼1 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.