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.


| INTRODUCTION
Neutrophils express pattern recognition receptors that allow them to discriminate between 'self' and 'non-self' by detecting specific pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).Among these receptors, many belong to the family of seven-transmembrane G proteincoupled receptors (GPCRs) that enable the cells to detect environmental changes and transfer this information into the appropriate intracellular signalling and cellular functions, in order to adapt to such changes.In addition to the well-known GPCRs, such as the formyl peptide receptors FPR1 and FPR2, the platelet-activating factor (PAF) receptor, the ATP receptor -the P2Y 2 purinoceptor -and receptors for the chemokine IL-8 -CXCR1 and CXCR2, neutrophils also express two GPCRs -GPR84 and FFA2 -that can sense free fatty acids of different carbon lengths.Irrespective of the specific structure of a GPCR, ligand binding leads to a conformational change of the receptor, which mediates activation of the heterotrimeric G protein, located on the cytosolic side of the receptor expressing membrane (Figure 1, shown as GPR84).The activated heterotrimeric G protein disassociates into an α subunit and a βγ protein dimer, which initiates intracellular signalling downstream of the ligandoccupied GPCR (Dahlgren et al., 2022;Flock et al., 2017).In a targeted search for potential endogenous ligands, GPR84 was "deorphanized" as the receptor for medium-chain fatty acids (MCFAs) (Wang et al., 2006).However, very high concentrations of MCFAs are needed to activate GPR84 in vitro and it is still not clear if such high concentrations are ever present in relevant tissues in vivo.
Hence, it remains to be clarified whether MCFAs are the 'true' endogenous ligands for GPR84 or if this receptor should still be classed as an orphan GPCR (Luscombe et al., 2020).To overcome the poor activity of the MCFAs in activating GPR84, we and others have used a variety of synthetic tool compounds, targeting GPR84, that have been developed during the last few years, in an attempt to characterize and understand this receptor in more detail (Marsango et al., 2022).Using this approach, our knowledge of the activation and signalling profile mediated through GPR84 has increased considerably.It is now clear that GPR84 is functionally active in human neutrophils and primarily promotes pro-inflammatory responses.Furthermore, multiple mechanisms involved in the regulation of both the induction and termination of GPR84-mediated responses in human neutrophils have been identified and will be discussed in this review.

| NEUTROPHILS
Neutrophils are professional phagocytic cells that belong to the innate immune system and constitute the most abundant leukocyte in human peripheral blood (Hidalgo et al., 2019;Summers et al., 2010).Based on the multilobular form of the nucleus and the heavily packed subcellular organelles (granules or vesicles), neutrophils are also called polymorphonuclear leukocytes (PMN) or granulocytes (McKenna et al., 2021).Neutrophil maturation from haematopoietic stem cells occurs in the bone marrow through a process referred to as granulocytic differentiation or granulopoiesis (Cowland & Borregaard, 2016;Theilgaard-Mönch et al., 2022).Upon maturation and release from the bone marrow into the circulation, the lifespan of neutrophils is limited to a few days.During this time, neutrophils patrol the bloodstream in a quiescent state prepared for recruitment to sites of infection or traumatic tissue destruction, as needed.Under normal, healthy conditions, a large number of aged neutrophils return from circulation to the bone marrow or are cleared by Kupffer cells in the liver (Hidalgo et al., 2019;Martin et al., 2003).

| The neutrophil granules
Neutrophils contain three types of intracellular granules and one type of secretory vesicles that are filled with pre-made proteins.These F I G U R E 1 Summary of GPR84mediated activation and regulation of the NADPH oxidase in human neutrophils.The regulatory roles of G protein-coupled receptor kinase 2 (GRK2) and the actin cytoskeleton on GPR84-mediated neutrophil reactive oxygen species (ROS) production and reactivation are shown as the degree of NADPH oxidase activity in the absence (control) or presence of selective inhibitors (À, no activity; +, weak activity; ++, strong activity).
proteins facilitate various neutrophil functions, including migration, phagocytosis and activation of the NADPH-oxidase, that are essential for host defence.The cytoplasmic granules are formed at different time points during granulopoiesis in the bone marrow and are sorted to different granules through a process referred to as 'targeting by timing'.In response to certain stimuli, the intracellular granules and secretory vesicles can move to, and fuse with, the plasma membrane, in a specific order, which is the inverse of the order to how they are formed.That is, the secretory vesicles are the last formed and first to be mobilized, whereas the azurophil granules are the first formed and last to be mobilized.This tightly regulated stepwise formation and mobilization process of granules is termed degranulation and ensures the delivery of granule proteins and membrane receptors to the correct compartments that are required for proper functions at different stages during the lifetime of the neutrophil (Cowland & Borregaard, 2016;Sengeløv et al., 1993Sengeløv et al., , 1995)).The primary function of neutrophils is to provide the host with a first line of defence against invading microorganisms.The classical killing process is initiated when an invading microbe is engulfed in a phagosome which then fuses with the neutrophil granules forming a phagolysosome containing the granule proteins required for destruction of the engulfed microbes (Nordenfelt & Tapper, 2011).Activation of GPR84 has been shown to modulate phagocytosis of human neutrophils (Peters et al., 2022), murine macrophages (Recio et al., 2018) and U937 macrophage-like cells (Lucy et al., 2019).However, the underlying mechanism for this modulation remains to be clarified.

| The neutrophil NADPH oxidase
In addition to granule proteins involved in classical killing process, neutrophils rely also on an oxygen-dependent mechanism consisting of reactive oxygen species (ROS), derived from the NADPH oxidase.
Depending on the type of stimuli, assembly of a functional NADPH oxidase can occur either at the membranes of phagosomes or the intracellular granules resulting in intracellular ROS production or at the plasma membrane resulting in an extracellular ROS release (Bylund et al., 2010;Karlsson & Dahlgren, 2002).Most of the GPCRs expressed in neutrophils, including GPR84, can upon agonist activation, mediate an assembly of the NADPH oxidase that primarily results in production of extracellular ROS (see below).The critical role of the NADPH oxidase in host defence is clearly illustrated by the increased susceptibility to severe fungal and bacterial infections linked to chronic granulomatous disease (CGD), a primary deficiency disorder caused by a mutation in one of the NADPH oxidase subunits (Kuijpers & Lutter, 2012).Such a mutation leads to a non-functional NADPH oxidase complex with a total or partial inability to generate ROS (Bylund et al., 2005;Yu et al., 2021).The ROS generated by the NADPH oxidase are also, in response to certain stimuli, of importance for neutrophils' ability to form yet another killing mechanism called neutrophil extracellular traps (NETs) (Schoen et al., 2022).By undergoing NETosis, neutrophils release de-condensed nuclear chromatin and granular proteins to the extracellular space, which not only trap but also kill pathogens (Brinkmann & Zychlinsky, 2007).Hence, neutrophils from patients with CGD, dependent on the trigger, do not form NETs (Anjani et al., 2020;Davidsson et al., 2020).It is worth mentioning that in addition to being susceptible to infections, CGD patients also display a hyperinflammatory phenotype (Bylund et al., 2005;Dahlgren et al., 2019;Yu et al., 2021).The latter may be attributed to redox dysregulation in their professional phagocytes (Sundqvist et al., 2017) and strongly implies that the ROS derived from NADPH oxidase exhibit important immune modulatory functions.With regard to GPR84, one recent study has shown that neutrophils undergo NETosis upon GPR84 activation (Peters et al., 2022), but whether the GPR84 agonists used were dependent on NADPH oxidase-derived ROS for initiating NETosis remains to be clarified.

| Neutrophil priming
The level of ROS production induced by agonists for many GPCRs including GPR84 largely depends on the neutrophil state, naïve or primed.In this context, primed neutrophils are more prone to respond to a secondary stimulation, and to produce higher levels of ROS in response to most GPCR agonists (Dahlgren et al., 2022).
Transfer of neutrophils from a naïve (resting) to a primed state can be achieved either in vivo by transmigration from the blood to an inflamed tissue or in vitro by exposure to different priming agents, such as LPS or TNF-α.One of the proposed mechanisms underlying priming is increased expression of cell surface receptors through degranulation, that is, movement of intracellular receptors, stored in secretory vesicles or granules, to the plasma membrane.Many surface receptors including the FPRs and the complement receptors CR1 (CD35) and CR3 (CD11b/CD18), involved in neutrophil migration, are present in the secretory vesicle or granule membranes and are mobilized to the plasma membrane upon priming (Condliffe et al., 1998;Cowland & Borregaard, 2016;Hallett & Lloyds, 1995;Miralda et al., 2017).Accordingly, tissue neutrophils recruited to inflammatory sites in vivo or circulating neutrophils treated with a priming agent in vitro would be expected to be degranulated, but this is not always the case, as the priming process is very complex.For example, neutrophils recruited from the blood to the synovial fluid in inflamed joints of patients with inflammatory arthritis can display a phenotype similar to the naïve neutrophils in the blood, that is, they show minimal signs of degranulation (Björkman et al., 2019).In addition, certain agents in vitro can modify neutrophils into a primed state, measured as an increased production of ROS compared to resting neutrophils, but without any signs of degranulation (Niemietz et al., 2020;Sundqvist et al., 2020).Regarding GPR84, we have demonstrated that primed neutrophils produce greater amounts of ROS in response to GPR84 agonists (Fredriksson et al., 2022;Mårtensson et al., 2021;Sundqvist et al., 2018).However, whether or not this primed response is accompanied by an up-regulation of surface GPR84, as a result of the degranulation process, has yet to be determined.

| CHARACTERISTICS OF NEUTROPHIL GPCRs
Different approaches have been used to map the GPCR atlas in primary human cells.Data obtained from transcriptional studies have shown that only a handful out of more than 10,000 active genes identified in different neutrophil populations classify as GPCRs.A similar pattern is obtained when the protein expression profile is analysed; only a handful out of more than 1000 proteins identified belong to the GPCR family (Hohenhaus et al., 2013;Insel et al., 2019;Theilgaard-Mönch et al., 2005), and many receptors shown to be functionally active in neutrophils are not present in the transcriptional or proteomic data (Rørvig et al., 2013).This suggests that neither gene expression nor proteomic analysis reflects the correct pattern of functionally expressed neutrophil GPCRs and that more advanced techniques are needed to identify additional neutrophil GPCRs.Our functional studies with known GPCR-selective tool compounds have demonstrated the expression of numerous GPCRs, including GPR84, in human neutrophils.At a functional level, these GPCRs recognize a broad range of ligands including free fatty acids of different carbon chain lengths, peptides, proteins/protein fragments, lipids and nucleotides and are of main importance for regulation of several basic neutrophil functions including degranulation or secretion, chemotactic migration and production of NADPH oxidase-derived ROS (Dahlgren et al., 2022).

| Initiation of GPCR signalling in neutrophils
Upon GPCR ligand binding, basically all downstream signalling is initiated by activation of the heterotrimeric G protein containing a βγ protein dimer (Kobilka, 2007) and an α subunit, which binds GTP/GDP and possesses an intrinsic GTPase activity (Ross & Wilkie, 2000).The GTP/GDP exchange leads to dissociation of the βγ subunit from the α subunit, and each subunit interacts with different downstream effector molecules.Based on α-subunit homology and function, the G proteins are grouped into four major families (Gα q , Gα i/o , Gα s and Gα 12/13 ) (Hurowitz et al., 2000).The different Gα subunits differ in signalling characteristics, which include activation of membraneassociated adenylyl cyclases, enzymes catalysing the conversion of ATP to cAMP (Altarejos & Montminy, 2011).GPR84 as well as FFA2 and the FPRs can all couple to Gα i/o -containing G proteins for signalling in human neutrophils.Upon ligand binding to these receptors, the dissociated βγ complex activates phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to diacylglycerol (DAG) and inositol trisphosphate (IP 3 ).The latter molecule then binds to specific receptors on the endoplasmic reticulum (ER, the Ca 2+storing organelle), which promote a transient rise in the intracellular calcium concentration ([Ca 2+ ] i ).This signalling pathway, induced by the βγ complex of Gα i/o -containing G proteins, is also activated by the α subunit of the Gα q -containing G proteins (Dahlgren et al., 2022).
Although the heterotrimeric G proteins were discovered about 40 years ago, very few pharmacological agents have been developed that allow determination of G protein involvement in primary cells.
The two commonly used bacterial toxins (cholera toxin and Pertussis toxin) act via covalent modification of the Gα subunits Gα s and Gα i/o , respectively.The recent identification of specific Gα q inhibitors (Nishimura et al., 2010;Schrage et al., 2015) has allowed for studies of also Gα q involvement in primary cells.This has revealed that in human neutrophils, the P2Y 2 and the PAF receptors rely on Gα q for signalling, whereas GPR84, FFA2 and the FPRs are (as expected) insensitive to Gα q inhibitors but inhibited by Pertussis toxin, as they couple to Gα i/o -containing G proteins (Dahlgren et al., 2022).However, it is worth noting that, in human neutrophils, the PAF receptor response is sensitive not only to Gα q inhibitors but also to Pertussis toxin (Becker et al., 1986;Holdfeldt et al., 2017).It should also be mentioned that GPCRs insensitive to Gα q inhibitors for signalling could acquire such a sensitivity through receptor cross-talk mechanisms (Holdfeldt et al., 2017(Holdfeldt et al., , 2020)).Recently, a new tool molecule (larixol, extracted from Euphorbia formosana) was described to interfere with responses mediated by neutrophil FPR1 and this signalling block was attributed to effects on the βγ subunit coupled to α subunit of Gα i/o -containing G proteins (Liao et al., 2022).However, this finding has not yet been confirmed by other independent researchers.

| Termination of GPCR signalling in neutrophils
After a short period of GPCR agonist stimulation, the neutrophil response is terminated through a process called receptor homologous desensitization.For most GPCRs including GPR84, recruitment of β-arrestin in coordination with the actin cytoskeleton physically/sterically blocks further G protein binding to the receptor, thereby terminating G protein-dependent neutrophil signalling (Dahlgren et al., 2022).In addition, recruitment of β-arrestin initiates receptor endocytosis/internalization by binding to adaptor protein 2 (AP2) and the heavy chain of clathrin (Goodman et al., 1996;Kelly et al., 2014;Luttrell & Lefkowitz, 2002).Furthermore, β-arrestin recruitment also induces activation of non-canonical and endosomal signalling pathways involving ERK (Lefkowitz & Whalen, 2004;Luttrell & Lefkowitz, 2002).However, in human neutrophils, phosphorylation of ERK can be triggered by some GPCR agonists that lack ability to recruit β-arrestin, suggesting that GPCR-mediated ERK activation can occur independent of β-arrestin recruitment (Sundqvist et al., 2019).
Recently, an inhibitor (barbadin) that blocks the interaction between AP2 and β-arrestin was introduced as a tool compound for inhibiting the endocytosis and internalization of GPCRs, using the clathrin pathway (Beautrait et al., 2017).However, this inhibition has been shown not to apply to FPR2, implying that some GPCRs may undergo endocytosis through an AP2/β-arrestin-independent process (Sundqvist et al., 2020).Much is still unknown about the role of β-arrestin in regulating GPCR-mediated neutrophil functions.Our studies on the regulation of GPR84 signalling in neutrophils have revealed that the modulatory fine-tuning determining the functional outcome, requires the involvement of not only β-arrestin but also the plasma membranecoupled actin cytoskeleton (Figure 1) (Fredriksson et al., 2022;Mårtensson et al., 2021;Sundqvist et al., 2018).In line with these findings, the basis for both termination of signalling and receptor desensitization of neutrophil FPRs, relies on the interaction of the agonist-occupied FPRs with the actin cytoskeleton, rather than with β-arrestin.The involvement of the actin cytoskeleton in the termination/desensitization of GPCRs in neutrophils has primarily been established using drugs that inhibit polymerization of G-actin to F-actin.

| Neutrophil FPRs
The human genome encodes for three members of the family of FPRs.
All three members are expressed in monocytes, whereas neutrophils express only two members (FPR1 and FPR2).Both FPR1 and FPR2 recognize formyl peptides with a formylated methionine (fMet) at the N-terminus, but the FPR preference as well as the activating effect of these agonistic peptides are determined by the peptide size and amino acid composition.For example, short formyl peptides prefer FPR1, whereas community-associated methicillin-resistant Staphylococcus aureus produced formylated phenol-soluble modulin (PSM) peptides prefer FPR2 (Forsman et al., 2015;Kretschmer et al., 2015;Sundqvist et al., 2019).In addition, among the 13 mitochondrial DNAencoded proteins (mitocryptides [MCTs]), around half activate the neutrophil FPRs.Although some of these MCTs display different preference for either FPR1 or FPR2, others act as dual agonists (activating both FPR1 and FPR2) (Gabl et al., 2018;Lind, Gabl, et al., 2019;Mukai et al., 2009).Both FPR1 and FPR2 can also recognize several non-formylated peptides, compounds, pepducins and peptidomimetics, but how they distinguish between formyl peptides and non-formyl peptides is not yet known (Dahlgren et al., 2016).However, the recent structural data on both FPR1 and FPR2 should provide more insights into how these receptors recognize different ligands (Liao & Ye, 2022).

| GPCR cross-talk in neutrophils
In neutrophils, there is a defined GPCR hierarchy in which FPRs are 'higher in rank' than some of the other receptors.For example, activated FPRs either suppress or amplify a secondary response induced by other GPCR agonists, and this has led to the general assumption that FPR1 and FPR2 cross-talk with other GPCRs in an identical manner (Dahlgren et al., 2022).However, our recent study with GPR84 has challenged this view, as FPR1 and FPR2 regulated the GPR84 response in opposite directions, with FPR2 activation amplifying secondary responses mediated by GPR84 and FPR1 activation suppressing secondary responses mediated by GPR84 (Figure 2).These opposing effects are most probably achieved by FPR2 reactivation induced by GPR84 agonists and FPR1-mediated heterologous desensitization of GPR84, respectively (Mårtensson et al., 2021).In addition to homologous desensitization, activated FPRs can also induce heterologous desensitization of hierarchically lower ranked GPCRs.For example, neutrophils that first received FPR agonists are nonresponsive to a second stimulation with IL-8, which is caused by that the activated FPRs heterologously desensitize CXCR1 and CXCR2 (Fu F I G U R E 2 Diagram of formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2) cross-talk with GPR84 in regulating the reactive oxygen species (ROS) production by human neutrophils.Stimulation of neutrophils with FPR agonists induces ROS production and subsequent receptor homologous desensitization.In comparison to the GPR84-mediated ROS production by naïve neutrophils, the ROS production induced by GPR84 agonists in cells containing desensitized FPR1 is suppressed (upper panel), whereas it is amplified in cells with desensitized FPR2 (lower panel).These opposite effects of the GPR84-mediated ROS production in FPR1/FPR2-desensitized neutrophils is achieved through different receptor cross-talk mechanisms.et al., 2004).Moreover, desensitized FPRs can, in addition to heterologous desensitization, also be reactivated through a form of receptor cross-talk by signals generated downstream of some GPCRs, such as the PAF-and P2Y 2 -receptors (Forsman et al., 2013;Gabl et al., 2014;Holdfeldt et al., 2017;Önnheim et al., 2014;Sundqvist et al., 2019).
The precise details of the signalling downstream of the PAF-and P2Y 2 -receptors that lead to reactivation of the desensitized FPRs have not yet been identified.But no reactivation signals are triggered by these receptors when the activity of the Gα q subunit of the G protein is inhibited, suggesting an involvement of Gα q signalling in their cross-talk with the FPRs (Holdfeldt et al., 2017(Holdfeldt et al., , 2020)).

| GPCR-biased signalling in neutrophils
Binding of receptor-specific GPCR ligands (orthosteric agonists or allosteric modulators) can stabilize the occupied receptor in distinct different conformations.In contrast to the responses induced by 'balanced' ligands, biased ligands stabilize the receptor in a conformation in which one of the receptor's downstream signalling pathways is favoured over another pathway.Hence, biased signalling may give rise to a functional selective response that can range from a complete avoidance of one signalling pathway to a skewed efficacy for different functions.Consequently, the use of GPCR-biased ligands has become a strategy and attracted great interest in drug discovery, aiming to develop new drugs that mediate solely beneficial responses and/or avoid undesired side effects.In line with the emerging concept of GPCR-biased signalling and functional selectivity, we have found a distinct signalling profile and functional selectivity mediated by the FPRs expressed by neutrophils.Thus, FPR-activating biased ligands that lack the ability to recruit β-arrestin, are unable to induce chemotactic migration, suggesting a link between β-arrestin recruitment and neutrophil chemotaxis.However, the precise role of β-arrestin in neutrophil migration has not yet been clarified (Gabl et al., 2017;Lind, Dahlgren, et al., 2021;Lind, Holdfeldt, et al., 2019;Sundqvist et al., 2019).Furthermore, our studies on FFA2 using two allosteric modulators and GPR84 using a biased agonist have provided insights into biased signalling/functional selectivity for these FFA receptors in human neutrophils (see below).

| Neutrophil FFA receptors
Free fatty acids were traditionally believed to exert their metabolic responses only through interactions with intracellular targets such as the PPARs (Nakamura et al., 2014).However, it is now evident that free fatty acids mediate their effects also through the group of GPCRs that together are termed FFA receptors.This receptor group includes FFA1 (earlier known as GPR40), FFA2 (earlier known as GPR43), FFA3 (earlier known as GPR41), FFA4 (earlier known as GPR120) and GPR84, which differ in that they recognize free fatty acids of different carbon chain lengths.The FFA receptors are involved in regulation of both inflammatory responses and energy metabolism and they have therefore received attention as potential drug targets in both metabolic and inflammatory conditions (Alvarez-Curto & Milligan, 2016;Kimura et al., 2020;Miyamoto et al., 2017;Tan et al., 2017).Among the five FFA receptors, FFA2 (sensing short-chain fatty acids [SCFAs]) and GPR84 (sensing MCFAs) are functionally expressed by primary human neutrophils, whereas GPR84, but not FFA2, is functionally expressed by primary human monocytes and human monocytederived macrophages (Sundqvist et al., 2018).

| EXPRESSION AND FUNCTION OF FFA2 IN NEUTROPHILS
Fermentation by gut bacteria of fibre diet carbohydrates generates large amounts of SCFAs, the most abundant being acetate (C2), propionate (C3) and butyrate (C4).Neutrophils express FFA2 for sensing these SCFAs, a molecular pattern produced as the final metabolites by gut microbes during fermentation (Brown et al., 2003;Le Poul et al., 2003).Recent studies have provided new aspects of FFA2 activation, including the finding that the anaerobic oral bacterial flora, regarded to be of importance for the development of periodontal diseases, release SCFAs that mediate neutrophil migration through FFA2 in vitro (Dahlstrand Rudin et al., 2020, 2021).In addition, tissue neutrophils isolated from an aseptic inflammatory site are desensitized to FFA2 agonists, suggesting that FFA2 is involved in neutrophil migration in vivo (Björkman et al., 2016;Sundqvist et al., 2018).More recently, Mårtensson et al., (2022) showed that acetoacetate (one of the ketone bodies) was an endogenous ligand for FFA2, activating neutrophils with a profile similar to that of the SCFAs.These data not only highlight the role of FFA2 expressed by neutrophils as a link between metabolism and inflammation but also imply that novel roles of FFA2 in other tissues/organs than the intestine should be explored.
The identification and characterization studies of potent FFA2 selective tool compounds in the form of allosteric modulators, agonist and antagonists have increased our understanding of the signalling and function of this receptor (Grundmann et al., 2021;Milligan et al., 2017;Suckow & Briscoe, 2017).Several neutrophil functions such as activation of the ROS-generating NADPH oxidase, chemotaxis and generation of cytokines are regulated by SCFAs (Björkman et al., 2016;Rodrigues et al., 2016).Mechanistic insights into FFA2 activation and allosteric modulation have disclosed both similarities, differences and some unique features of this FFA receptor compared to both the closely related GPR84 and the FPRs (see below).

| Role of cytosolic calcium in FFA2 signalling and allosteric modulation
Studies with positive allosteric FFA2 modulators of show that these modulators enhance the neutrophils response to orthosteric agonists recognized by FFA2.Regarding signalling and neutrophil activation, the transient rise in [Ca 2+ ] i is one of the earliest events following GPCR agonist binding.But the NADPH oxidase-derived ROS can be produced without any rise in [Ca 2+ ] i (Dahlgren et al., 2020(Dahlgren et al., , 2022)), suggesting that raised [Ca 2+ ] i is not essential for the activation of NADPH oxidase in neutrophils.This suggestion is supported by the fact that two allosteric FFA2 modulators, recognized by different allosteric binding sites of neutrophil-expressed FFA2, together potently induce NADPH oxidase activity without inducing any rise in [Ca 2+ ] i (Lind et al., 2020;Lind, Holdfeldt, et al., 2021).Such biased signalling downstream FFA2 (signalling that leads ROS production without any rise in [Ca 2+ ] i ) induced by two allosteric modulators is not triggered by balanced orthosteric FFA2 agonists that activate both the PLC-PIP 2 -IP 3 -Ca 2+ and NADPH oxidase routes.In addition, allosterically modulated FFA2 cross-talk with other GPCRs in neutrophils and converts agonists for the FPRs, the PAF-and the P2Y 2 -receptors into potent ROS inducers (Dahlgren et al., 2022).Our recent observation suggests that a rise in [Ca 2+ ] i may activate allosterically modulated FFA2 in the absence of an orthosteric agonist, suggesting a novel regulatory mechanism operating from the cytosolic side of the receptor that directly can activate FFA2 (Lind et al., 2022).

| EXPRESSION AND FUNCTION OF GPR84 IN NEUTROPHILS
GPR84 was cloned in 2001 from RNA isolated from human peripheral blood neutrophils, and studies show that resting neutrophils express high level of GPR84 (Yousefi et al., 2001).In addition, GPR84 mRNA has been identified in eosinophils, monocytes/macrophages, bone marrow, lungs, activated microglia and splenic B and T cells (Luscombe et al., 2020).The low basal expression of GPR84 in immune cells can be further increased by treatment with an inflammatory stimulus, such as LPS (Mancini et al., 2019;Recio et al., 2018;Wang et al., 2006), suggesting an important role of GPR84 in different inflammatory disorders, including ulcerative colitis (Marsango et al., 2022;Zhang et al., 2022).Compared to the extensively studied FPRs, very little is known about the activation pattern and regulation mechanisms of GPR84 in human neutrophils and in the context of inflammation.Through a targeted search for potential endogenous ligands, GPR84 was, in 2006, de-orphanized as a receptor that, in vitro, was activated by MCFAs containing between 9 and 14 carbon atoms, with capric acid/decanoic acid (C10), undecanoic acid (C11) and lauric acid (C12) being the most potent activators (Wang et al., 2006).With regard to human neutrophils, MCFAs mediated chemotaxis (Mikkelsen et al., 2022;Suzuki et al., 2013), induced NADPH oxidase activity in primed neutrophils (Sundqvist et al., 2018), initiated formation of NETs and modulated neutrophil phagocytosis (Peters et al., 2022).However, because high concentrations of MCFAs are needed to activate rather modest GPR84-mediated neutrophil responses in vitro and that it is unclear if such high concentrations are present in vivo, it remains to be elucidated if there are other (more potent) endogenous ligand/ligands for GPR84 (Luscombe et al., 2020).

| GPR84 activity in in vitro and in vivo primed neutrophils
Functional studies of GPR84 and FFA2 in human phagocytes have revealed that both these receptors are expressed in primary neutrophils, but only GPR84 is functionally expressed also in primary monocytes and monocyte-derived macrophages (Lucy et al., 2019;Sundqvist et al., 2018).The difference in the functional expression of these two classes of FFA receptors in neutrophils and monocytes/ macrophages suggests that the role of FFA2 may be restricted to the very early phase of an acute inflammation dominated by neutrophils, whereas GPR84 may have a broader functional spectrum in modulating inflammatory processes where both neutrophils and monocytes are involved.We have shown that the GPR84 agonist ZQ16 induces a rise in [Ca 2+ ]i, is a weak secretagogue (triggered a low level of degranulation), a modest chemoattractant and a weak activator of NADPH oxidase-derived ROS in naïve neutrophils (Sundqvist et al., 2018).All these neutrophil functions were abolished by the GPR84 selective antagonist GLPG1205 (Labéguère et al., 2020;Vanhoutte et al., 2015), confirming the involvement of GPR84 in neutrophil activation.Furthermore, when we examined the GPR84-mediated ROS production induced by ZQ16 in more detail, we found that the level of ROS could be significantly enhanced by pre-treatment of the cells with either the priming agent TNF-α or the actin cytoskeleton disrupting agent latrunculin A (Sundqvist et al., 2018).One possible mechanism underlying priming is induction of degranulation resulting in increased expression of secretory vesicle/granule membrane-stored receptors on the plasma membrane (Condliffe et al., 1998;Cowland & Borregaard, 2016;Hallett & Lloyds, 1995;Miralda et al., 2017).If degranulation is the cause for the increased GPR84-mediated NADPH oxidase activity in primed neutrophils can only be speculated on, as it is not yet known if neutrophils contain an intracellular storage of this receptor.However, it is known that TNF-α also primes the response mediated by some GPCRs that lack an intracellular and mobilizable pool of receptors (Fu et al., 2004), suggesting that there are priming mechanisms involved, other than degranulation (Miralda et al., 2017).This suggestion also gains support from the fact that both barbadin (an inhibitor of the β-arrestin/AP2 complex) and hyaluron (a nonsulfated glycosaminoglycan) prime neutrophils for increased FPRmediated ROS production without inducing degranulation and subsequent receptor up-regulation on the plasma membrane (Niemietz et al., 2020;Sundqvist et al., 2020).Further examination with more specific antibodies for GPR84 and for FFA2 to (i) determine the surface receptor expression upon priming by flow cytometry and (ii) determine the subcellular localization of the receptors with use of purified organelles and Western blotting should facilitate the understanding of the underlying mechanism(s) behind the primed GPR84 and FFA2 response in neutrophils.
Functional characterization of neutrophils is often performed with easily obtained peripheral blood neutrophils, but in vivo neutrophils primarily exert their functions in inflamed tissues after they have left the blood and migrated through the endothelial cell layer (Faurschou & Borregaard, 2003).Using human neutrophils collected from a skin chamber model representing an aseptic inflammation (Christenson et al., 2014), we have been able to study GPCR-mediated ROS production in vivo obtained tissue neutrophils, and compare them with the peripheral blood neutrophils, obtained from the same donor.Our data demonstrate that the ROS production was attenuated in tissue neutrophils, following stimulation with IL-8 or FFA2 agonists, whereas it was elevated upon stimulation with agonists selective for the FPRs or GPR84 (Björkman et al., 2016;Follin et al., 1991;Sundqvist et al., 2018).The exact mechanism behind why the tissue neutrophils are primed for GPR84 and FPR but not FFA2 activation remains to be investigated.

| Regulation of GPR84 by intracellular signalling molecules
Studies with a Gα q -specific inhibitor and Pertussis toxin (inhibits Gα i/o ) to analyse how GPR84 downstream signals are initiated suggest that GPR84 most probably relies on a heterotrimeric G protein that contains a Gα i/o subunit to initiate intracellular signalling.However, although Pertussis toxin completely inhibited the ZQ16 response in neutrophils (Sundqvist et al., 2018), we cannot exclude the possibility that other signalling proteins are involved due to the non-specific inhibition of this toxin on Gα i/o (Becker et al., 1986;Holdfeldt et al., 2017).Shortly after activation, GPCR signalling is terminated and the receptors are desensitized through a process involving β-arrestin binding to the cytosolic parts of the activated GPCR (Lefkowitz et al., 2006;Shenoy & Lefkowitz, 2011).However, in neutrophils, the actin cytoskeleton can replace β-arrestin and form the basis, not only for termination of signalling but also for desensitization of the FPRs (Bylund et al., 2003;Dahlgren et al., 2016Dahlgren et al., , 2022;;Jesaitis & Klotz, 1993;Klotz & Jesaitis, 1994).Regarding the role of the actin cytoskeleton in regulating GPR84 signalling, our data have shown that in naïve neutrophils, the NADPH oxidase activity induced by GPR84 agonists is blocked by the actin cytoskeleton.Hence, disrupting the actin cytoskeleton prior to GPR84 activation leads to an increased production of ROS, similar to that previously observed after activating the P2Y 2 receptor with ATP (Gabl et al., 2015;Sundqvist et al., 2018).However, it is not known whether this cytoskeletonregulated change in responsiveness is due to a direct conformational change, leading to a switch of GPR84 from a low-affinity to a highaffinity state, or if it is due to a changed access to downstream signalling partners.
Data with DL-175, a biased GPR84 agonist that lacks the ability to induce recruitment of β-arrestin (Lucy et al., 2019), a protein of importance for GPCR signalling termination including receptor desensitization/internalization (Cheng et al., 2022;Gurevich & Gurevich, 2019;Luttrell et al., 2018;Luttrell & Lefkowitz, 2002), have revealed some clues about how GPR84-mediated responses are regulated in neutrophils.For example, the DL-175-induced NADPH oxidase activity is as rapidly terminated as that induced by a GPR84 agonist (ZQ16) that recruits β-arrestin, which suggests that additional mechanisms than β-arrestin recruitment are in use to terminate GPR84 signalling (Fredriksson et al., 2022).In addition, and in contrast to the FPRs, desensitized GPR84 and FFA2 in neutrophils are not reactivated upon disruption of the actin cytoskeleton, as the latrunculin A reactivation effect was abolished if added after a GPR84/FFA2 agonist.This finding also implies the actin cytoskeleton not to be involved in terminating the neutrophil responses mediated through GPR84/FFA2 (Björkman et al., 2016;Sundqvist et al., 2018).Based on this, we have proposed that, depending on the type of GPR84 agonist that is used (balanced or biased), at least two pathways are involved in terminating GPR84 signalling, one being regulation by GPCR kinase 2 (GRK2) and β-arrestin recruitment, following activation by a balanced agonist, and the other being regulation by the actin cytoskeleton upon activation by a biased (non-β-arrestin recruiting) agonist (Figure 1).The latter pathway has gained support by showing that a disruption of the actin cytoskeleton induced a robust reactivation of the ROS production in DL-175-desensitized neutrophils (i.e., in the absence of β-arrestin recruitment), as compared to no reactivation response upon disruption of the actin cytoskeleton in ZQ16-desensitized neutrophils (i.e., in the presence of β-arrestin recruitment) (Fredriksson et al., 2022).The recruitment of β-arrestin is facilitated by phosphorylation of the activated GPCR, and the corresponding kinases (GRKs) play key roles in this process.Among the seven GRK isoforms described, GRK2 is the predominant isoform expressed by leukocytes (Cheng et al., 2022).Using sub-cellularly fractionated neutrophils (azurophil granules, the specific/gelatinase granules, plasma membranes/secretory vesicles and cytosol), our recent study demonstrated GRK2 to predominantly be present in the cytosol.
Furthermore, this study also showed that neutrophils pre-treated with GRK2 inhibitors display (i) an enhanced GPR84-mediated ROS production and (ii) a reduced GPR84-mediated β-arrestin recruitment, whereas (iii) the receptor downstream PLC-PIP 2 -IP 3 -Ca 2+ signalling pathway was unaffected.These results thus highlight a role of GRK2 in regulating GPR84 signalling that may be specific for this receptor, as the same responses, when mediated through FPR2, were unaffected by GRK2 inhibition (Fredriksson et al., 2022).At the mechanistic level, it is reasonable to assume that the difference in the potential phosphorylation sites between GPR84 and FPR2 may determine the effects of GRK2 inhibition.However, although these data agree with that GRK2 and β-arrestin work together to regulate GPCRs, the molecular mechanism for how GRK2 is activated and subsequently regulates and interacts with GPR84-activated neutrophils is not yet known.To add complexity, the precise roles of β-arrestin in regulating GPCR-mediated neutrophil functions also remain largely unknown.
Recent studies have proposed an important role of β-arrestin in neutrophil chemotaxis (Gabl et al., 2017;Lind, Dahlgren, et al., 2021;Lind, Holdfeldt, et al., 2019;Sundqvist et al., 2019).The exact molecular mechanism underlying the regulation of neutrophil chemotaxis by β-arrestin is currently not known.However, it has been suggested that β-arrestin directly binds to regulatory proteins that are involved in the reorganization of the actin cytoskeleton (DeFea, 2007).Such binding might be able to modulate the activity of these regulatory proteins and ultimately influence the chemotactic response of neutrophils.
However, further research is needed to fully understand the underlying mechanism(s) of this process.

| GPR84 hierarchy and cross-talk with FPRs in neutrophils
The neutrophil response triggered by GPCRs relies not only on the specific agonists that activate their respective receptor but also on a complex receptor downstream signalling network.Different hierarchical receptor cross-talk mechanisms, which regulate receptor activities, are involved in fine-tuning the functions of neutrophils.In the neutrophil receptor hierarchy, the position of FPR1 and FPR2 is in most cases identical, that is, activation of the FPRs either amplifies or suppresses the responses induced by other GPCR agonists (Dahlgren et al., 2022).However, in terms of GPR84, FPR1 and FPR2 regulate the activity of this receptor in opposite directions (Figure 2).For example, agonists of GPR84 are poor activators of the ROSgenerating NADPH oxidase in naïve neutrophils, but their potency as NADPH oxidase activators is much increased in FPR2-desensitized neutrophils.Furthermore, the GPR84-mediated ROS production in FPR2-desensitized neutrophils was blocked by either a GPR84 antagonist or a FPR2 antagonist.This suggests that the FPR2-mediated amplification of the GPR84-induced NADPH activity is achieved through a receptor cross-talk in which the FPR2-desensitized neutrophils are activated by signals generated downstream of GPR84.In contrast to FPR2-desensitized neutrophils, the GPR84-mediated ROS production in FPR1-desensitized neutrophils primarily resulted in suppression of NADPH oxidase activity, which may be a cause of heterologous GPR84 desensitization by FPR1-mediated signals (Mårtensson et al., 2021).Thus, these recent data on the interactions between the two FPRs and GPR84 in neutrophils revealed a signalling difference between the two FPRs.

| CONCLUSIONS AND FUTURE PERSPECTIVES
Due to easy accessibility and high quantity, human peripheral blood neutrophils have proven to be valuable model cells for examining GPCR regulation in primary cells.Studies of neutrophil GPCRs with recently developed tool compounds have indeed led to the generation of a vast amount of information on ligand binding and downstream signalling.
However, the classical view of the 'on-off' mode of GPCR activation has been challenged as these studies also have provided novel insights into GPCR signalling cross-talk and reactivation.The data generated highlight also more complex regulation mechanisms and targeting difficulties of GPCRs expressed on cells in other tissues than peripheral blood.Although GPR84 was discovered over 20 years ago and shown to be highly expressed in human neutrophils, the precise biological role of this receptor remains unclear.The general assumption is that GPR84 is a pro-inflammatory receptor that binds MCFAs, a link that has highlighted a role for GPR84 in both metabolism and inflammation.However, as it still remains to be shown that the in vivo concentrations of MCFAs reach the levels shown to activate GPR84 in vitro, the search for other potential physiological ligands is ongoing.Hence, more research and new tools are needed to fully understand the pathophysiological role of GPR84.
Increased knowledge of the complex mechanisms of GPR84 activation in human neutrophils, where the receptor and downstream signalling molecules are endogenously expressed, will undoubtedly increase our understanding of GPCR signalling in general and provide novel GPR84-based prophylactic and secondary prevention strategies.