Insights into GPR84 Biology

Immune Cell GPCRs

Given the importance of GPCRs and their agonists in cell-cell communication it is not surprising that GPCRs play central roles in regulation of the immune system. In drawing up Table I we have sought to include examples from all aspects of innate and adaptative immunity. Recognition and response to antigens by B lymphocytes is dominated by the B cell receptor for antigen in the context of MHC restriction, both of which are dominated by kinase signalling rather than GPCRs.
In a recent review Jason Cyster delineates the multiple roles of specific GPCRs in humoral immunity and B lymphocyte biology (Lu & Cyster, 2019). Efficient humoral antibody responses rely on optimising relatively rare interactions between antigen presenting cells and patrolling B lymphocytes within secondary lymphoid organs, with the CCR7 and the CCR6 chemokine receptors playing an important role in this process and the S1P1R receptor being important for lymphocyte recirculation. B lymphocyte differentiation, and their tropism to different host tissues continues to be directed by Class A GPCRs including CXCR4, CXCR3, CCR9 and CCR10 (Lu & Cyster, 2019).
Class A GPCRs play multiple roles in T cell biology through chemotaxis and immune cell homeostasis. One such receptor is CCR4 which came to attention following single cell RNA sequencing of human adult T cell leukemia / lymphoma (ATLL) cells (Nakagawa, Schmitz et al. , 2014). All the ATLL mutant CCR4 receptors responded more strongly in chemotaxis assays and p-Akt signalling assays to CCR4 chemokines and showed a loss of receptor internalisation following ligand binding. Enhanced PI3K / Akt activation conferred a survival advantage on cells expressing mutant CCR4 and hence identified CCR4 as a therapeutic molecular target in ATLL.
Another Class A GPCR chemokine receptor whose multiple roles in leukocyte homeostasis were revealed by rare germline mutations is the CXCR4 receptor the C-terminus of which is mutated in WHIM syndrome. WHIM stands for warts, hypogammaglobulinemia, infections and myelokathexis (retention of neutrophils in the bone marrow). Patients also have fewer B and T lymphocytes which leads to increased human papilloma virus infections, the cause of warts.
The majority of GPCRs listed in Table I are Class A GPCRs and their ligands are considered inflammatory mediators be they secreted proteins (e.g. chemokines), peptides (e.g. fMLF), or lipid mediators (e.g. prostaglandins and leukotrienes). The biological effects of the GPCRs listed in Table I are most often recognised in innate immune cells but signalling via these receptors is manifest in multiple cell types for instance in the stromal cells of lymphoid tissues or in adaptive immune cells. Consideration of the breadth of molecules that can act as Class A GPCR ligands listed in Table I and further consideration of GPCR deorphanisation leaves open the possibility that the physiological ligand of the GPR84 receptor remains to be discovered and that it plays a role in host immunity.

Effects of GPR84 agonists on innate immune cells

We know that GPR84 is expressed predominantly in immune cell types and is highly upregulated by inflammatory stimuli (Luscombe, Lucy et al. , 2020). Once expressed, activation of GPR84 using surrogate agonists results in an enhancement of inflammatory processes in a cell-type dependent manner. In innate immune cells, particularly monocytes/macrophages and neutrophils, research using surrogate GPR84 agonists has uncovered a role for this receptor in the mobilisation of inflammatory mediators, chemotaxis, and phagocytosis (Table II).
A review of the effects of GPR84 agonists on specific cells involved in innate immunity highlights the inflammatory effects of this receptor. The secretion of inflammatory cytokines, chemokines, and prostanoids, as well as ROS production, NETosis, and degranulation are regulated by GPR84 (Table II). In almost all cases, the effect of GPR84 activation is an increase in the mobilisation of pro-inflammatory mediators, or a decrease in anti-inflammatory mediators. In just two cases to date were opposite effects observed. Firstly, a decrease in CCL2 secretion with 6-OAU treatment was observed by Reyes, Kim, et al . (2021) in RAW264.7 macrophages infected with Brucella abortus . Secondly, a decrease in Tnf expression was observed by Ohue-Kitano, Nonaka,et al . (2023) with 6-OAU treatment of palmitate-stimulated RAW264.7 cells. Conversely, 6-OAU increased CCL2 secretion in primary murine M-CSF differentiated and LPS-stimulated bone marrow-derived macrophages (BMDMs) (Recio, Lucy et al. , 2018), and 6-OAU increased TNFα secretion in PMA-differentiated LPS-stimulated U937 macrophage cells (Suzuki, Takaishi et al. , 2013). This seeming contradiction only serves to highlight the variability of responses between cell types and stimulation paradigms, and indeed, potential context-dependent responses of GPR84.
GPR84 agonists have been demonstrated to act as chemoattractants, promoting chemotaxis, migration, and motility. For example, stimulation with both 6-OAU and embelin has been shown to induce chemotaxis in primary human and murine neutrophils and monocytes (Table II). However, results with macrophages are less clear. Evidence from immortalised cell lines suggest 6-OAU can act as a chemoattractant in PMA-differentiated U937 macrophages (Lucy, Purvis et al. , 2019; Suzuki, Takaishiet al. , 2013) but not primary murine M-CSF-differentiated LPS-stimulated BMDMs (Recio, Lucy et al. , 2018). There is a growing appreciation that the underlying mechanisms of macrophage migration differs from those of its monocytic precursors (Rumianek & Greaves, 2020). However, given the relative scarcity of reports on GPR84-mediated chemotaxis, cell-type differences in GPR84-mediated chemotaxis remain unclear.
The observation of enhanced bacterial adhesion and phagocytosis has been a more recent development in the field, and GPR84 agonists have been shown to promote these pathways (Table II). In contrast to observations in chemotaxis, reports of phagocytosis are almost exclusively made in macrophages. Here, 6-OAU has been shown to enhance adhesion to bacteria, enhance phagocytic activity, and enhance the number of phagocytic cells (Recio, Lucy et al. , 2018). GPR84 was also identified in a CRISPR screen for regulators of cancer cell phagocytosis (Kamber, Nishigaet al. , 2021). Here, stimulation with 6-OAU enhanced the phagocytosis of PMA-differentiated LPS-stimulated U937 macrophages and LPS-stimulated J774 macrophages of Ramos lymphoma target cells (Kamber, Nishiga et al. , 2021). Only one report showed small decreases in adhesion and uptake of live bacteria when stimulating RAW264.7 macrophages with 6-OAU (Reyes, Kim et al. , 2021). As with the previously discussed chemokine readout, the live infection context with pathogenic and immune-evasive Brucella spp. and Salmonellaspp. may relate to the observations of decreased CCL2 secretion and bacterial adhesion and uptake and certainly warrants further investigation.
The use of specific GPR84 agonists has undoubtedly proven highly useful in interrogating the physiological role of this receptor. While there is still ongoing debate over the endogenous agonist, particularly whether it is some form of MCFA, we believe it is still necessary to use potent surrogate agonists to support conclusions made with MCFAs which are low potency, are often used at concentrations above 100 µM, and consequently lack selectivity for GPR84. Furthermore, the discovery and characterisation of biased and allosteric agonists has opened new avenues to probe the biology of innate immune cells and should be widely adopted alongside the use of ‘balanced’ agonists such as 6-OAU. For example, Lucy, Purvis, et al . (2019) compared 6-OAU and DL-175 and found that only 6-OAU promotes chemotaxis in PMA-differentiated LPS-stimulated U937 macrophages, while both agonists promote chemotaxis in human monocytes. Similar comparisons were made by Mikkelsen, Arora,et al . (2022), noting that 3-hydroxy capric acid (3-OH-C10; Fig 2) promotes neutrophil but not monocyte migration, and that 2- or 3-hydroxylated MCFAs appear to promote migration while their non-hydroxy conterparts did not.
Delineating the effects of GPR84 signalling bias at the level of physiological readouts is a challenging task and requires systematic investigation. As can be seen, the variety of cell-types and stimulation paradigms completely obscures comparisons that could be made about agonist-specific results in the secretion of cytokines, chemotaxis, or phagocytosis. Studying the function of GPR84 in primary cells poses three challenges; first, the receptor requires transcriptional upregulation that subsides over time (e.g. LPS-stimulation upregulatesGpr84 with a peak at 8 h in BMDMs), second, some responses require priming (e.g. TNFα-priming prior to GPR84-mediated ROS release in neutrophils), and thirdly, activation of GPR84 will sometimes only augment existing responses (e.g. 6-OAU only augments C5a-mediated migration in BMDMs, or fMLP- or C5a-mediated ROS production in neutrophils). We therefore encourage researchers to attempt multiple stimulation paradigms when testing for agonist-mediated effects.