Using embelin and its analogs as surrogate GPR agonists we

Using embelin and its analogs as surrogate GPR84 agonists we discovered that GPR84 couples to G12 and G13 signaling pathways in addition to Gi, linking receptor function to Rho/Rac signaling and modulation of the cytoskeleton. In primary human macrophages, GPR84 activation leads to Gβγ signaling, Erk1/2 and Akt phosphorylation, PI-3 kinase activation, and initiation of intracellular calcium flux. Additionally, we observed noncanonical Giβγ dependent elevation of cAMP in conjunction with Gs agonists, acting in a paracrine or autocrine fashion. Thus, GPR84 modulates pathways known to regulate cell motility/chemotaxis, such as initiating and maintaining cell polarity and controlling actin/myosin mediated cell contractility. Chemokine, S1P and other GPCRs which induce cell chemotaxis share similar signaling features. Indeed, we demonstrated that GPR84 is a chemoattractant receptor and its activation produces robust migration of human neutrophils across agonist gradients. We speculate that an endogenous ligand for GPR84 is likely to be a modulator released during inflammation, possibly of prostaglandin/eicosanoid nature, which is involved in attracting macrophages and/or neutrophils to the site of inflammation. This chemotactic nature and signaling pattern suggest that GPR84 possesses proinflammatory phenotypic features. These observations were strengthened by the findings that while GPR84 activation by itself does not lead to oxidative burst in neutrophils, but rather is involved in priming of neutrophils for oxidative burst initiated by other proinflammatory agents, such as FMLP and C5A. Involvement of GPR84 in proinflammatory cytokine release from immune UCM05 was observed in cells derived from the peritoneum of wild type and GPR84 deficient mice. While no differences in the cellular composition of both exudates were detected, cells from the GPR84 deficient mice produced significantly lower amounts of several proinflammatory cytokines and molecules in response to LPS challenge, namely IL-6, KC-GROα, VEGF, MIP-2 and NGAL. These findings are consistent with previous reports, which provided data for GPR84 involvement in LPS mediated release of IL-6, IL-1β and TNFα from mouse peritoneal macrophages [7]. The differences in the nature of the differentially expressed markers between the two studies are most likely due to the nature of cells used in the assays. Total peritoneal exudates containing B/T cells, macrophages and neutrophils from the naïve mice were used in this study, while polyacrylamide bead-attracted peritoneal macrophages were used in the study by Nicol et al. [7]. In addition, Nicol et al. used transcriptional profiling of cells, while in the current study soluble proteins/cytokines/factors released into the culture media were evaluated. While performing these studies we observed that many features of GPR84 signaling in macrophages resemble those of GPR109A, a receptor for niacin that is also highly expressed on macrophages. This includes very similar modalities of non-canonical cAMP regulation, calcium and Erk signaling, and regulation of receptor expression by inflammatory stimuli [11]. Thus, we investigated whether GPR84 agonists regulate the same aspects of macrophage function as niacin and other GPR109A agonists. Niacin is a cholesterol modulating agent, which is highly effective in treating atherosclerotic disease. It has anti-dyslipidemic properties and is effective in increasing HDL-cholesterol in vivo, accompanied by substantial reductions in LDL-cholesterol [32]. The initial identification of GPR109A as a niacin receptor raised the possibility that the therapeutic properties of niacin might be mediated by this receptor, which became an attractive target for pharmaceutical development [33]. Extensive efforts were devoted to the development of niacin-like therapeutics, perhaps devoid of its flushing side effect [34,35]. The HDL modulating properties of niacin were believed to be mediated through cAMP dependent antilipolytic activity in adipocytes [33,36]. Niacin’s flushing side effect was found to be mediated by prostaglandin D2 and E2 release mediated by GPR109A expressed in dermal Langerhans cells [13,37]. Unfortunately, pharmaceutical development of GPR109A agonists has largely been halted by a number of serious roadblocks, including the discovery that the HDL elevating activity of niacin is GPR109A independent [34,35,38]. Moreover, the role of HDL elevation in reducing atherosclerotic disease in general, as well as its role in niacin’s antiatherosclerotic properties has been questioned [39,40]. Following these developments, several studies have revealed that niacin exerts antiatherosclerotic activities via macrophage expressed GPR109A, which are independent of its anti-dyslipidemic properties [11,21,22]. Macrophages are important players in the development of atherosclerotic disease. They are recruited by various inflammatory mediators to sites of developing atherosclerotic lesions, where they transform into the lipid/cholesterol loaded “foam” cells, which can serve as nucleation points for thrombus formation and arterial rupture [41]. In a study by Lukasova et al. [22], niacin’s macrophage mediated antiatherosclerotic effects were shown to be disease modifying in an in vivo setting.