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 β-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.

The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca 2 + mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five inhouse compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch.

Neutrophils express many G protein-coupled receptors (GPCRs) including 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 GPCRs, i.e., the GPCR-mediated response can be either suppressed or amplified by signals generated by FPRs. In this study, we investigated the position of GPR84 in the FPR-defined hierarchy regarding the activation of neutrophil NADPH-oxidase, an enzyme system designed to generate reactive oxygen species (ROS). When naïve neutrophils are activated by GPR84 agonists a modest ROS release was induced. However, vast amounts of ROS production was induced by these GPR84 agonists in FPR2-desensitized neutrophils, and the response is inhibited not only by a GPR84 antagonist but also by an FPR2 specific antagonist. This suggests that the amplified GPR84 agonist response is achieved through a reactivation of the desensitized FPR2. In addition, the GPR84-mediated FPR2 reactivation was independent of β-arrestin recruitment and sensitive to a protein phosphatase inhibitor. In contrast, the modest ROS production induced by GPR84 agonists was primarily suppressed in FPR1-desensitized neutrophils through hierarchical desensitization of GPR84 by FPR1 generated signals.In summary, our data show that FPRs control the NADPH-oxidase activity mediated through GPR84 in human neutrophils. While an amplified ROS generation is achieved by GPR84 agonists through reactivation of desensitized FPR2, FPR1 heterologously desensitizes GPR84 and by that suppresses the release of ROS induced by GPR84 agonists.