Activation and signaling characteristics of the hydroxy-carboxylic acid 3 receptor identified in human neutrophils through a microfluidic flow cell technique

Human neutrophils express numerous G protein-coupled receptors (GPCRs) of importance for immune regulation. However, several functionally characterized neutrophil GPCRs, are not included within the human neutrophil proteome. To identify GPCRs not previously demonstrated to be expressed in human neutrophils, we utilized a microfluidic flow cell technique in conjunction with subcellular granule fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach led to the identification of hydroxy-carboxylic acid 3 receptor (HCA₃R, also known as GPR109B) as a novel component of the human neutrophil proteome. The β-oxidation intermediate 3-hydroxy-octanoic acid (3-OH-C8) is the primary endogenous agonist of the HCA₃R and expressed at high levels in adipocytes where it exerts anti-lipolytic effects. However, literature describing the role and function of HCA₃R in human neutrophils is scarce. We show that 3-OH-C8, as well as the synthetic HCA₃R agonist IBC 293, activate human neutrophils determined as an increase in the intracellular concentration of free calcium ions ([Ca²⁺]_i) and activation of the NADPH oxidase. However, in contrast to the rise in [Ca²⁺]_i, which could be triggered in naïve neutrophils, pre-treatment of neutrophils was required for the HCA₃R agonists to activate the NADPH oxidase. That is, the HCA₃R-mediated NADPH oxidase activation occurred only in neutrophils pre-treated with either an actin cytoskeleton disrupter or an allosteric modulator targeting the GPCR termed free fatty acid receptor 2 (FFA2R). Our findings demonstrate that HCA₃R is not only a new member of the human neutrophil proteome but also exhibits functional activity with complex signaling pathways when stimulated with endogenous and synthetic HCA₃R agonists.