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. Several of the functionally known neutrophil GPCRs, are not part of the human neutrophil proteome. To identify GPCRs not earlier shown to be expressed in human neutrophils, we utilized a microfluidic flow cell technique in combination with subcellular granule fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach added the hydroxy-carboxylic acid 3 receptor (HCA ₃ R) to the human neutrophil proteome. The β-oxidation intermediate 3-hydroxy-octanoic acid (3-OH-C8) is the primary endogenous agonist for this receptor which is highly expressed in adipocytes where it has anti-lipolytic effects. Literature describing the role and function of HCA ₃ R in human neutrophils is scarce. We now show that 3-OH-C8 as well the synthetic HCA ₃ R agonist IBC 293 activate human neutrophils determined as an increase in the intracellular concentration of free calcium ions ([Ca ²⁺ ] _i ) and activation of the NADPH oxidase. However, in contrast to the rise in [Ca ²⁺ ] _i which could be triggered in naïve neutrophils, pre-treatment of neutrophils was required for HCA ₃ Rs to activate the NADPH oxidase. That is, the HCA ₃ R-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 HCA ₃ R 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 HCA ₃ R agonists.