The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids ( HCAR ) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA 1 , HCA 2 ) but humans and other hominids contain a third member (HCA 3 ) in their genomes. A plausible hypothesis why HCA 3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA 3 in vitro and in vivo . The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA 3 . D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA 3 -dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA 3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.

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Background: Medium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria. They activate G protein-coupled receptors, including GPR84 and HCA 3 , which regulate metabolism and immune functions. Although both receptors are coupled to G i proteins, share at least one agonist and show overlapping tissue expression, GPR84 exerts pro-inflammatory effects whereas HCA 3 is involved in anti-inflammatory responses. Here, we analyzed signaling kinetics of both HCA 3 and GPR84, to unravel signal transduction components that may explain their physiological differences. Methods: To study the signaling kinetics and components involved in signal transduction of both receptors we applied the label-free dynamic mass redistribution technology in combination with classical cAMP, ERK signaling and β-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models. Results: We present strong evidence for a natural biased signaling of structurally highly similar agonists at HCA 3 and GPR84. We show that HCA 3 signaling and trafficking depends on dynamin-2 function. Activation of HCA 3 by 3hydroxyoctanoic acid but not 3-hydroxydecanoic acid leads to β-arrestin-2 recruitment, which is relevant for cellcell adhesion. GPR84 stimulation with 3-hydroxydecanoic acid causes a sustained ERK activation but activation of GPR84 is not followed by β-arrestin-2 recruitment. Conclusions: In summary, our results highlight that biased agonism is a physiological property of HCA 3 and GPR84 with relevance for innate immune functions potentially to differentiate between endogenous, non-pathogenic compounds and compounds originating from e.g. pathogenic bacteria.

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