A molecular mechanism to diversify Ca2+ signaling downstream of Gs protein-coupled receptors.

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.

Cell swelling enhances ligand-driven ß-adrenergic signaling.

G protein-coupled receptors' conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the ß2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors' function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the ß2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.

Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting ß-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels.

Arginine-vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Development and basic performance verification of a rapid homogeneous bioassay for agonistic antibodies against the thyroid-stimulating hormone receptor.

Graves' disease is a type of autoimmune hyperthyroidism caused by thyroid-stimulating antibodies (TSAb).1 The combination of a porcine thyroid cell bioassay and cyclic adenosine monophosphate (cAMP) immunoassay (TSAb-enzyme immunoassay; EIA) is a clinically approved TSAb measurement method. Due to the requirement of multiple procedures and a long assay time of 6 h in the TSAb-EIA, a simplified and rapid assay is desired. Herein, we developed a rapid homogeneous TSAb bioassay (rapid-TSAb assay) using the human embryonic kidney cell line (HEK293), engineered to express the human thyroid-stimulating hormone receptor (TSHR), along with a cAMP-dependent luminescence biosensor. The measurement consists of three steps: thawing frozen cells, blood sample addition, and luminescence detection. The procedures can be conducted within 1 h. The World Health Organization International Standard TSAb (NIBSC 08/204) stimulated the cells co-expressing TSHR and cAMP biosensor. The intra- and inter-assay coefficients of variance were < 10%. Stimulation activity using wild-type TSHR and chimeric TSHR (Mc4) almost completely correlated with the tested Graves' disease and normal samples. In the rapid-TSAb assay, the evaluation of 39 samples, including TSHR antibody-positive sera, yielded a sensitivity of 100.0% and a specificity of 90.9%, compared to the TSAb-EIA control. The rapid-TSAb assay enables simple and rapid measurement of TSAb and is promising for improving the diagnosis of autoimmune thyroid diseases.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

Eriodictyol and thymonin act as GPR35 agonists.

Although herbs and spices have been used in traditional medicine for more than a century owing to their health benefits, the associated underlying mechanism is still not clear. Since the G protein-coupled receptor 35 (GPR35) has been linked to exert various antioxidant and anti-inflammatory effects, we screened 19 different herbs and spices for possible GPR35 agonist(s) to understand the GPR35-dependent functions of herbs and spices. Among the screened extracts, the ethyl acetate extract of thyme exhibited a remarkable GPR35 agonistic activity. Activity-guided separations allowed us to identify 2 polyphenolic phytochemicals, eriodictyol and thymonin, acting as GPR35 agonists. Both eriodictyol and thymonin showed a potent and specific agonist activity toward GPR35 with half maximal effective concentration values of 5.48 and 8.41 µm, respectively. These findings indicate that these phytochemicals may have beneficial health effects upon GPR35 activation.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Structural basis of CXC chemokine receptor 1 ligand binding and activation.

Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.

A2B adenosine receptor activation and modulation by protein kinase C.

Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A2B adenosine receptor (AR)-mediated, but not ß2-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated A2BAR with low Emax (H9C2 and NIH3T3 cells endogenously expressing A2BAR), or with high Emax (A2BAR-overexpressing HEK293 cells) to induce cAMP accumulation. A2BAR activation induced by PKC was inhibited by A2BAR and PKC inhibitors but enhanced by A2BAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of A2BAR function and A2BAR activation. Thus, we establish PKC as an endogenous modulator and activator of A2BAR, involving Giα and PKCγ. Depending on signaling pathway, PKC could activate and enhance, or alternatively inhibit A2BAR activity. These findings are relevant to common functions of A2BAR and PKC, e.g. cardioprotection and cancer progression/treatment.

Stabilization of pre-existing neurotensin receptor conformational states by ß-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. ß-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A ß-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels.

Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding.

Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex.

Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.

Enhanced membrane binding of oncogenic G protein αqQ209L confers resistance to inhibitor YM-254890.

Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of αqQ209L by YM, we developed and examined N-terminal mutants of αqQ209L, termed PM-restricted αqQ209L, in which the addition of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted αqQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both αqQ209L and PM-restricted αqQ209L, resulting in increased binding to ßγ and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-ß. GPCR-dependent signaling by PM-restricted WT αq is strongly inhibited by YM, demonstrating that resistance to YM inhibition by membrane-binding mutants is specific to constitutively active αqQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit αqQ209L and suggest that YM contributes to inhibition of αqQ209L by promoting its relocalization.

Endogenous ligand recognition and structural transition of a human PTH receptor.

Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.

Agonist-Dependent Coupling of the Promiscuous Adenosine A2B Receptor to Gα Protein Subunits.

The adenosine A2B receptor (A2BAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family. It is upregulated under hypoxic conditions, in inflammation and cancer. Previous studies indicated the coupling of the A2BAR to different G proteins, mainly Gs, but in some cases Gq/11 or Gi, depending on the cell type. We have now utilized novel technologies, (i) heterologous expression of individual members of the Gαq/11 protein family (Gαq, Gα11, Gα14, and Gα15) in Gαq/11 knockout cells, and (ii) the TRUPATH platform, allowing the direct observation of Gα protein activation for each of the Gα subunits by bioluminescence resonance energy transfer (BRET) measurements. Three structurally diverse A2BAR agonists were studied: the cognate agonist adenosine, its metabolically stable analog NECA, and the non-nucleosidic partial agonist BAY 60-6583. Adenosine and NECA activated most members of all four Gα protein families (Gαs, Gαq/11, Gαi, and Gα12/13). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the Gα15, Gαs, and Gα12 proteins, and for NECA additionally at the Gαi2 protein. In contrast, the partial agonist BAY 60-6583 only activated Gα15, Gαs, and Gα12 proteins. Adenosine deaminase, an allosteric modulator of ARs, selectively increased the potency and efficacy of NECA and BAY 60-6583 at the Gα15 protein, while it had no effect or decreased efficacy at the other Gα proteins. We conclude that the A2BAR is preferably coupled to the Gα15, Gαs, and Gα12 proteins. Upon upregulation of receptor or Gα protein expression, coupling to further Gα proteins likely occurs. Importantly, different agonists can display different activation profiles.

Secreted phospholipase A2 modifies extracellular vesicles and accelerates B cell lymphoma.

Extracellular vesicles (EVs) including exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of EV lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived EVs is augmented via secreted phospholipase A2 (sPLA2)-driven lipid metabolism. Hydrolysis of EV phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids, and their metabolites. sPLA2-treated EVs were smaller and self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor-associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice, while treatment with sPLA2-modified EVs reversed this phenotype. Furthermore, sPLA2 expression in human large B cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated EV modification promotes tumor development, highlighting a non-canonical mechanistic action of EVs as an extracellular hydrolytic platform of sPLA2.

Phenotypic evaluation of constitutive GPCR/G-protein signaling in zebrafish embryos and larvae.

Signal transduction initiation by G-protein-coupled receptors (GPCRs) normally begins upon extracellular ligand binding. Some oncogenic GPCR mutants are capable of inducing G-protein signaling without ligand stimulation, thus behaving as constitutively active receptors. Evaluation of disease-causing capacity of constitutively active mutations in animal models requires months of time-consuming experiments, which hampers research progress. Here, using zebrafish embryos transiently expressing with constitutively active mutations via mRNA microinjection, we describe G-protein-subtype-specific phenotypes that can be evaluated over several days. Exogenous expression of the cysteinyl leukotriene receptor type II (CysLT2R) with an oncogenic L1293.43Q mutation by mRNA injection into a fertilized embryo induced developmental arrest during epiboly and eventual embryonic lethality, which were suppressed by treatment with the Gq inhibitor, YM-254890. Embryos with a constitutively active Gαq mutant exhibited an analogous phenotype. Interestingly, expression of constitutively active Gαs, Gαi, and Gα13 mutants induced distinct phenotypes. These phenotypes may thus serve as useful indicators for rapid in vivo evaluation of signaling activity of GPCR and G-protein mutants.

Intestinal microbe-dependent ω3 lipid metabolite αKetoA prevents inflammatory diseases in mice and cynomolgus macaques.

Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.