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.


INTRODUCTION
The incidence of allergic and inflammatory skin diseases and metabolic disorders, including type 2 diabetes, is increasing. [1][2][3] Accumulating evidence suggests that quantity of dietary lipid is a critical determinant in the development of inflammatory diseases. 4,5 In addition to the quantity of dietary lipids, their fatty acid composition plays important roles in the regulation of inflammatory diseases. In fact, the potential benefits of ω3 fatty acids in prevention of inflammatory vascular disease were discovered in a cohort study more than 40 years ago. 6 Yet, the beneficial effects of ω3 fatty acids in clinical studies remain debated. [7][8][9] Recent evidence suggests that the metabolism of dietary ω3 fatty acids is a key factor which influences their effectiveness in the regulation of health and diseases. The conversion of ω3 fatty acids into bioactive metabolites is mediated by mammalian enzymes including cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP). 10,11 Eicosapentaenoic acid (EPA), n-3 docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) are representative ω3 fatty acids, which exert pro-resolution and antiinflammatory properties through their conversion into bioactive lipid mediators, including EPA-derived resolvins, 17,18-epoxyeicosatetraenoic acid (EpETE) and 15-hydroxyeicosapentaenoic acid, n-3 DPA-derived 14-hydroxy DPA, and DHA-derived protectins and maresins. [12][13][14][15][16][17][18] These studies highlight that conversion of ω3 fatty acids into bioactive metabolites is essential for the regulatory roles of these lipids.
In this study, we found that αKetoA exerted potent antiinflammatory activities for the control of contact hypersensitivity in both mice and non-human primates and for the amelioration of diabetes in mice fed a high-fat diet (HFD) through regulating macrophage activity in a peroxisome proliferator-activated receptor (PPAR)γ-dependent manner. These results extend our knowledge by revealing the important roles of bacteria in accomplishing the health-promoting effects of ω3 fatty acids by generating the unique intestinal microbial lipid metabolite αKetoA.

RESULTS
αKetoA and αHYA are ω3 α-linolenic acid-derived and intestinal bacteria-dependent lipid metabolites We first sought to examine whether dietary intake of linseed oil, which is high in α-linolenic acid, increases the amount of αHYA and αKetoA in mouse feces. We fed mice a diet containing either soybean oil (Soy-mice) or linseed oil (Lin-mice) for 2 months and collected feces for the analysis of fatty acid metabolites. Consistent with the fatty acid composition of the dietary oils, lipidomic analysis through liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that the amount of α-linolenic acid was higher in the feces of Lin-mice than in those of Soy-mice (Fig. 1a). We also found that the amounts of αHYA and αKetoA were increased in the feces of Lin-mice (Fig. 1a). Furthermore, Linmice also showed increased serum levels of α-linolenic acid, αHYA, and αKetoA (Fig. 1a).
We next fed a linseed oil-containing diet to mice maintained under either specific-pathogen-free (SPF) or germ-free (GF) housing conditions for 2 months. The lipidomic analysis revealed that the amount of α-linolenic acid in the serum was comparable between SPF and GF mice (Fig. 1b). In contrast, the amounts of αHYA and αKetoA were lower or absent in the feces and serum of GF mice than in those of SPF mice (Fig. 1b). These results demonstrate that αHYA and αKetoA are lipid metabolites derived from ω3 α-linolenic acid and that their generation in the intestine and subsequent absorption into the body is dependent on the presence of intestinal bacteria.
Contact hypersensitivity is ameliorated by αKetoA through PPARγ-dependent inhibition of the development of inducible skin-associated lymphoid tissue (iSALT) We then examined whether αHYA and αKetoA exert antiinflammatory properties. To address this issue, we applied the 2,4-dinitrofluorobenzene (DNFB)-induced murine contact hypersensitivity model, a representative type IV skin allergic inflammation model that comprises sensitization and elicitation phases. In the sensitization phase, skin exposure to DNFB activates skin dendritic cells to migrate to the regional lymph nodes and activate naive T cells and consequently induce Th1 and Tc1 cells. 30 In the elicitation phase, re-exposure to DNFB induces the development of iSALT, which enhances the production of IFNγ by skin effector T cells in situ. 30 We orally administered the fatty acid metabolite to mice from before sensitization and during the experimental protocol, and we evaluated ear swelling as a representative inflammatory sign of contact hypersensitivity. We found that DNFB-induced ear swelling was ameliorated by oral administration of αKetoA but not αHYA (Fig. 2a). We next examined the therapeutic effects of αKetoA by administering it orally to mice at 1 day after elicitation with DNFB and measuring ear swelling the day after αKetoA administration. We found that αKetoA treatment effectively ameliorated ear swelling (Fig. 2b). Topical treatment with αKetoA also exerted anti-inflammatory activity in the inhibition of ear swelling (Fig. 2c).
We next sought to examine molecular mechanisms of αKetoA in the amelioration of contact hypersensitivity. The carbon length of fatty acids is an important determinant of receptor specificity: short-chain fatty acids are recognized by GPR41 and GPR43, whereas long-chain fatty acids are recognized by GPR40 and GPR120. 31 In addition, long-chain fatty acids are directly recognized by PPARγ. 32 To identify the functional receptor of αKetoA, we applied specific antagonist treatment in the contact hypersensitivity model, using GW1100, AH7614, and GW9662 as selective antagonists of GPR40, GPR120, and PPARγ, respectively. We found that the anti-inflammatory effect of αKetoA was dependent on PPARγ but not GPR40 or GPR120, according to ear swelling at 24 h after elicitation (Fig. 2d). This effect continued 48 h after the elicitation ( Supplementary Fig. S1). Consistent with the independence of αKetoA from GPR40 and GPR120 in exerting anti-inflammatory activity, transforming growth factor (TGF)αshedding assays revealed that αKetoA had little activity in inducing GPR40-and GPR120-mediated signaling when compared with the positive control, 13-oxo-cis-9,cis-15-octadecadienoic acid ( Supplementary Fig. S2). 33 We next sought to examine cellular dynamics in the treatment with αKetoA. Flow cytometry analysis revealed that αKetoA decreased the number of IFNγ + T cells and dendritic cells in the ear skin in a PPARγ-dependent manner (Fig. 2e). Histologic analysis revealed that αKetoA disrupted the iSALT structure; this disruption was dependent on PPARγ but independent from GPR40 and GPR120 (Fig. 2f). These results indicate that the αKetoA-PPARγ axis ameliorates contact hypersensitivity by inhibiting IFNγ production by T cells through the disruption of iSALT formation.
We have developed a hapten-induced contact hypersensitivity model in cynomolgus macaques. 13 In this model, macaques are sensitized with DNFB on the abdominal skin and then stimulated with DNFB on the back, thus inducing skin inflammatory signs, including epidermal hyperplasia and inflammatory cell infiltration ( Supplementary Fig. S3). 13 In the current study, we used this model to address whether αKetoA exerts anti-inflammatory activity in non-human primates and found that, as in the murine model, DNFB-induced skin inflammatory signs such as epidermal hyperplasia and the accumulation of CD3 + T cells were inhibited by topical application of αKetoA ( Supplementary Fig. S3). These results show that αKetoA is effective for the treatment of skin inflammation not only in rodents but also in non-human primates and therefore is a promising candidate for drug development.
αKetoA inhibited chemokine expression by interfering with nuclear translocation of NF-kB in macrophages To identify the target cells of αKetoA, we compared the gene expression level of Pparg among dendritic cells, T cells, and macrophages in the skin; dendritic cells and T cells are essential constituents of iSALT, and macrophages act as iSALT inducer cells. 34 We found that macrophages expressed the highest level of Pparg among these cells (Fig. 3a). This finding prompted us to examine whether αKetoA affected the expression of macrophage-derived chemokines that recruit CXCR2 + dendritic cells for the formation of iSALT. 34 Treatment of mice with αKetoA reduced the amount of CXCL1 in ear homogenates, with minimal effects on CXCL2 levels ( Fig. 3b and Supplementary Fig. S4a). In addition, treatment with GW9662 abrogated the effects of αKetoA on CXCL1 levels, thus indicating their dependency on PPARγ ( Fig. 3b and Supplementary Fig. S4a).
We then prepared bone marrow-derived macrophages and stimulated them with IL-1α to induce CXCR2 ligands. 34 We found that αKetoA inhibited IL-1α-mediated induction of Cxcl1 and Cxcl2 ( Fig. 3c and Supplementary Fig. S4b). To induce Cxcl1 and other pro-inflammatory cytokines and chemokines, the signaling pathway from IL-1α activates NF-κB, 35 therefore we asked whether αKetoA inhibited the nuclear translocation of NF-κB, which is an essential step for NF-κB-mediated gene expression. We found that   hypersensitivity is ameliorated by αKetoA, but not αHYA, through PPARγ-dependent inhibition of iSALT development. a Mice orally received αHYA (dose: 1 μg/mouse), αKetoA (dose: 1 μg/mouse), or 0.5% (vol/vol) ethanol dissolved in PBS (vehicle control) on days −10 to −6, days −3 to 1, and days 4−6; DNFB-induced ear swelling was evaluated on day 7. Data are combined from two independent experiments. b Mice orally received αKetoA (dose: 10 μg/mouse) or 0.5% (vol/vol) ethanol dissolved in PBS (vehicle control) on the days indicated 90 min before DNFB stimulation on days 0 and 5; DNFB-induced ear swelling was evaluated on day 7. Data are combined from two independent experiments. c Mice were topically treated with αKetoA (dose: 10 μg/mouse) or 50% (vol/vol) ethanol dissolved in PBS (vehicle control), 30 min before DNFB stimulation on days 0 and 5, and DNFB-induced ear swelling was evaluated on day 7. Data are combined from two independent experiments. d Mice were intraperitoneally injected with either GW1100, AH7614, or GW9662 30 min before oral administration of αKetoA on days 0 and 5. Mice were challenged with DNFB 90 min after oral administration of αKetoA, and ear swelling was evaluated on day 6. Data are combined from three independent experiments. e Mice were intraperitoneally injected with either GW1100, AH7614, or GW9662 30 min before oral administration of αKetoA on days 0 and 5. Mice were challenged with DNFB 90 min after oral administration of αKetoA, and the numbers of IFNγ + T cells (7-AAD − CD45 + TCRβ + IFNγ + ) and dendritic cells (7-AAD − CD45 + CD11c + F4/80 -I-A b + CD11b + ) were calculated on the basis of total cell numbers and flow cytometric data on days 6 and 7, respectively. Data of IFNγ + T cells and dendritic cells are combined from 4 and 2 independent experiments, respectively. f Mice were intraperitoneally injected with either GW1100, AH7614, or GW9662 30 min before oral administration of αKetoA on days 0 and 5. Mice were challenged with DNFB 90 min after the oral administration of αKetoA. Ears were obtained on day 7 and frozen sections were stained with hematoxylin and eosin or the indicated antibodies and reagent. Elicitation (-) indicates mice that were not stimulated with DNFB on day 5 and used as a control. Data are representative of three independent experiments. Scale bars, 100 μm. Each point represents data from individual mice Statistical significance was evaluated by using one-way ANOVA; ****p < 0.0001; ***p < 0.001; ** p < 0.01; * p < 0.05; N.S. not significant.
αKetoA inhibited IL-1α-mediated nuclear translocation of NF-κB in a PPARγ-dependent manner (Fig. 3d). These results collectively indicate that the αKetoA-PPARγ axis ameliorated contact hypersensitivity by disrupting iSALT formation through inhibiting NF-κB activation and chemokine expression in macrophages.
HFD-induced glucose intolerance is ameliorated by αKetoA through inhibiting adipose tissue inflammation Given that αKetoA targets macrophages, we examined whether αKetoA ameliorates other macrophage-associated inflammatory diseases. Several lines of evidence indicate that obesity is associated with adipose tissue inflammation due to recruitment of pro-inflammatory M1 macrophages and contributes to the development of metabolic disorders, including diabetic glucose intolerance. [36][37][38] When we fed mice an HFD combined with oral administration of either αKetoA or vehicle (as a control) for several months, neither body weight increase nor the weight of the epididymal adipose tissue differed between the 2 groups, suggesting that αKetoA did not affect the development of obesity ( Supplementary Fig. S5a-c).
In contrast to obesity-associated phenotypes, we found that the number of macrophages infiltrated into the epididymal adipose tissue was decreased by oral administration with αKetoA (Fig. 4a). Consistently, αKetoA decreased the expression level of the M1 macrophage marker Nos2 and increased that of the M2 macrophage marker Fizz1 in macrophages isolated from epididymal adipose tissues (Fig. 4b). In addition, in vitro assays using bone marrow-derived macrophages revealed that αKetoA influenced the polarization of M1 and M2 macrophages. Indeed, αKetoA decreased the expression levels of the M1 markers Nos2 and Cd86 yet promoted those of the M2 markers Fizz1, Chi3l3, and Arg1 ( Fig. 4c, d). The effects of αKetoA on the gene expression levels of Nos2, Fizz1, and Arg1 were canceled by inhibition of PPARγ (Fig. 4c, d). These findings were consistent with a previous study showing the involvement of PPARγ in macrophage polarization to M2 phenotypes. 39 In obesity-associated inflammation, adipocytes produce CCL2 and S100A8 for the recruitment of macrophages. [36][37][38]40 Treatment with αKetoA had little effect on the expression of Ccl2 and S100a8 in adipocytes ( Supplementary Fig. S6), suggesting that αKetoA acted directly on macrophages to inhibit their infiltration into adipose tissues and to alter macrophage polarization to M2 phenotypes.
In accordance with these findings, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) revealed that αKetoA decreased HFD-induced glucose intolerance (Fig. 5a,  b). We then examined HFD-induced adipose tissue remodeling, such as the development of crown-like structures and fibrosis, which play key roles in promoting chronic inflammation and metabolic disorders. 41 Histologic analysis revealed that αKetoA ameliorated cellular infiltration into epididymal adipose tissues (Fig. 5c). Furthermore, immunohistochemical analysis using an anti-F4/80 mAb to visualize macrophages revealed that αKetoA inhibited the development of crown-like structures (Fig. 5c).
Chronic inflammation in adipose tissue eventually leads to the development of interstitial fibrosis, which causes adipose tissue and macrophages (7-AAD − CD45 + Gr1 Low CD11b + ) were isolated from mouse ear skin on day 7 of the contact hypersensitivity model, and the gene expression level of Pparg was measured through reverse transcription and quantitative PCR analysis and normalized to that of Actinb. Data are combined from two independent experiments. b Ear homogenates were prepared on day 6 and 7 of the contact hypersensitivity model and examined by ELISA to determine the amount of CXCL1. Data are combined from five independent experiments. c, d In vitro assay of bone marrow-derived macrophages. Bone marrow cells were incubated as described in the Methods section and stimulated with IL-1α with or without αKetoA to examine the gene expression level of Cxcl1 (c) and nuclear translocation of NF-κB (d). c The gene expression levels were normalized to that of Actinb. Data are representative of three independent experiments with similar results (triplicate assay). d NF-κB and macrophages were visualized by staining with anti-p65 mAb (green) and anti-F4/80 mAb (red), respectively; nuclei were stained with DAPI (blue). Nuclear translocation of p65 is indicated as a change in the color of the nucleus to turquoise (arrowheads). Data are representative of three independent experiments. Scale bars, 20 μm. Each point represents data from individual mice. Statistical significance was evaluated by using one-way ANOVA; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; N.S. not significant, N.D. not detected.
dysfunction and ectopic lipid accumulation; these conditions subsequently lead to non-alcoholic steatohepatitis, which shows hepatic insulin resistance due to reduced expression of insulin receptor β. 42 Consistent with the finding that αKetoA inhibited adipose tissue inflammation and decreased glucose intolerance, we found that αKetoA prevented the development of adipose tissue fibrosis, as evaluated by Masson's trichrome staining (Fig. 5c). In line with our current findings regarding the contact hypersensitivity model, the activities of αKetoA in inhibiting the formation of crown-like structures and fibrosis were dependent on PPARγ because these activities were abrogated by treatment with GW9662 (Fig. 5c). These results collectively demonstrate that the αKetoA-PPARγ axis ameliorates HFD-induced adipose tissue remodeling without affecting obesity-associated increases in body weight and epididymal adipose tissues.

Detection of αKetoA in human feces
Given that αKetoA showed anti-diabetic effects in mice, we next asked whether αKetoA levels are decreased in human diabetic patients. As it is generally known that ordinary intake of dietary ω3 oil is low in normal life, the fecal αKetoA levels were low and comparable between healthy people and diabetic patients ( Supplementary Fig. S7a). To assess the correlation between αlinolenic acid and αKetoA in feces, we then established another cohort that included participants who consumed various amounts of dietary α-linolenic acid due to ad libitum intake of α-linolenic acid-rich linseed-related products. We found that the amount of αKetoA was positively correlated with that of α-linolenic acid ( Supplementary Fig. S7b). Although the precise effects of αKetoA on human diabetes are a subject for future study, these findings collectively suggest that dietary intake of α-linolenic acid promotes the production of αKetoA in humans.

DISCUSSION
Accumulating evidence suggests that the intestinal microbiome influences host health and diseases, not only in the intestine but also in other tissues, including the respiratory tract, central nervous system, and skin, through the regulation of inflammation, allergy, and metabolic disorders. [43][44][45][46] Microbial metabolites of food materials are known as 'postbiotics'. Currently postbiotics are attracting attention as bioactive molecules that likely are important in the underlying mechanisms through which the intestinal microbiome can control multiple host organs remotely. Indeed, we detected αKetoA not only in feces but also in serum in mice. In addition, αKetoA exerted its anti-inflammatory activities through regulation of macrophage activities in the skin and adipose tissue to ameliorate contact hypersensitivity and metabolic disorder. A recent study similarly showed that the microbedependent ω6 linoleic acid metabolite HYA ameliorated metabolic disorders. 47 HYA induced GPR40-and GPR120-dependent GLP1 secretion from enteroendocrine cells, facilitated glucose metabolism, and inhibited the development of obesity. 47 Therefore, αKetoA and HYA are both microbe-dependent metabolites of essential fatty acids that improve glucose metabolism through the different molecular and cellular bases of the αKetoA-PPARγ axis in macrophages and the HYA-GPR40 and -GPR120 axes in enteroendocrine cells. These intestinal microbial metabolites, which are generated through reduction reactions, are chemically much more stable than the oxidation metabolites produced by Fig. 4 αKetoA induces M2 macrophage polarization in the adipose tissue of HFD mice. a Mice were fed either a control diet containing soybean oil or HFD for 3 months with or without oral administration of αKetoA (dose: 10 μg/mouse, 3 times/week), and epididymal adipose tissues were analyzed through flow cytometry. The number of macrophages (7-AAD − CD45 + Ly6G − F4/80 + CD11b + ) was calculated on the basis of total cell numbers and flow cytometric data. Data are combined from four independent experiments. b After the HFD was fed for 4 months with or without oral administration of αKetoA (dose: 10 μg/mouse, 3 times/week), macrophages were isolated from epididymal adipose tissues and examined for gene expression of Nos2 and Fizz1 as markers of M1 and M2 macrophages, respectively. Data are combined from four independent experiments. c, d In vitro assay of bone marrow-derived macrophages. Gene expression levels were normalized to that of Actinb and expressed as ratios to control data. Data are combined from six independent experiments. Statistical significance was evaluated by using one-way ANOVA for comparison of multiple groups and the Mann-Whitney test for two groups; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; N.S. not significant.
the host, because the reduction metabolites lack the unstable conjugated double-bond structure. This stability enhances the usefulness of these intestinal microbial metabolites as postbiotics.
With current dietary habits, people tend to consume only low amounts of ω3 fatty acids. In agreement with this trend, our cohort study indicated that Japanese adults generally ingest small quantities of ω3 fatty acids, which resulted in barely detectable levels of αKetoA even in the feces of healthy participants. αKetoA could be increased to more than 1000 pg per 50 mg feces in humans when they consumed a diet rich in α-linolenic acid, thus suggesting that these levels would result in an anti-inflammatory effect. Although αKetoA is not a critical determinant in the development of diabetes, these findings suggest that increasing αKetoA levels through increased intake of α-linolenic acid might ameliorate diabetic inflammation in human patients. We plan to establish another cohort to directly evaluate the effect of dietary intake of α-linolenic acid and its metabolism to αKetoA in regard to the development of diabetes.
It is worth noting that the population having the same amount of α-linolenic acid contains both αKetoA-high and -low producers, indicating that the composition of the intestinal microbiota would affect the level of αKetoA. Conversion of α-linolenic acid to αHYA is potentially mediated by several types of bacteria, including Lactobacillus plantarum, L. acidophilus, Streptococcus pyogens, Stenotrophomonas nitritireducens, and Flavobacterium spp., 11 and that of αHYA to αKetoA is mediated by L. plantarum and Flavobacterium spp. 19,48 Therefore, rather than dietary supplementation with precursor compounds, a better strategy might be to take αKetoA itself to obtain suitable anti-inflammatory effects, because the microbiota differs among people. Several fermented Fig. 5 HFD-induced glucose intolerance is ameliorated by αKetoA through inhibiting adipose tissue inflammation. a IPGTT was performed after HFD feeding for 3 months with or without oral administration of αKetoA (dose: 10 μg/mouse, 3 times/week). b ITT was performed after HFD feeding for 3.5 months with or without oral administration of αKetoA (dose: 10 μg/mouse, 3 times/week). c After HFD feeding for 4 months with or without oral administration of αKetoA (dose: 10 μg/mouse, 3 times/week) and with or without intraperitoneal injection of GW9662, epididymal adipose tissues were examined histologically. Mice fed with control diet containing soybean oil were used as a control. Data are representative of four independent experiments (n = 12/group). Scale bars, 100 μm. Statistical significance was evaluated by using the Mann-Whitney test; **p < 0.01; *p < 0.05.
foods, including Japanese pickles, Korean kimchi, and German sauerkraut, are enriched with L. plantarum. Because these foods are produced through fermentation, they might contain bioactive microbial metabolites. Therefore, another prospective strategy involves adding the precursors of bioactive metabolites (e.g., αlinolenic acid) during fermentation to increase the amounts of desired bioactive microbial metabolites in food products.
In the contact hypersensitivity model, macrophages play important roles in the induction of iSALT formation by expressing CXCR2 ligands, which induces clustering of dermal dendritic cells. 34 We found that αKetoA inhibited iSALT formation by decreasing the level of the inflammatory chemokine CXCL1. Consistently, we found that the nuclear translocation of NF-κB, which plays central roles in triggering inflammation by inducing the gene expression of pro-inflammatory cytokines and chemokines, including Cxcl1, 49 was inhibited by αKetoA in macrophages in a PPARγ-dependent fashion. This scenario is in accordance with previous reports indicating that the activation of PPARγ suppresses NF-κB activation and consequent inflammatory responses. 50,51 NF-κB-mediated gene induction of Tnfa and Il1b is a hallmark of M1 macrophage polarization. 52 Consistent with the finding that the αKetoA-PPARγ axis inhibited NF-κB activity, αKetoA suppressed polarization to M1 macrophages. In addition, PPARγ activators are known to induce the polarization of macrophages to the M2 phenotype, 53,54 thus supporting our finding that αKetoA promoted M2 macrophage polarization. αKetoA simultaneously inhibited M1 macrophage polarization and promoted M2 macrophage polarization, such that both activities contributed to the inhibition of adipose tissue inflammation. From the viewpoint of fibrosis, it is worth noting that macrophage production of nitric oxide plays a key role in the induction of adipose tissue fibrosis. 55 Conversely, αKetoA reduced the gene expression of Nos2 yet promoted that of Arg1 in macrophages, thereby decreasing tissue levels of nitric oxide.
It is widely accepted that obesity is the critical determinant in inducing adipose tissue inflammation, which is the mechanism underlying the development of metabolic disorders. [36][37][38] However, we found that αKetoA decreased glucose intolerance without affecting body weight gain, suggesting that obesity does not always lead to the development of metabolic disorders. The infiltration of macrophages is a primary event in obesity-induced adipose tissue inflammation. [36][37][38] In obesity, adipocytes produce CCL2 and S100A8, which recruit CCR2-expressing pro-inflammatory M1 macrophages and monocytes to adipose tissue. [36][37][38]40 We found that αKetoA did not alter the expression levels of Ccl2 and S100a8; instead, αKetoA-in a PPARγ-dependent manner-prevented macrophages from infiltrating the adipose tissues of obese mice and inhibited fibrosis and the formation of crown-like structures. In addition to their effects on macrophage polarization, agonists of PPARγ (e.g., rosiglitazone and thiazolidinediones) suppress the chemotaxis of monocyte-macrophages. 56,57 Macrophage-specific deletion of PPARγ increases the chemotactic response to CCL2. 58 Mechanisms of PPARγ-mediated suppression of the chemotactic response include the down-regulation of CCR2 expression. 57,58 Given that CCR2 expression is upregulated through NF-κB signaling in macrophages, 59 PPARγ-mediated down-regulation of CCR2 might occur through the suppression of NF-κB activity. Together, these previous studies suggest that the αKetoA-PPARγ-NF-κB axis negatively regulates chemotaxis of pro-inflammatory M1 macrophages and monocytes into adipose tissue.
GW9662 used in this study is widely employed as a potent, irreversible, and selective PPARγ antagonist, which acts by covalently modifying a cysteine residue in the PPARγ ligandbinding domain. [60][61][62][63] Indeed, GW9662 inhibits radioligand binding to PPARγ, PPARα, and PPARδ with pIC 50  However, a recent study identified that GW9662 triggered gene expression via PPARδ, therefore, the off-target effect existed. 64 Based on the fact, we should further address the specific role of PPARγ by using other methods, including genetically modified animals and gene knock-down system in a future study.
In summary, αKetoA is found as α-linolenic acid-derived postbiotics and as such is only extracted from dietary ω3 fatty acids in the presence of intestinal microbiota. We found that, by regulating various activities of macrophages, αKetoA exerted potent anti-inflammatory effects in mice and cynomolgus macaques, ameliorated skin inflammation, and decreased diabetic glucose intolerance. These results pave the way for the development of new drugs and probiotics, functional fermented foods, and postbiotics for the treatment of macrophage-associated inflammatory diseases, including skin inflammation and diabetes.

MATERIALS AND METHODS Animals
For lipidomics, female C57BL/6J wild-type mice (6 weeks old) were purchased from Japan SLC (Hamamatsu, Japan), and were maintained for 2 months on chemically defined diets containing 4% (wt/wt) dietary oil (soybean oil or linseed oil, Oriental Yeast, Tokyo, Japan) 65 in the SPF animal facility at National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN; Osaka, Japan). Male GF mice (ICR background), and their control ICR mice (age, 6 weeks) were purchased from Japan SLC (Hamamatsu, Japan); these mice were maintained for 2 months on chemically defined diets containing 4% (wt/wt) dietary oil (soybean oil or linseed oil) under GF or SPF conditions, respectively, at NIBIOHN and Oriental Bioservice, Inc. For contact hypersensitivity studies, female C57BL/6J wild-type mice (age, 7 weeks) were purchased from Japan SLC and maintained for 1 week before use in experiments in the SPF animal facility at NIBIOHN. These mice were maintained on a commercially available FR2 regular diet (Funabashi Farm, Chiba Japan). For diabetes experiments, male C57BL/6J wild-type mice (age, 8 weeks) were purchased from Japan SLC and CLEA Japan (Tokyo, Japan), and maintained in the SPF animal facility at NIBIOHN for 3-4 months on HFD composed of chemically defined materials. 65 Mice were maintained under conditions (16:8 h light/dark cycle, 22-24°C, and 50-60% humidity), with ad libitum access to food and distilled water. Mice were euthanized by cervical dislocation under isoflurane (Forane, AbbVie, North Chicago, Illinois, USA) anesthesia.
Male cynomolgus macaques (Macaca fascicularis; age, 2 years; weight; 2 kg) were maintained at the Tsukuba Primate Research Center, NIBIOHN, according to the "Rules for Animal Care and Management of Tsukuba Primate Center" and the "Guiding Principles for Animal Experiments using Non-human Primates" formulated by the Primate Society of Japan. All experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of NIBIOHN (DS25-2, DS26-41, DS27-47, DSR01-2, and DSR01-3). The study was carried out in compliance with the ARRIVE guidelines.

LC-MS/MS
LC-MS/MS was performed as reported. 66 Data analysis was performed by using the software Xcalibur 2.2 (ThermoFisher Scientific).

Induction of contact hypersensitivity in cynomolgus macaques
Contact hypersensitivity in cynomolgus macaques was induced as described previously. 13 αKetoA (1500 μg/350 μL) or 50% (vol/vol) ethanol dissolved in PBS (350 μL) as a vehicle control were applied topically to the right and left sides of the back, respectively, 30 min before DNFB challenge on days 5 and 7. αKetoA and vehicle control were additionally applied topically on days 6 and 11. Skin samples were obtained by means of biopsy on day 12.

Induction of diabetes in mice
Mice were kept for 3-4 months on HFD composed of chemically defined materials in the SPF animal facility at NIBIOHN. 65 During this period, mice were orally treated with αKetoA (dose, 10 μg/mouse) or 0.5% (vol/vol) ethanol dissolved in PBS as a vehicle control 3 times each week. In some experiments, mice intraperitoneally received GW9662 (1 mg/kg body weight) 30 min before oral administration of αKetoA.

Cell isolation and flow cytometric analysis
Cell isolation and flow cytometry were performed as described previously. 13,40 To avoid non-specific staining, cell samples were blocked with anti-CD16/32 monoclonal antibody (mAb) (dilution, 1:100; catalog no.

Histologic analysis
Frozen and paraffin tissues were analyzed histologically as described previously. 13 For staining of paraffin tissues with anti-CD3 mAb (Clone: CD3-12, GeneTex), antigen retrieval was conducted by heating sections in 1 mM EDTA solution (pH 9.0) for 15 min in a microwave oven after deparaffinization. The following antibodies and reagents were used for immunohistologic analysis: purified-anti-CD3ε mAb (

ELISA for CXCL1 and CXCL2
The amounts of CXCL1 and CXCL2 proteins in ear homogenates were analyzed by using Mouse CXCL1/KC Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota, USA) and Mouse CXCL2 Quantikine ELISA Kit (R&D Systems), respectively, according to the manufacturer's protocol. In brief, ear samples were homogenized for 30 sec with one 4.8-ϕ and three 3.2-ϕ beads in PBS containing protease inhibitor (P8340, Sigma) and centrifuged (10,000 rpm, 20 min, 4°C); supernatants were collected and used for ELISA (protein concentration; 4 mg/mL). Absorbance at OD 450 and OD 570 was measured by using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).

In vitro assay of bone marrow-derived macrophages
Bone marrow cells were prepared from the femurs and tibias of 5-to 8week-old C57BL/6J wild-type mice, and the differentiation of macrophages was induced as described previously 34 with modification.
To analyze polarization of M1 and M2 macrophages, bone marrow cells were cultured in 6-cm dishes (1 × 10 5 cells/mL, 5 mL/well; RepCell dishes, CellSeed) containing Dulbecco's modified Eagle medium (high glucose) supplemented with macrophage colony-stimulating factor (50 ng/mL), 10% (vol/vol) fetal bovine serum, and 1% (vol/vol) penicillin and streptomycin at 37°C and 5% CO 2 . Culture medium was replaced on days 3 and 5. On day 5, cells were stimulated with IFNγ (10 ng/mL; Peprotech) or IL-4 (20 ng/mL) to induce their differentiation to M1 and M2 macrophages, respectively. 34 Cells were incubated with αKetoA (30 nM) or 0.1% (vol/vol) ethanol as a vehicle control for 30 min before cytokine stimulation. On day 7, mRNA samples were prepared and used for reverse transcription and quantitative PCR analysis of the expression of Nos2 and CD86 as M1 polarization markers and of Fizz1, Arg1, and Chi3l3 as M2 polarization markers.

IPGTT and ITT
IPGTT and ITT were performed as described previously 71 with modification. In brief, for IPGTT, mice were fasted overnight (16 h) and then injected intraperitoneally with D-(+)-glucose (20% solution; 2 g/kg body weight; Nacalai Tesque). For ITT, mice in the randomly fed state were injected intraperitoneally with human regular insulin (1.0 U/kg body weight; Eli Lilly, Indiana, USA). To measure blood glucose levels, blood was obtained from the tail veil by cutting with a single-edged blade (Feather, Osaka, Japan) and measured by using One Touch Ultra Vue (LifeScan Japan, Tokyo, Japan) before and after glucose injection at indicated time points.

Human samples and ethics
Fecal samples were collected from participants in two human cohort studies. One includes healthy and diabetic patients who were recruited from Shunan City Shinnanyo Hospital (Shunan City, Yamaguchi, Japan) and surrounding communities; the other study involves healthy adult volunteers who were recruited from the communities around NIBIOHN (Ibaraki City, Osaka, Japan). All experiments were approved by the Ethics Committee of NIBIOHN (approval numbers: 177-07 and 154-10) and were conducted in accordance with their guidelines; informed consent was obtained from all participants. All samples were stored at −80°C until use. All participants had no history of cancer, cardiovascular, liver, or gastrointestinal disease; candidates who took antibiotics, laxatives, or antiflatulents within a month before sample collection were excluded.

Statistical analysis
Statistical significance was evaluated through one-way ANOVA for comparison of multiple groups and the Mann-Whitney test for two groups (Prism 6, GraphPad Software, La Jolla, CA, USA). A P value less than 0.05 was considered to be significant.