Ingesting probiotic yogurt containing Lactiplantibacillus plantarum OLL2712 improves glycaemic control in adults with prediabetes in a randomized, double-blind, placebo-controlled trial.

AIM: The ingestion of Lactiplantibacillus plantarum OLL2712 (OLL2712) cells has been shown to improve glucose metabolism by suppressing chronic inflammation in murine models and clinical studies. This study aimed to clarify the effect of OLL2712 on glycaemic control in healthy adults with prediabetes. MATERIALS AND METHODS: The study was a randomized, double-blind, placebo-controlled, parallel-group design. Adult participants with prediabetes [n = 148, glycated haemoglobin (HbA1c) range: 5.6%-6.4%, age range: 20-64 years] were assigned randomly to placebo or OLL2712 groups (n = 74/group) and administered daily for 12 weeks either conventional yogurt or yogurt containing >5 × 109 heat-treated OLL2712 cells, respectively. In addition, the participants were followed for 8 weeks after the discontinuation of either yogurt. The primary outcome was the changes in HbA1c levels at weeks 12 and 16 by analysis of covariance. RESULTS: The levels of HbA1c and glycoalbumin decreased significantly in both groups at week 12 in comparison with those at week 0, but only in the OLL2712 group at week 16. HbA1c levels decreased significantly at weeks 12 and 16 in the OLL2712 group in comparison with the placebo group (p = .014 and p = .006, respectively). No significant inter- and intragroup differences in HbA1c levels were observed at week 20. CONCLUSIONS: The ingestion of OLL2712 prevents the deterioration of glycaemic control and maintains the HbA1c levels within the normal range in adults with prediabetes; yogurt probably exhibits similar effects, which may contribute to reducing the risk of developing type 2 diabetes.

Orally administered Lactiplantibacillus plantarum OLL2712 decreased intestinal permeability, especially in the ileum: Ingested lactic acid bacteria alleviated obesity-induced inflammation by collaborating with gut microbiota.

Introduction: Chronic inflammation caused by dietary obesity has been considered to induce lifestyle-related diseases and functional ingredients with anti-inflammatory effects are attracting attention. Although multiple studies on obesity had proved the anti-inflammatory effects of ingestion of lactic acid bacteria (LAB) and other functional ingredients on adipose tissue, the precise effects on the intestine, especially on the individual intestinal segments have not been made clear. In this study, we elucidated the mechanisms of Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) OLL2712 in suppressing obesity-induced inflammation using high fat diet (HFD)-fed mice obesity model. Methods: We orally administered heat-treated LAB to HFD-fed mice model, and investigated the inflammatory changes in adipose tissue and intestinal immune cells. We also analyzed gut microbiota, and evaluated the inflammation and permeability of the duodenum, jejunum, ileum and colon; four intestinal segments differing in gut bacteria composition and immune response. Results: After 3-week LAB administration, the gene expression levels of proinflammatory cytokines were downregulated in adipose tissue, colon, and Peyer's patches (PP)-derived F4/80+ cells. The LAB treatment alleviated obesity-related gut microbiota imbalance. L. plantarum OLL2712 treatment helps maintain intestinal barrier function, especially in the ileum, possibly by preventing ZO-1 and Occludin downregulation. Discussion: Our results suggest that the oral administration of the LAB strain regulated the gut microbiota, suppressed intestinal inflammation, and improved the gut barrier, which could inhibit the products of obesity-induced gut dysbiosis from translocating into the bloodstream and the adipose tissue, through which the LAB finally alleviated the inflammation caused by dietary obesity. Barrier improvement was observed, especially in the ileum, suggesting collaborative modulation of the intestinal immune responses by ingested LAB and microbiota.

Suppressive Effect of Lactococcus lactis subsp. cremoris YRC3780 on a Murine Model of Japanese Cedar Pollinosis.

Accumulating evidence suggests that Lactococcus lactis subsp. cremoris YRC3780 isolated from kefir has the potential to alleviate allergic responses. Herein, we investigated the effect of YRC3780 on a murine model of Japanese cedar pollinosis (JCP). BALB/c mice immunized with cedar pollen extract (CPE) exhibited an increase in serum immunoglobulin E and developed nasal inflammatory responses including sneezing, nasal hyperresponsiveness, and nasal eosinophil accumulation upon intranasal allergen challenge. These responses were suppressed by the oral administration of YRC3780, although the effects on CPE-induced sneezing response and eosinophil infiltration were not statistically significant. Total fecal microbiota diversity was not affected by allergen immunization and challenge or by YRC3780 administration. However, the abundances of Bifidobacteriales, Veillonellaceae, Lactococcus, and Lactococcus lactis were larger and that of Bacteroides was smaller in YRC3780-treated mice compared with those in CPE-challenged and YRC3780-untreated mice. Our findings suggest the usefulness of YRC3780 for alleviating JCP.

Immunomodulation by Food: Novel Collaborations between Food Components and Microbiota.

Recent studies have revealed that various food components affect the immune response. It has been shown that such components could act on the intestinal immune system. On the other hand, intestinal microbiota and their metabolites affect intestinal immunity. Such findings suggest the possibility that food components could act on the intestinal immune system directly, indirectly through intestinal microbiota, or through collaborative immunomodulation by both.

Extracts of Gluconacetobacter hansenii GK-1 induce Foxp3+T cells in food-allergic mice by an IL-4-dependent or IL-4-independent mechanism.

The biological activities of acetic acid bacteria (AAB) as Gram-negative bacteria have attracted our interests, especially in their inhibitory effects on allergic responses. To clarify the underlying mechanism that improves allergic symptoms by ingestion of the AAB Gluconacetobacter hansenii, we examined whether different extracts of heat-killed G. hansenii GK-1 could reduce the interleukin (IL)-4 production of immune cells from food-allergic model of OVA23-3, transgenic mice with ovalbumin (OVA)-specific T-cell-receptor genes. A hot-water extract fraction (FII) of G. hansenii GK-1 significantly decreased the in vitro IL-4 production of spleen cells of OVA23-3 mice compared with those stimulated with OVA alone. The IL-4 inhibitory effect was also observed for FIV (purified lipopolysaccharide (LPS) fraction), but the activity was lower than for FII or LPS from Escherichia coli. Unlike LPS from Escherichia coli, FIV significantly inhibited the LPS-induced IL-6 production of the spleen cells. The addition of FII or FIV to a Foxp3+T cell-inducing culture showed that FII significantly promoted the rate of Foxp3+CD4+T cells of OVA-stimulated mesenteric lymph node cells from recombination-activating-gene (RAG)-2-deficient food-allergic inflammatory OVA23-3 (R23-3) mice with suppression of IL-4 production, while FIV induced Foxp3+T cells from RAG-2-deficient DO11.10 non-inflammatory mice. Structure analysis showed a lack of O-antigen in FIV, which seemed to lead to the weak biological activities of FIV observed. The present study suggests that extracts of G. hansenii GK-1 to inhibit IL-4 production of immune cells and/or promote regulatory T cell differentiation synergistically play important roles in improving allergic symptoms safely as well as normal condition.

Cecal Patches Generate Abundant IgG2b-Bearing B Cells That Are Reactive to Commensal Microbiota.

Gut-associated lymphoid tissue (GALT), such as Peyer's patches (PPs), are key inductive sites that generate IgA+ B cells, mainly through germinal center (GC) responses. The generation of IgA+ B cells is promoted by the presence of gut microbiota and dietary antigens. However, the function of GALT in the large intestine, such as cecal patches (CePs) and colonic patches (CoPs), and their regulatory mechanisms remain largely unknown. In this study, we demonstrate that the CePs possess more IgG2b+ B cells and have fewer IgA+ B cells than those in PPs from BALB/c mice with normal gut microbiota. Gene expression analysis of postswitched transcripts supported the differential expression of dominant antibody isotypes in B cells in GALT. Germ-free (GF) mice showed diminished GC B cells and had few IgA+ or IgG2b+ switched B cells in both the small and large intestinal GALT. In contrast, myeloid differentiation factor 88- (MyD88-) deficient mice exhibited decreased GC B cells and presented with reduced numbers of IgG2b+ B cells in CePs but not in PPs. Using ex vivo cell culture, we showed that CePs have a greater capacity to produce total and microbiota-reactive IgG2b, in addition to microbiota-reactive IgA, than the PPs. In line with the frequency of GC B cells and IgG2b+ B cells in CePs, there was a decrease in the levels of microbiota-reactive IgG2b and IgA in the serum of GF and MyD88-deficient mice. These data suggest that CePs have a different antibody production profile compared to PPs. Furthermore, the innate immune signals derived from gut microbiota are crucial for generating the IgG2b antibodies in CePs.

ß-elemene regulates M1-M2 macrophage balance through the ERK/JNK/P38 MAPK signaling pathway.

Macrophages are classified into classically activated M1 macrophages and alternatively activated M2 macrophages, and the two phenotypes of macrophages are present during the development of various chronic diseases, including obesity-induced inflammation. In the present study, ß-elemene, which is contained in various plant substances, is predicted to treat high-fat diet (HFD)-induced macrophage dysfunction based on the Gene Expression Omnibus (GEO) database and experimental validation. ß-elemene impacts the imbalance of M1-M2 macrophages by regulating pro-inflammatory cytokines in mouse white adipose tissue both in vitro and in vivo. In addition, the RAW 264 cell line, which are macrophages from mouse ascites, is used to identify the effects of ß-elemene on inhibiting bacterial endotoxin lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activated protein kinase (MAPK) pathways. These pathways both induce and are activated by pro-inflammatory cytokines, and they also participate in the process of obesity-induced inflammation. The results highlight that ß-elemene may represent a possible macrophage-mediated therapeutic medicine.

Physical and chemical properties of nabak (Zizyphus spina-christi) seed kernel and sweet pepper (Capsicum annuum L.) seed oils.

BACKGROUND: Nabak seed kernels and sweet pepper seeds, which are separated from the fruits and discarded as waste after processing or consumption, contain high levels of oils (30.19% and 19.57%, respectively). The chemical and thermal characteristics of nabak seed kernel oil (NSO) and sweet pepper seed oil (PSO) were investigated in this study. RESULTS: The NSO and PSO contained high levels of unsaturated fatty acids (84.1% and 86.5%, respectively), and the major fatty acid was oleic acid (57.3%) in NSO, but it was linoleic acid (69.4%) in PSO. The triacylglycerol (TAG) profiles show that NSO contained ten TAG species, three of which represented 87.1%, namely C54:3, C52:2 and C54:4, and triolein was the dominant (OOO, 47.0%). Pepper seed oil contained nine TAG molecular species, four of which represented 93.6%, namely C54:6, C52:4, C54:4 and C52:5, and trilinolein was dominant (LLL, 44.0%). The differential scanning calorimetry (DSC) analysis of NSO revealed that three exothermal peaks were detected during cooling, two endothermal peaks were detected during melting, and the major peak occurred at a low temperature. For PSO, three exothermal peaks were detected during cooling, three peaks were detected (one of them was exothermal) during melting, and the major peaks were observed at low temperatures. Fourier transform infrared (FTIR) spectra indicated that NSO and PSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids. CONCLUSION: This study offers a scientific basis for the use of NSO and PSO as new sources of edible oils for food applications. © 2021 Society of Chemical Industry.

Crystallization and melting properties studied by DSC and FTIR spectroscopy of goldenberry (Physalis peruviana) oil.

The chemical and thermal characteristics of goldenberry pomace oil (GPO) and goldenberry seed oil (GSO) were investigated. GPO and GSO contained high levels of unsaturated fatty acids (90.1% and 85.1%, respectively), and the major fatty acid was linoleic (62.0% and 72.8%, respectively). Additionally, GPO contained eleven triacylglycerol (TAG) species, three of which represented 82.7%, namely C54:6, C54:4 and C52:4, and trilinolein was the dominant one (35.5%). GSO contained nine TAG species, two of which represented 80.3%, namely C54:6 and C52:4, and trilinolein was dominant (53.3%). The DSC analysis of GPO and GSO revealed that three exothermal peaks were detected during cooling. Three endothermal peaks (one of which is exothermal for GSO) were detected during melting, and the most significant peaks occurred at low temperatures. FTIR spectra indicated that GPO and GSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids.

Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.

Effects of orally administered Euglena gracilis and its reserve polysaccharide, paramylon, on gastric dysplasia in A4gnt knockout mice.

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, ß-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

ß-Elemene Suppresses Obesity-Induced Imbalance in the Microbiota-Gut-Brain Axis.

As a kind of metabolically triggered inflammation, obesity influences the interplay between the central nervous system and the enteral environment. The present study showed that ß-elemene, which is contained in various plant substances, had effects on recovering the changes in metabolites occurring in high-fat diet (HFD)-induced obese C57BL/6 male mice brains, especially in the prefrontal cortex (PFC) and hippocampus (HIP). ß-elemene also partially reversed HFD-induced changes in the composition and contents of mouse gut bacteria. Furthermore, we evaluated the interaction between cerebral metabolites and intestinal microbiota via Pearson correlations. The prediction results suggested that Firmicutes were possibly controlled by neuron integrity, cerebral inflammation, and neurotransmitters, and Bacteroidetes in mouse intestines might be related to cerebral aerobic respiration and the glucose cycle. Such results also implied that Actinobacteria probably affected cerebral energy metabolism. These findings suggested that ß-elemene has regulatory effects on the imbalanced microbiota-gut-brain axis caused by obesity and, therefore, would contribute to the future study in on the interplay between cerebral metabolites from different brain regions and the intestinal microbiota of mice.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Ingesting Yogurt Containing Lactobacillus plantarum OLL2712 Reduces Abdominal Fat Accumulation and Chronic Inflammation in Overweight Adults in a Randomized Placebo-Controlled Trial.

BACKGROUND: Chronic inflammation and insulin resistance are factors that are related to obesity. We have suggested that the administration of heat-treated Lactobacillus plantarum OLL2712 (OLL2712) cells can improve glucose and lipid metabolism by suppressing chronic inflammation in mouse models and a preliminary clinical study. OBJECTIVE: The aim of this study was to investigate whether ingesting OLL2712 cells can reduce body fat accumulation and improve metabolic risk factors, in overweight, healthy adults. METHODS: This study was a randomized, double-blind, placebo-controlled, parallel-group trial conducted at a single center in Japan. The study participants included 100 overweight (BMI range, ≥25 to <30 kg/m2) adults aged 20-64 y. They were randomly assigned to either the placebo or OLL2712 group (n = 50 each) and were administered conventional yogurt or yogurt containing >5 × 109 heat-treated OLL2712 cells, respectively, daily for 12 wk. The primary outcome was the 12-wk change in the abdominal fat area, as assessed by computed tomography, and the secondary outcomes were glucose and lipid metabolism-related parameters and chronic inflammation markers, which were analyzed using a linear mixed model. RESULTS: The 12-wk change of abdominal fat area (difference: 8.5 cm2; 95% CI: 0.3, 16.6 cm2; P = 0.040) and fasting plasma glucose (difference: 3.2 mg/dL; 95% CI: 0.8, 5.6 mg/dL; P = 0.021) were significantly less in the OLL2712 group than the placebo group. The overall trend of serum IL-6 was significantly decreased in the OLL2712 group compared with baseline and the placebo group. CONCLUSIONS: The ingestion of heat-treated OLL2712 cells reduces body fat accumulation and the deterioration of glycemic control and chronic inflammation, in overweight, healthy adults. We hypothesize that OLL2712 cells may prevent obesity by regulating chronic inflammation. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN000027709.

Intestinal regulatory T cell induction by ß-elemene alleviates the formation of fat tissue-related inflammation.

The role of the intestinal immune system in the inhibition of fat tissue-related inflammation by dietary material is yet to be elucidated. Oral administration of ß-elemene, contained in various foodstuffs, downregulated expressions of inflammatory cytokines and increased Foxp3+CD4+ T cells in adipose tissue of obese mice. However, ß-elemene did not affect the inflammatory response of adipose tissue in vitro, suggesting that the inhibition observed in vivo was not due to direct interactions of adipose tissue with ß-elemene. Instead, ß-elemene increased Foxp3+CD4+ T cell population enhancing gene expressions of transforming growth factor ß 1, retinaldehyde dehydrogenase 2, integrin αvß8, and interleukin-10 in intestinal dendritic cells (DCs) in vivo and in vitro. Taken together, this study suggested the therapeutic effects of ß-elemene on treating experimental obesity-induced chronic inflammation by adjusting the balance of immune cell populations in fat tissue through the generation of regulatory T cells in the intestinal immune system by modulating DC function.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

Age-Dependent Decrease in the Induction of Regulatory T Cells Is Associated With Decreased Expression of RALDH2 in Mesenteric Lymph Node Dendritic Cells.

A decline in immune function with aging has been reported. Regulatory T cell (Treg) induction is known to decrease with age, and elucidating the underlying mechanism is important for preventing age-related diseases due to age-related chronic inflammation. In the intestine, dendritic cells (DCs) play an important role in inducing Tregs specific to oral antigens, and they efficiently induce Tregs via production of retinoic acid (RA), a vitamin A metabolite, catalyzed by the enzyme retinaldehyde dehydrogenase 2 (RALDH2). We have previously reported that in the mesenteric lymph node (MLN), a secondary lymphoid tissue in which immune responses to oral antigens are induced, four DC subsets express different levels of CD11b, CD103, and PD-L1, and we have reported that the CD11b-CD103+PD-L1high subset expresses the highest levels of the RALDH2 gene and induces Tregs in vitro. We examined Treg induction in young and aged mice using a Treg induction model by administering a food antigen, and we found that antigen-specific Treg induction was decreased in aged mice. We further investigated the MLN DCs, and a significant decrease in RALDH2 gene expression was observed in MLN DCs from aged mice. As factors, we found that the proportion of the CD11b-CD103+PD-L1high subset was decreased in aged mice compared with that in young mice and that RALDH enzyme activity was decreased in the CD11b-CD103+PD-L1high and CD11b+CD103+PD-L1high subsets. Furthermore, analysis of the methylation of the RALDH2 gene promoter region revealed that CpG motifs were more methylated in the MLN DCs of aged mice, suggesting that RALDH2 expression was suppressed by epigenetic changes. Finally, we found that RA treatment tended to increase Treg induction. These results suggest that the regulation of RA production may be involved in the age-related decrease in antigen-specific Treg induction.