Ingestion of miso regulates immunological robustness in mice

Kunihiko Kotake; Toshihiko Kumazawa; Kiminori Nakamura; Yu Shimizu; Tokiyoshi Ayabe; Takahiro Adachi

doi:10.1371/journal.pone.0261680

Abstract

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69⁺ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca²⁺ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Citation: Kotake K, Kumazawa T, Nakamura K, Shimizu Y, Ayabe T, Adachi T (2022) Ingestion of miso regulates immunological robustness in mice. PLoS ONE 17(1): e0261680. https://doi.org/10.1371/journal.pone.0261680

Editor: Deyu Fang, Northwestern University Feinberg School of Medicine, UNITED STATES

Received: August 3, 2021; Accepted: December 7, 2021; Published: January 21, 2022

Copyright: © 2022 Kotake et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This research was partly funded by ICHIBIKI Co Ltd (https://www.ichibiki.co.jp/). The funder provided the source. This work was supported in part by grants from the Japan Society for the Promotion of Science (https://www.jsps.go.jp/; No. 17H06170 and 19H04036 to T.Adachi, No. 20H04098 to K.N.) and Nipponham Foundation for the Future of Food (https://www.miraizaidan.or.jp/), Tojuro Iijima Foundation for Food Science and Technology(http://www.iijima-kinenzaidan.or.jp/), and Naoki Tsuchida Memorial Research Grant (https://www.tmd.ac.jp/tmd-research/studysupport/grant/tsuchidagrant/) (to T.Adachi).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Miso is a traditional fermented food consumed in Japan. It is classified according to the type of mold (koji), fermented by Aspergillus or other microorganisms. Soybean-miso (mame-miso) is prepared by mixing fermented beans and salt. It primarily consists of soybeans and is rich in proteins (including amino acids and peptides); thus, it is characterized by a strong umami and a dark color.

There are various health benefits associated with eating miso. Daily consumption of miso soup may improve skin moisture, and some ingredients in miso stimulate the increase of ceramides in the stratum corneum [1]. Miso also has an anti-hypertensive effect [2,3] and is known to act on the brain to suppress salt-sensitive sympathoexcitation [4,5]. Moreover, it has been reported that miso has anti-cancer activity against breast [6], gastric [7] and colon cancers [8]. The consumption of miso has also been reported to exert health effects, such as fat suppression [9], anti-inflammation [10] and stroke prevention [11].

Miso contains various microorganisms, such as Aspergillus, yeast and lactic acid bacteria (LAB). In recent years, these microorganisms have attracted attention as a source of probiotics. Aspergillus oryzae has been shown to alter the intestinal bacterial flora through the production of glycosylceramide [12]. There are many types of LABs, and several strains have been reported to show beneficial health effects, such as activating immunoglobulin (Ig) A production [13,14] and alleviating allergy symptoms [15]. Recently, we have identified miso-derived LABs that promote interleukin (IL)-22 production in B cells and improve skin barrier [13,16].

Therefore, miso is expected to have many beneficial effects on human health, but it is not clear how immune cells are regulated by miso intake. In this study, we evaluated immune cells in mice fed a diet containing miso.

Materials and methods

Ethics statement

All mice were maintained at our animal facility under specific-pathogen-free (SPF) conditions in accordance with the animal care guidelines of Tokyo Medical and Dental University. All animal experiments were approved by the Committee for Animal Care at Tokyo Medical and Dental University (approval number A2019-207C4).

Miso and raw materials

We used the following food materials: soybean-miso (Ichibiki), rice-miso (Ichibiki), white hilum soybean cultivar Tanrei made in Japan (Kushida), black hilum soybean made in the USA (Kanematsu), white hilum soybean made in the USA (Kanematsu), rice cultivar Calrose made in the USA (Kantokokuhun), rice made in Japan (Kimura-Shoten) and rice cultivar Koshihikari (YAMATO SANGYO). Soybeans and rice were crushed and used as a powder.

Cells and mice

C57BL/6 mice were fed a diet supplemented with 5% miso (soybean-miso) under SPF conditions. The ingredients of the miso are provided in Table 1. Miso mixed feed was prepared by combining with CLEA CE-2. As a control, feed was prepared by adding salt to CLEA CE-2. These feeds were standardized at a salt content of 1.4 w/w%. IL-22 deficient mice were generated by a genome editing system of the CRISPR/Cas9 as described previously [17]. A transgenic mouse line expressing YC3.60 for intravital imaging was described previously [18]. After euthanasia due to cervical vertebral dislocation, spleens were removed from mice and spleen cells were prepared as described previously [19].

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Table 1. Ingredient of miso per 100 g.

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Flow cytometry

The cells were analyzed on a MACSQuant Flow Cytometer (MiltenyiBiotec) using the following specific antibodies: VioletFluor^TM 450-conjugated anti-B220 (TONBO Biosciences), Brilliant Violet 510^TM-conjugated anti-CD4 (BioLegend), Brilliant Violet 510^TM-conjugated anti-CD8a (BioLegend), phycoerythrin (PE)-conjugated anti-CD138 (BioLegend), Alexa Fluor 647-conjugated anti-GL7 (BioLegend), PE-conjugated anti-CD62L (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD44 (eBioscience), APC-conjugated anti-CD86 (TONBO Biosciences), PE-conjugated anti-CD69 (BioLegend), PE-conjugated anti-CD23 (BioLegend), Alexa Fluor 647-conjugated anti-CD21 (BD Pharmingen), PE-conjugated anti-PD-1 (TONBO Biosciences), Brilliant Violet 421^TM-conjugated anti-CXCR5 (BioLegend), Alexa Fluor 647-conjugated ICOS (BioLegend), eFour450-conjugated anti-CD127 (eBioscience), PE-conjugated anti-CD25 (TONBO Biosciences) and APC-conjugated anti-CTLA4 (TONBO Biosciences). Data analysis was conducted with FlowJo software (v7.6.5; FLOWJO, LLC).

DNA extraction

Two-hundred mg of fecal samples was subjected to total DNA extraction using QIAamp Fast DNA Stool Mini Kit (QIAGEN) following the manufacturer’s instruction. Final DNA concentrations were estimated based on 260 nm absorbance using a Nanodrop 2000 spectrometer (Thermo Fischer Scientific).

Preparation of 16S rRNA gene amplicon sequence library

V3-V4 variable region of 16S rDNA in each fecal DNA sample was amplified by polymerase chain reaction (PCR) using universal primer set of Bakt 341F (5-cctacgggnggcwgcag) and Bakt 805R (5-gactachvgggtatctaatcc) [20]. 1^st PCR was conducted in 25 μL of reaction mixtures containing 12.5 ng of fecal DNA, 200 nM of each primer, and 1x KAPA HiFi Hot Start Ready Mix (Kapa Biosystems) under the following parameters: 95°C for 3 min, 25 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, followed by 72°C for 5 min. 1^st PCR products were purified by AMPure XP beads (Beckman Coulter) and subjected to 2^nd PCR for adding sequencing adapters containing sample specific 8 bp barcodes to the 3’- and 5’- ends by using the Nextera XT Index Kit v2 Set B (Illumina). 2^nd PCR was conducted in 50 μL of reaction mixtures containing 5 μL of 1st PCR products, 5 μL of each indexing primer and 1x KAPA HiFi Hot Start Ready Mix under the following conditions: 95°C for 3 min, 8 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, followed by 72°C for 5 min. Each 2^nd PCR amplicon was purified, quantified by the Qubit dsDNA HS Assay Kit (Thermo Fischer Scientific), and adjusted to 4 nM. Then, 4μL of each amplicon were pooled and quantified by KAPA Library Quantification Kit Lightcycler 480 qPCR Mix (Kapa Biosystems) and adjusted to 4 pM. The amplicon library was mixed with 5% of equimolar PhiX Control v3 (Illumina) and sequenced on a MiSeq instrument using the MiSeq 600-cycle v3 kit (Illumina) by pair-end sequencing mode.

Metagenomic analysis

Pair-end sequencing reads generated from Miseq were demultiplexed and imported into QIIME2 pipeline (v2019.7) [21]. Quality-filtering, denoising, and removal of chimeric sequences were conducted by DADA2 plugin [22] with following parameters;—p-trim-left-f 17,—p-trim-left-r 21,—p-trunc-len-f 280,—p-trunc-len-r 200,—p-max-ee-f 2 and—p-max-ee-r 2. Phylogenic tree was generated by FastTree [23] after alignment with MAFFT [24]. Taxonomy of each feature were assigned using a naïve-bayes classifier trained on 16S rRNA gene OTUs clustered at 99% similarities within the Silva database (v132). Estimation of α-diversity (Simpson index) and β-diversity (unweighted UniFrac distance) was conducted by QIIME2 workflow. Statistical significance of α-diversity was tested by Permutational multivariant analysis of variance (PERMANOVA) test in QIIME2 pipeline.

Measurement of serum Ig levels

Serum Ig levels were measured as described previously [25] using enzyme-linked immunosorbent assays (ELISAs). Sonicated herring sperm DNA (10 μg/ml, Sigma-Aldrich) was coated onto ELISA plates. Alkaline phosphatase-conjugated anti-IgM and anti-IgG (Southern Biotech) antibodies (Abs) were used.

Statistics

Experimental data are indicated as the mean ± standard deviation (S.D.). Statistical significance was evaluated using a two-tailed Student’s t-test for unpaired data. P-values < 0.05 were considered statistically significant.

Transcriptome analyses

A total of 4 × 10⁶ spleen cells were cultured in 1 mL RPMI 1640 medium containing 10% FCS with 10 μg of miso or miso ingredients powders for 2 d. Total RNA was prepared from spleen cells using ISOGEN II (Nippon Gene) and subjected to RNA sequencing analysis (GENEWIZ). RNA-Seq library construction, next generation sequencing, and bioinformatics analyses were done using GENEWIZ. Briefly, total RNA was quantified and qualified using the Qubit RNA Assay (Invitrogen) and RNA ScreenTape (TapeStation; Agilent Technologies). Poly(A) mRNA was enriched using magnetic bead-conjugated oligo(dT), and library preparation for high-throughput sequencing was conducted according to the manufacturer’s instructions (NEBNext Ultra II RNA Library Prep Kit for Illumina; New England BioLabs). Approximately 250 ng of total RNA was used for mRNA selection, and adapter-ligated double stranded cDNA fragment was amplified using 12 cycles of PCR, which incorporated Illumina P5/P7 adapters and a sample-specific barcode sequence. Fragment size and quantity of the libraries were confirmed using Qubit DNA Assay (Invitrogen) and DNA ScreenTape (TapeStation; Agilent Technologies). Libraries with unique sample barcodes were pooled together and loaded onto an Illumina HiSeq/NovaSeq instrument according to the manufacturer’s instructions (Illumina). Sequencing was carried out using a 150-bp paired-end configuration. Image analysis, base calling, and demultiplex were performed using the Illumina standard software. Approximately 20 M paired-end reads (6-Gb output in 150-bp paired-end configuration) per sample were obtained. The raw sequencing reads were filtered to remove adapter and low-quality reads. The resulting clean reads were used for mapping against a reference genome (Mus musculus; Ensembl/GRCm38), quantifying gene expression, identifying differential gene expression, and other downstream analyses. Gene expression ratio over 1.5 indicate significant differences.

Intravital microscopy

Intestinal epithelial cells (IECs) from anesthetized mice were imaged. Mice were anesthetized by intraperitoneal administration of 50 mg/kg pentobarbital, and the small intestinal tract was surgically opened lengthwise, placed on a slide glass with a pump attached, and immobilized on a microscope stage. For image acquisition, a Nikon A1 laser-scanning confocal microscope with a 20× objective and NIS-Elements AR software were used as previously described [25]. We used a dichroic mirror (DM457/514) and two bandpass emission filters (482/35 for cyan fluorescent protein [CFP] and 540/30 for yellow fluorescent protein [YFP]). The YFP/CFP ratio was obtained by excitation at 458 nm. Acquired images were analyzed using NIS-Elements software (Nikon).

Results

Miso acts on immune cells as an activator and suppressor

Since miso and miso-derived microorganisms have been shown to modulate the immune response [13,16], we examined the effect of miso on B cells and T cells in mice. Mice were fed a diet containing 5% miso for 4 or 12 weeks, and the ratio of B cells and T cells in the spleen were analyzed, specifically, B cells (B220⁺), CD4⁺ and CD8⁺ T cells (S1A and S1B Fig). Regardless of the duration of feeding, the ratio between B cells and T cells in the spleen of miso-treated mice did not differ from those of the control mice (Fig 1A–1D).

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Fig 1. The effect of miso on B cells and T cells in mouse spleens.

(A) The ratio of B220⁺/CD4⁺ cells fed for 4 weeks. (B) The ratio of B220⁺/CD4⁺ cellsfed for 12 weeks. (C) The ratio of B220⁺/CD8a⁺ cells fed for 4 weeks. (D) The ratio of B220⁺/CD8a⁺ cells fed for 12 weeks. (E) The rate of CD86⁺/B cells fed for 4 weeks. (F) The rate of CD86⁺/B cells fed for 12 weeks. (G) The rate of CD69⁺/B cells fed for 4 weeks. (H) The rate of CD69⁺/B cells fed for 12 weeks. (I) The rate of GL7⁺/B cells fed for 4 weeks. (J) The rate of GL7⁺/B cells fed for 12 weeks. (K) The rate of CD138⁺/B cells fed for 4 weeks. (L) The rate of CD138⁺/B cells fed for 12 weeks. Diet containing 5% soybean-miso was fed to C57BL/6 mice for 4 (n = 12) or 12 (n = 3) weeks. Then, spleen samples were harvested and analyzed using flow cytometry. Bars indicate mean ± S.D. P-values relative to the control using a t-test.

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Next, we analyzed subsets of B cells. There are several subsets of B cells in the spleen: marginal zone B cells (MZ B cells: CD19⁺CD21⁺CD23⁻), follicular B cells (Fo B cells: CD19⁺CD21⁺CD23⁺) and transitional type 1 B cells (T1 B cells: CD19⁺CD21^-CD23^-). We examined subsets of these B cells and found no changes associated with miso intake (S2 Fig).

B cells are activated and differentiate into plasma cells through germinal center (GC) B cells or directly. We examined the activation markers, CD86 and CD69, on spleen B cells (S1C and S1D Fig). Although the populations of CD86⁺ B cells were unaltered, CD69⁺ B cells were increased (Fig 1E–1H). Since activated B cells were increased, we next examined the status of GC B cells (B220⁺GL7⁺) and plasma cells (B220^-CD138⁺) (S1E and S1F Fig). Ingestion of miso increased the population of GC B cells in the spleen, whereas it did not alter the population of plasma cells (Fig 1I–1L).

We also analyzed subsets of T cells. First, we measured the number of memory T cells (CD4⁺CD44^highCD62L⁻) and naive T cells (CD4⁺CD44^lowCD62L⁺) in the spleen, however, the population of these cell types was not altered (S3 Fig). Next, we analyzed follicular helper T (Tfh) cells crucial for B cell activation. The Tfh (CD4⁺PD-1⁺CXCR5⁺ or CD4⁺PD-1⁺ICOS⁺) cell population was not altered (Fig 2A–2D). Furthermore, the population of activated CD4 T cells (CD69⁺CD4⁺ T cells) was not altered (Figs 2E, 2F and S1G). In addition, we analyzed regulatory T (Treg) cells crucial for immune tolerance. Splenic Treg (CD4⁺CD127⁻CD25⁺CTLA4⁺) cells were increased in miso-fed mice for 4 weeks compared with that of the control mice (Fig 2G and 2H). Overall, these results show that miso increased both immune-stimulatory and -regulatory cells in mice under steady-state conditions.

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Fig 2. The effect of miso on subsets of T cells in mouse spleens.

(A) Examples of the measurement of Tfh (CD4⁺PD-1⁺CXCR5⁺) by flow cytometry are shown. (B) Examples of the measurement of Tfh (CD4⁺PD-1⁺ICOS⁺) by flow cytometry are shown. (C) The rate of Tfh (CD4⁺PD-1⁺CXCR5⁺)/CD4⁺ T cells in spleens of mice fed for 4 weeks. (D) The rate of Tfh (CD4⁺PD-1⁺ICOS⁺)/CD4⁺ T cells in spleens of mice fed for 4 weeks. (E) The rate of CD69⁺/CD4⁺ T cells in mice fed for 4 weeks. (F) The rate of CD69⁺/CD4⁺ T cells in mice fed for 12 weeks. (G) Examples of the measurement of Treg cells by flow cytometry are shown. (H) The rate of Treg/CD4⁺ T cells in mice fed for 4 weeks. Diet containing 5% soybean-miso was fed to C57BL/6 mice for 4 (n = 12) or 12 (n = 3) weeks. Spleen samples were collected and analyzed using flow cytometry. Bars indicate mean ± S.D. P-values relative to the control using a t-test.

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Treg cells increased in miso-fed mice. Butyrate which is a metabolite derived from microbiota is known to induce Treg cells [26]. Furthermore, Treg cells induction is known to mediated by food antigen and gut microbiota [27]. To clarify whether increase of Treg cells is mediated by microbiota metabolite, butyrate, we examined fecal microbiota of miso-fed mice. So far, the effect of miso on gut microbiota is limited. Therefore, we used IL-22 deficient mice which is sensitive to diet due to its weak gut barrier function [28]. Fecal microbiota composition of miso-fed mice was almost no differences in α-diversity and β-diversity from that of control diet-fed mice (S4 Fig). Furthermore, relative abundance of Clostridiales including most of butyrate-producing bacteria was almost the same between the two groups. These results suggested that effect of metabolite on miso-mediated immune regulation seems to be marginal.

Long-term ingestion of miso increases anti-DNA IgM Abs

Serum IgM is crucial for preventing autoimmunity [29]. Abs of the IgM class prevent further immune response and production of IgG. Autoantibodies of the IgG class specific for DNA have been shown to be pathogenic and trigger autoimmune symptoms, such as lupus nephritis [30]. To confirm the effect of miso on immune tolerance, we measured anti-DNA Abs in the serum. Anti-DNA IgM Abs and anti-DNA IgG Abs in the serum were detected in normal mice and were unchanged in mice fed miso for 3 weeks (Fig 3A and 3B). In mice fed miso for 12 weeks, anti-DNA IgM Abs in the serum were increased, whereas anti-DNA IgG Abs were not altered (Fig 3C and 3D). Thus, protective IgM Abs against autoimmunity were increased upon continued ingestion of miso, suggesting that miso augments beneficial immune reactions for self-tolerance after long-term ingestion of miso.

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Fig 3. The effect of miso on Anti-DNA IgM and IgG production in serum.

(A) Anti-DNA IgM levels in serum from mice fed for 3 weeks. (B) Anti-DNA IgG level in serum from mice fed for 3 weeks. (C) Anti-DNA IgM level in serum of mice fed for 12 weeks. (D) Anti-DNA IgG level in serum of mice fed for 12 weeks. Diet containing 5% soybean-miso was fed to C57BL/6 mice for 3 (n = 3) or 12 (n = 3) weeks. Then, spleen samples were collected and analyzed using flow cytometry. Bars indicate mean ± S.D. P-values relative to the control using a t-test.

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Miso directly regulates the expression of genes involved in the immunity of spleen cells in mice

We previously found that Tetragenococcus halophilus No.1, which was isolated from miso, regulates gene expression in spleen cells in vitro [13]. To confirm whether the miso itself or miso ingredients have the same effect, we performed a transcriptome analysis of spleen cells cultured with these extracts (Tables 2 and 3 and S5 Fig). Similar to T. halophilus No.1 isolated from miso [13], the expression of CD86, IL-10, IL-22 and Interferon gamma (IFN-γ) were upregulated by miso. In addition, the expression of CD69, which is an activation marker for B cells and T cells, and IL-1β, which is an inflammatory cytokine, were all upregulated by miso. In contrast, soybeans and rice, which are the raw materials of miso, did not show significant upregulation of these genes. These results indicate that miso, but not soybeans and rice, directly regulates the expression of genes involved in the immunity of spleen cells in mice.

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Table 2. Soybean and rice information.

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Table 3. Gene expression ratio (sample/control).

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Effect of miso on Ca²⁺ signaling in the IECs of mice with ubiquitous YC3.60 expression in vivo

As mentioned above, the ingestion of miso affects the immune system. The IECs also secrete cytokines and their receptors [31]. For instance, the IECs secrete IL-10 in response to toll-like receptor 4 stimulation [32]. Furthermore, recently the enteric epithelial tuft cells produce IL-25 which is crucial for protection against parasite infection [33]. Thus, the IECs communicating with immune cells play crucial roles in the immune system. Therefore, we examined whether miso stimulates directly the enteric epithelial cells or not. To clarify this, we performed intravital imaging to determine whether miso directly stimulates IECs. We previously established an intravital imaging system for Ca²⁺ signaling in IECs and evaluated the effect of probiotics, including Lactococcus lactis and Bacillus subtilis [18]. To improve the imaging system, we created a special device that can inject a stimulus solution along with intravital imaging. As shown in S5 Fig, this device together with a cover glass is used to hold the target tissue by compression with a pump. Meanwhile, the solution can reach the tissue through a tube from an injection syringe. This enables stable observation of moving tissues such as that of the intestine.

Using an improved imaging system, we tested whether miso induces Ca²⁺ signaling in IECs. The miso extract was prepared by diluting 10 times with water and then filtered. As a control, we adjusted the NaCl concentration to 1% with water. We surgically opened the small intestinal tract of the mice exhibiting ubiquitous YC3.60 expression [18], placed it on the stage of the confocal microscope, and added PBS, 1% NaCl and miso extract to the intestinal epithelium (S6 Fig). Adding 1% NaCl temporarily increased intracellular Ca²⁺ concentration, whereas the addition of PBS did not affect intracellular Ca²⁺ concentration in the IECs (Fig 4A and 4B and S1 Video). However, adding miso strikingly increased intracellular Ca²⁺ concentration in the IECs (Fig 4C and 4D and S2 Video). Miso induced a gradual elevation of intracellular Ca²⁺ concentration, which is similar to that of probiotics such as L. lactis and B. subtilis [18]. Thus, miso directly affected IECs to a similar extent as probiotics.

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Fig 4. Images of intravital Ca²⁺ signaling in the IECs of a mouse expressing YC3.60.

(A) Representative Ca²⁺ signaling of PBS and 1% NaCl images in the intestinal tract of a mouse with ubiquitous YC3.60 expression and SPF conditions. Ratiometric images (YFP/CFP at excitation of 458 nm) are shown. A total of 0.1 ml of PBS or 1% NaCl were added at the indicated time points. (B) Time course of fluorescence intensities of YFP/CFP upon excitation at 458 nm when the intestinal tract was stimulated with PBS and 1% NaCl. (C) Representative Ca²⁺ signaling of PBS and 10% soybean-miso images in the intestinal tract of a mouse with ubiquitous YC3.60 expression and SPF conditions. Ratiometric images (YFP/CFP at excitation of 458 nm) are shown. A total of 0.1 ml of 10% miso in PBS was added at the indicated time point. (D) Time course for fluorescence intensities of YFP/CFP upon excitation at 458 nm when the intestinal tract was stimulated with miso and measured in randomly selected regions (n = 3). Scale bar, 100 μm; frames = 131. Spontaneous Ca²⁺ signals are indicated by arrows.

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Discussion

In this study, we found that the ingestion of miso increased the expression of CD69 on B cells and GC B cells, suggesting that miso enhances part of the process by which B cells differentiate into plasma cells. Furthermore, consuming miso increased the amount of Treg cells and increased anti-DNA IgM Abs levels. Miso upregulated the expression of IL-10, IL-22 and CD86 mRNA. Miso evoked Ca²⁺ signaling in the IEC similar to probiotics. These results suggest that miso regulates immunity by affecting immune activation and immune tolerance.

As part of the daily diet in Japan, miso has traditionally been consumed and suggested to be beneficial to human health. Indeed, it was shown that the consumption of a large amount of soy products in the Asian population can effectively control breast cancer, but this effect has not been observed in Westerners who consume a small amount of soy products [34]. Soybean protein has also been reported to inhibit blood pressure [35] and exhibit antioxidant activity [36], whereas soybean isoflavones exhibit anti-cancer effects [37,38] and promote bone formation [39]. Furthermore, intake of miso soup before pregnancy significantly reduces the risk of early preterm birth [40]. In this study, we showed that miso augmented both immunity and immune tolerance under steady-state conditions as activated B cells and Treg cells were increased upon ingestion of miso. These results suggest that miso strengthens immune robustness and prevents both autoimmunity and infectious diseases, resulting in the maintenance of good health.

LAB isolated from miso has been reported to suppress allergies [41,42], and other studies have reported that it enhances immunity, in part, by activating B cells [13]. In this study, we found that miso regulates immunity in the same way as LAB isolated from miso. Furthermore, we conducted a transcriptome analysis of miso and its raw materials, which revealed that miso regulates the expression of genes involved in immunity. This result is similar to that observed with T. halophilus No.1, which we previously isolated from miso rather than the soybeans and rice that are the raw materials for miso. From these results, we conclude that the LAB in miso contributes, in part, to the health effects of miso.

Intravital Ca²⁺ imaging of the small intestinal epithelium showed that miso stimulates IECs. The kinetics of Ca²⁺ signaling mediated by miso resembles that of probiotics [18]. Based on this similarity, it is likely that probiotics contained in miso may induce Ca²⁺ signaling. Miso seems to affect the immune system by directly stimulating immune cells and epithelial cells. In the small intestine, there is an abundance of endocrine cells, immune cells and nerve cells (neurons and glial cells), which functionally interact with one another [43]. Besides microbiotics, miso contains abundant amino acids. Some amino acids such as glutamate have been shown to induce the production of hormones in enteroendocrine cells [44,45]. It is also possible that these components contribute to Ca²⁺ signaling via enteroendocrine cells, although further analyses of this process are needed.

Collectively, we showed the effect of miso on immunological robustness in mice. This may contribute to Japanese longevity and health. Our study may help to clarify the beneficial effects of miso on human health in the near future.

Supporting information

S1 Fig. Examples of mice spleen cells measurement by flow cytometry.

(A) B220 and CD4. (B) B220 and CD8a. (C) B220 and CD86. (D) B220 and CD69. (E) B220 and GL7. (F) B220 and CD138. (G) CD4 and CD69.

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S2 Fig. The effect of miso on subsets of B cells in mouse spleen.

(A) Examples of the measurement by flow cytometryare shown. (B) The rate of CD21⁺CD23⁻/B cells in mice fed for 4 weeks. (C) The rate of CD21⁺CD23⁻/B cells in mice fed for 12 weeks. (D) The rate of CD21⁺CD23⁺/B cells in mice fed for 4 weeks. (E) The rate of CD21⁺CD23⁺/B cells in mice fed for 12 weeks. (F) The rate of CD21^-CD23^-/B cells in mice fed for 4 weeks. (G) The rate of CD21^-CD23^-/B cells in mice fed for 12 weeks. Diet containing 5% soybean-miso was fed to C57BL/6 mice for 4 (n = 12) or 12 (n = 3) weeks. Spleen samples were collected and were analyzed using flow cytometry. Bars indicate mean ± S.D. P-values relative to the control using a t-test.

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S3 Fig. The effect of miso on memory and naive T cells in spleen.

(A) Examples of the measurement by flow cytometry. (B) The rate of CD44^highCD62L⁻/CD4⁺ T cells in mice fed for 4 weeks. (C) The rate of CD44^highCD62L⁻/CD4⁺ T cells in mice fed for 12 weeks. (D) The rate of CD44^highCD62L⁺/CD4⁺ T cells in mice fed for 4 weeks. (E) The rate of CD44^highCD62L⁺/CD4⁺ T cells in mice fed for 12 weeks. (F) The rate of CD44^lowCD62L⁺/CD4⁺ T cells in mice fed for 4 weeks. (G) The rate of CD44^lowCD62L⁺/CD4⁺ T cells in mice fed for 12 weeks. (H) The rate of CD44^lowCD62L^-/CD4⁺ T cells in mice fed for 4 weeks. (I) The rate of CD44^lowCD62L^-/CD4⁺ T cells in mice fed for 12 weeks. Diet containing 5% miso was fed to C57BL/6 mice for 4 (n = 12) or 12 (n = 3) weeks. Spleen samples were collected and were analyzed using flow cytometry. Bars indicate mean ± S.D. P-values relative to the control using a t-test.

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S4 Fig. Comparison of intestinal microflora analyzed from feces of mice fed control or 5% miso diets.

(A) β-diversity (weighted UniFrac distance). (B, C, D, E) Four α-diversity indexes, (B) PD whole tree, (C) Observed OTUs, (D) Shannon index, (E) Simpson index. (F) Overall composition of microbiota at the phylum level.

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S5 Fig. Comparison of gene expression levels in spleen cells treated with the miso or other ingredients.

Heat maps of representative gene expression of (A) transcription factors, (B) cytokines and (C) CD molecules.

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S6 Fig. Injection apparatus.

This device together with a cover glass is used to hold the target tissue by compression with a pump. The solution can be injected though a tube from an injection syringe.

https://doi.org/10.1371/journal.pone.0261680.s006

(TIF)

S1 Video. Intravital Ca²⁺ signaling by PBS or NaCl in the IECs of a mouse expressing YC3.60.

A total of 0.1 ml of PBS was added at the time point of 1 min and a total of 0.1 ml of 1% NaCl in water was added at the time point of 2 min 30 s. A rainbow parameter indicates relative Ca²⁺ concentration.

https://doi.org/10.1371/journal.pone.0261680.s007

(MP4)

S2 Video. Intravital Ca²⁺ signaling by miso in the IECs of a mouse expressing YC3.60.

A total of 0.1 ml of 10% miso in PBS was added at the time point of 1 min was added at the time point of 2 min 30 s. A rainbow parameter indicates relative Ca²⁺ concentration.

https://doi.org/10.1371/journal.pone.0261680.s008

(MP4)

References

1. Maeda K, Nakata K, Nakamura A, Kitagawa M, Ito S (2018) Improvement in Skin Conditions by Consumption of Traditional Japanese Miso Soup and Its Mechanism. Journal of Nutrition & Food Sciences 08.
- View Article
- Google Scholar
2. Watanabe H, Kashimoto N, Kajimura J, Kamiya K (2006) A miso (Japanese soybean paste) diet conferred greater protection against hypertension than a sodium chloride diet in Dahl salt-sensitive rats. Hypertens Res 29: 731–738. pmid:17249529
- View Article
- PubMed/NCBI
- Google Scholar
3. Yoshinaga M, Toda N, Tamura Y, Terakado S, Ueno M, et al. (2012) Japanese traditional miso soup attenuates salt-induced hypertension and its organ damage in Dahl salt-sensitive rats. Nutrition 28: 924–931. pmid:22261579
- View Article
- PubMed/NCBI
- Google Scholar
4. Ito K, Hirooka Y, Sunagawa K (2014) Miso (Japanese soybean paste) soup attenuates salt-induced sympathoexcitation and left ventricular dysfunction in mice with chronic pressure overload. Fukuoka igaku zasshi = Hukuoka acta medica 105: 48–56. pmid:24908908
- View Article
- PubMed/NCBI
- Google Scholar
5. Ito K, Hirooka Y, Sunagawa K (2015) Japanese soybean paste miso lessens sympathovagal imbalance and attenuates brain sodium sensitivity in mice with pressure overload. Fukuoka igaku zasshi = Hukuoka acta medica 106: 54–63. pmid:26050502
- View Article
- PubMed/NCBI
- Google Scholar
6. Kobayashi M, Sasaki S, Yamamoto S, Tsugane S, Sobue T, et al. (2003) RESPONSE: Re: Soy, Isoflavones, and Breast Cancer Risk in Japan. JNCI: Journal of the National Cancer Institute 95: 1881–1882. pmid:14679159
- View Article
- PubMed/NCBI
- Google Scholar
7. Hirayama T (1982) Relationship of soybean paste soup intake to gastric cancer risk. Nutr Cancer 3: 223–233. pmid:6890671
- View Article
- PubMed/NCBI
- Google Scholar
8. Ohara M, Lu H, Shiraki K, Ishimura Y, Uesaka T, et al. (2002) Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats. Oncology reports 9: 69–73. pmid:11748458
- View Article
- PubMed/NCBI
- Google Scholar
9. Okouchi R, Sakanoi Y, Tsuduki T (2019) Miso (Fermented Soybean Paste) Suppresses Visceral Fat Accumulation in Mice, Especially in Combination with Exercise. Nutrients 11. pmid:30845686
- View Article
- PubMed/NCBI
- Google Scholar
10. Yang X, Nakamoto M, Shuto E, Hata A, Aki N, et al. (2018) Associations between intake of dietary fermented soy food and concentrations of inflammatory markers: a cross-sectional study in Japanese workers. The journal of medical investigation: JMI 65: 74–80. pmid:29593198
- View Article
- PubMed/NCBI
- Google Scholar
11. Watanabe H, Sasatani M, Doi T, Masaki T, Satoh K, et al. (2017) Protective Effects of Japanese Soybean Paste (Miso) on Stroke in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP). Am J Hypertens 31: 43–47. pmid:28985324
- View Article
- PubMed/NCBI
- Google Scholar
12. Hamajima H, Matsunaga H, Fujikawa A, Sato T, Mitsutake S, et al. (2016) Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic. SpringerPlus 5: 1321. pmid:27563516
- View Article
- PubMed/NCBI
- Google Scholar
13. Kumazawa T, Nishimura A, Asai N, Adachi T (2018) Isolation of immune-regulatory Tetragenococcus halophilus from miso. PLOS ONE 13: e0208821. pmid:30586377
- View Article
- PubMed/NCBI
- Google Scholar
14. Kawashima T, Ikari N, Kouchi T, Kowatari Y, Kubota Y, et al. (2018) The molecular mechanism for activating IgA production by Pediococcus acidilactici K15 and the clinical impact in a randomized trial. Scientific reports 8: 5065. pmid:29567956
- View Article
- PubMed/NCBI
- Google Scholar
15. Nishimura I, Igarashi T, Enomoto T, Dake Y, Okuno Y, et al. (2009) Clinical efficacy of halophilic lactic acid bacterium Tetragenococcus halophilus Th221 from soy sauce moromi for perennial allergic rhinitis. Allergology international: official journal of the Japanese Society of Allergology 58: 179–185. pmid:19240374
- View Article
- PubMed/NCBI
- Google Scholar
16. Kumazawa T, Kotake K, Nishimura A, Asai N, Ugajin T, et al. (2020) Isolation of food-derived bacteria inducing interleukin-22 in B cells. Bioscience of microbiota, food and health 39: 1–9. pmid:32010538
- View Article
- PubMed/NCBI
- Google Scholar
17. Nagaishi T, Watabe T, Kotake K, Kumazawa T, Aida T, et al. (2021) Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum. Gut. pmid:33963042
- View Article
- PubMed/NCBI
- Google Scholar
18. Adachi T, Kakuta S, Aihara Y, Kamiya T, Watanabe Y, et al. (2016) Visualization of Probiotic-Mediated Ca(2+) Signaling in Intestinal Epithelial Cells In Vivo. Frontiers in immunology 7: 601. pmid:28018362
- View Article
- PubMed/NCBI
- Google Scholar
19. Hokazono Y, Adachi T, Wabl M, Tada N, Amagasa T, et al. (2003) Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells. Journal of immunology 171: 1835–1843.
- View Article
- Google Scholar
20. Herlemann DP, Labrenz M, Jurgens K, Bertilsson S, Waniek JJ, et al. (2011) Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. The ISME journal 5: 1571–1579. pmid:21472016
- View Article
- PubMed/NCBI
- Google Scholar
21. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, et al. (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature biotechnology 37: 852–857. pmid:31341288
- View Article
- PubMed/NCBI
- Google Scholar
22. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, et al. (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nature methods 13: 581–583. pmid:27214047
- View Article
- PubMed/NCBI
- Google Scholar
23. Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PloS one 5: e9490. pmid:20224823
- View Article
- PubMed/NCBI
- Google Scholar
24. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 30: 772–780. pmid:23329690
- View Article
- PubMed/NCBI
- Google Scholar
25. Yoshikawa S, Usami T, Kikuta J, Ishii M, Sasano T, et al. (2016) Intravital imaging of Ca(2+) signals in lymphocytes of Ca(2+) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset. Scientific reports 6: 18738. pmid:26732477
- View Article
- PubMed/NCBI
- Google Scholar
26. Hui W, Yu D, Cao Z, Zhao X (2019) Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis. International immunopharmacology 68: 226–233. pmid:30660077
- View Article
- PubMed/NCBI
- Google Scholar
27. Kim KS, Hong SW, Han D, Yi J, Jung J, et al. (2016) Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 351: 858–863. pmid:26822607
- View Article
- PubMed/NCBI
- Google Scholar
28. Dudakov JA, Hanash AM, van den Brink MR (2015) Interleukin-22: immunobiology and pathology. Annual review of immunology 33: 747–785. pmid:25706098
- View Article
- PubMed/NCBI
- Google Scholar
29. Ehrenstein MR, Cook HT, Neuberger MS (2000) Deficiency in serum immunoglobulin (Ig)M predisposes to development of IgG autoantibodies. The Journal of experimental medicine 191: 1253–1258. pmid:10748243
- View Article
- PubMed/NCBI
- Google Scholar
30. Ehrenstein MR, Katz DR, Griffiths MH, Papadaki L, Winkler TH, Kalden JR, et al (1995) Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice. Kidney Int 48: 705–711. pmid:7474655
- View Article
- PubMed/NCBI
- Google Scholar
31. Andrews C, McLean MH, Durum SK (2018) Cytokine Tuning of Intestinal Epithelial Function. Frontiers in immunology 9: 1270. pmid:29922293
- View Article
- PubMed/NCBI
- Google Scholar
32. Hyun J, Romero L, Riveron R, Flores C, Kanagavelu S, et al. (2015) Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk. Journal of innate immunity 7: 87–101. pmid:25171731
- View Article
- PubMed/NCBI
- Google Scholar
33. von Moltke J, Ji M, Liang HE, Locksley RM (2016) Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit. Nature 529: 221–225. pmid:26675736
- View Article
- PubMed/NCBI
- Google Scholar
34. Maskarinec G, Ju D, Morimoto Y, Franke AA, Stanczyk FZ (2017) Soy Food Intake and Biomarkers of Breast Cancer Risk: Possible Difference in Asian Women? Nutrition and cancer 69: 146–153. pmid:27918846
- View Article
- PubMed/NCBI
- Google Scholar
35. Xiao Y, Zhang S, Tong H, Shi S (2018) Comprehensive evaluation of the role of soy and isoflavone supplementation in humans and animals over the past two decades. Phytotherapy research: PTR 32: 384–394. pmid:29193539
- View Article
- PubMed/NCBI
- Google Scholar
36. Illesca PG, Alvarez SM, Selenscig DA, Ferreira MDR, Gimenez MS, et al. (2017) Dietary soy protein improves adipose tissue dysfunction by modulating parameters related with oxidative stress in dyslipidemic insulin-resistant rats. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 88: 1008–1015. pmid:28178612
- View Article
- PubMed/NCBI
- Google Scholar
37. Chen M, Rao Y, Zheng Y, Wei S, Li Y, et al. (2014) Association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women: a meta-analysis of epidemiological studies. PLOS ONE 9: e89288. pmid:24586662
- View Article
- PubMed/NCBI
- Google Scholar
38. Pusparini, Dharma R, Suyatna FD, Mansyur M, Hidajat A(2013) Effect of soy isoflavone supplementation on vascular endothelial function and oxidative stress in postmenopausal women: a community randomized controlled trial. Asia Pacific journal of clinical nutrition 22: 357–364. pmid:23945405
- View Article
- PubMed/NCBI
- Google Scholar
39. Zheng X, Lee SK, Chun OK (2016) Soy Isoflavones and Osteoporotic Bone Loss: A Review with an Emphasis on Modulation of Bone Remodeling. Journal of medicinal food 19: 1–14. pmid:26670451
- View Article
- PubMed/NCBI
- Google Scholar
40. Ito M, Takamori A, Yoneda S, Shiozaki A, Tsuchida A, Matsumura K, et al (2019) Fermented foods and preterm birth risk from a prospective large cohort study: the Japan Environment and Children’s study. Environ Health Prevent Med 24: 25. pmid:31039736
- View Article
- PubMed/NCBI
- Google Scholar
41. Ohata E, Yoshida S, Masuda T, Kitagawa M, Nakazawa T, et al. (2011) Tetragenococcus halophilus MN45 Ameliorates Development of Atopic Dermatitis in Atopic Dermatitis Model NC/Nga Mice. Food Science and Technology Research 17: 537–544.
- View Article
- Google Scholar
42. Ohata E, Yoshida S, Masuda T, Kitagawa M, Nakazawa T, et al. (2011) Tetragenococcus halophilus MN45 Isolated from Miso Inhibits IgE Production. Food Science and Technology Research 17: 129–138.
- View Article
- Google Scholar
43. Fung TC, Olson CA, Hsiao EY (2017) Interactions between the microbiota, immune and nervous systems in health and disease. Nature neuroscience 20: 145–155. pmid:28092661
- View Article
- PubMed/NCBI
- Google Scholar
44. Nelson G, Chandrashekar J, Hoon MA, Feng L, Zhao G, et al. (2002) An amino-acid taste receptor. Nature 416: 199–202. pmid:11894099
- View Article
- PubMed/NCBI
- Google Scholar
45. Daly K, Al-Rammahi M, Moran A, Marcello M, Ninomiya Y, et al. (2013) Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion. American journal of physiology Gastrointestinal and liver physiology 304: G271–282. pmid:23203156
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Maeda K, Nakata K, Nakamura A, Kitagawa M, Ito S (2018) Improvement in Skin Conditions by Consumption of Traditional Japanese Miso Soup and Its Mechanism. Journal of Nutrition & Food Sciences 08.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Watanabe H, Kashimoto N, Kajimura J, Kamiya K (2006) A miso (Japanese soybean paste) diet conferred greater protection against hypertension than a sodium chloride diet in Dahl salt-sensitive rats. Hypertens Res 29: 731–738. pmid:17249529
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Yoshinaga M, Toda N, Tamura Y, Terakado S, Ueno M, et al. (2012) Japanese traditional miso soup attenuates salt-induced hypertension and its organ damage in Dahl salt-sensitive rats. Nutrition 28: 924–931. pmid:22261579
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Ito K, Hirooka Y, Sunagawa K (2014) Miso (Japanese soybean paste) soup attenuates salt-induced sympathoexcitation and left ventricular dysfunction in mice with chronic pressure overload. Fukuoka igaku zasshi = Hukuoka acta medica 105: 48–56. pmid:24908908
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Ito K, Hirooka Y, Sunagawa K (2015) Japanese soybean paste miso lessens sympathovagal imbalance and attenuates brain sodium sensitivity in mice with pressure overload. Fukuoka igaku zasshi = Hukuoka acta medica 106: 54–63. pmid:26050502
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Kobayashi M, Sasaki S, Yamamoto S, Tsugane S, Sobue T, et al. (2003) RESPONSE: Re: Soy, Isoflavones, and Breast Cancer Risk in Japan. JNCI: Journal of the National Cancer Institute 95: 1881–1882. pmid:14679159
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Hirayama T (1982) Relationship of soybean paste soup intake to gastric cancer risk. Nutr Cancer 3: 223–233. pmid:6890671
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Ohara M, Lu H, Shiraki K, Ishimura Y, Uesaka T, et al. (2002) Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats. Oncology reports 9: 69–73. pmid:11748458
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Okouchi R, Sakanoi Y, Tsuduki T (2019) Miso (Fermented Soybean Paste) Suppresses Visceral Fat Accumulation in Mice, Especially in Combination with Exercise. Nutrients 11. pmid:30845686
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Yang X, Nakamoto M, Shuto E, Hata A, Aki N, et al. (2018) Associations between intake of dietary fermented soy food and concentrations of inflammatory markers: a cross-sectional study in Japanese workers. The journal of medical investigation: JMI 65: 74–80. pmid:29593198
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Watanabe H, Sasatani M, Doi T, Masaki T, Satoh K, et al. (2017) Protective Effects of Japanese Soybean Paste (Miso) on Stroke in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP). Am J Hypertens 31: 43–47. pmid:28985324
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Hamajima H, Matsunaga H, Fujikawa A, Sato T, Mitsutake S, et al. (2016) Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic. SpringerPlus 5: 1321. pmid:27563516
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref13] 13. Kumazawa T, Nishimura A, Asai N, Adachi T (2018) Isolation of immune-regulatory Tetragenococcus halophilus from miso. PLOS ONE 13: e0208821. pmid:30586377
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Kawashima T, Ikari N, Kouchi T, Kowatari Y, Kubota Y, et al. (2018) The molecular mechanism for activating IgA production by Pediococcus acidilactici K15 and the clinical impact in a randomized trial. Scientific reports 8: 5065. pmid:29567956
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Nishimura I, Igarashi T, Enomoto T, Dake Y, Okuno Y, et al. (2009) Clinical efficacy of halophilic lactic acid bacterium Tetragenococcus halophilus Th221 from soy sauce moromi for perennial allergic rhinitis. Allergology international: official journal of the Japanese Society of Allergology 58: 179–185. pmid:19240374
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Kumazawa T, Kotake K, Nishimura A, Asai N, Ugajin T, et al. (2020) Isolation of food-derived bacteria inducing interleukin-22 in B cells. Bioscience of microbiota, food and health 39: 1–9. pmid:32010538
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. Nagaishi T, Watabe T, Kotake K, Kumazawa T, Aida T, et al. (2021) Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum. Gut. pmid:33963042
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Adachi T, Kakuta S, Aihara Y, Kamiya T, Watanabe Y, et al. (2016) Visualization of Probiotic-Mediated Ca(2+) Signaling in Intestinal Epithelial Cells In Vivo. Frontiers in immunology 7: 601. pmid:28018362
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Hokazono Y, Adachi T, Wabl M, Tada N, Amagasa T, et al. (2003) Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells. Journal of immunology 171: 1835–1843.
View Article
Google Scholar

[73] View Article

[74] Google Scholar

[ref20] 20. Herlemann DP, Labrenz M, Jurgens K, Bertilsson S, Waniek JJ, et al. (2011) Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. The ISME journal 5: 1571–1579. pmid:21472016
View Article
PubMed/NCBI
Google Scholar

[76] View Article

[77] PubMed/NCBI

[78] Google Scholar

[ref21] 21. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, et al. (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature biotechnology 37: 852–857. pmid:31341288
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref22] 22. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, et al. (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nature methods 13: 581–583. pmid:27214047
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref23] 23. Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PloS one 5: e9490. pmid:20224823
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref24] 24. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 30: 772–780. pmid:23329690
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref25] 25. Yoshikawa S, Usami T, Kikuta J, Ishii M, Sasano T, et al. (2016) Intravital imaging of Ca(2+) signals in lymphocytes of Ca(2+) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset. Scientific reports 6: 18738. pmid:26732477
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref26] 26. Hui W, Yu D, Cao Z, Zhao X (2019) Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis. International immunopharmacology 68: 226–233. pmid:30660077
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref27] 27. Kim KS, Hong SW, Han D, Yi J, Jung J, et al. (2016) Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 351: 858–863. pmid:26822607
View Article
PubMed/NCBI
Google Scholar

[104] View Article

[105] PubMed/NCBI

[106] Google Scholar

[ref28] 28. Dudakov JA, Hanash AM, van den Brink MR (2015) Interleukin-22: immunobiology and pathology. Annual review of immunology 33: 747–785. pmid:25706098
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref29] 29. Ehrenstein MR, Cook HT, Neuberger MS (2000) Deficiency in serum immunoglobulin (Ig)M predisposes to development of IgG autoantibodies. The Journal of experimental medicine 191: 1253–1258. pmid:10748243
View Article
PubMed/NCBI
Google Scholar

[112] View Article

[113] PubMed/NCBI

[114] Google Scholar

[ref30] 30. Ehrenstein MR, Katz DR, Griffiths MH, Papadaki L, Winkler TH, Kalden JR, et al (1995) Human IgG anti-DNA antibodies deposit in kidneys and induce proteinuria in SCID mice. Kidney Int 48: 705–711. pmid:7474655
View Article
PubMed/NCBI
Google Scholar

[116] View Article

[117] PubMed/NCBI

[118] Google Scholar

[ref31] 31. Andrews C, McLean MH, Durum SK (2018) Cytokine Tuning of Intestinal Epithelial Function. Frontiers in immunology 9: 1270. pmid:29922293
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref32] 32. Hyun J, Romero L, Riveron R, Flores C, Kanagavelu S, et al. (2015) Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk. Journal of innate immunity 7: 87–101. pmid:25171731
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref33] 33. von Moltke J, Ji M, Liang HE, Locksley RM (2016) Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit. Nature 529: 221–225. pmid:26675736
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref34] 34. Maskarinec G, Ju D, Morimoto Y, Franke AA, Stanczyk FZ (2017) Soy Food Intake and Biomarkers of Breast Cancer Risk: Possible Difference in Asian Women? Nutrition and cancer 69: 146–153. pmid:27918846
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref35] 35. Xiao Y, Zhang S, Tong H, Shi S (2018) Comprehensive evaluation of the role of soy and isoflavone supplementation in humans and animals over the past two decades. Phytotherapy research: PTR 32: 384–394. pmid:29193539
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref36] 36. Illesca PG, Alvarez SM, Selenscig DA, Ferreira MDR, Gimenez MS, et al. (2017) Dietary soy protein improves adipose tissue dysfunction by modulating parameters related with oxidative stress in dyslipidemic insulin-resistant rats. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 88: 1008–1015. pmid:28178612
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref37] 37. Chen M, Rao Y, Zheng Y, Wei S, Li Y, et al. (2014) Association between soy isoflavone intake and breast cancer risk for pre- and post-menopausal women: a meta-analysis of epidemiological studies. PLOS ONE 9: e89288. pmid:24586662
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref38] 38. Pusparini, Dharma R, Suyatna FD, Mansyur M, Hidajat A(2013) Effect of soy isoflavone supplementation on vascular endothelial function and oxidative stress in postmenopausal women: a community randomized controlled trial. Asia Pacific journal of clinical nutrition 22: 357–364. pmid:23945405
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref39] 39. Zheng X, Lee SK, Chun OK (2016) Soy Isoflavones and Osteoporotic Bone Loss: A Review with an Emphasis on Modulation of Bone Remodeling. Journal of medicinal food 19: 1–14. pmid:26670451
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref40] 40. Ito M, Takamori A, Yoneda S, Shiozaki A, Tsuchida A, Matsumura K, et al (2019) Fermented foods and preterm birth risk from a prospective large cohort study: the Japan Environment and Children’s study. Environ Health Prevent Med 24: 25. pmid:31039736
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref41] 41. Ohata E, Yoshida S, Masuda T, Kitagawa M, Nakazawa T, et al. (2011) Tetragenococcus halophilus MN45 Ameliorates Development of Atopic Dermatitis in Atopic Dermatitis Model NC/Nga Mice. Food Science and Technology Research 17: 537–544.
View Article
Google Scholar

[160] View Article

[161] Google Scholar

[ref42] 42. Ohata E, Yoshida S, Masuda T, Kitagawa M, Nakazawa T, et al. (2011) Tetragenococcus halophilus MN45 Isolated from Miso Inhibits IgE Production. Food Science and Technology Research 17: 129–138.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref43] 43. Fung TC, Olson CA, Hsiao EY (2017) Interactions between the microbiota, immune and nervous systems in health and disease. Nature neuroscience 20: 145–155. pmid:28092661
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref44] 44. Nelson G, Chandrashekar J, Hoon MA, Feng L, Zhao G, et al. (2002) An amino-acid taste receptor. Nature 416: 199–202. pmid:11894099
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref45] 45. Daly K, Al-Rammahi M, Moran A, Marcello M, Ninomiya Y, et al. (2013) Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion. American journal of physiology Gastrointestinal and liver physiology 304: G271–282. pmid:23203156
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Ethics statement

Miso and raw materials

Cells and mice

Flow cytometry

DNA extraction

Preparation of 16S rRNA gene amplicon sequence library

Metagenomic analysis

Measurement of serum Ig levels

Statistics

Transcriptome analyses

Intravital microscopy

Results

Miso acts on immune cells as an activator and suppressor

Long-term ingestion of miso increases anti-DNA IgM Abs

Miso directly regulates the expression of genes involved in the immunity of spleen cells in mice

Effect of miso on Ca2+ signaling in the IECs of mice with ubiquitous YC3.60 expression in vivo

Discussion

Supporting information

S1 Fig. Examples of mice spleen cells measurement by flow cytometry.

S2 Fig. The effect of miso on subsets of B cells in mouse spleen.

S3 Fig. The effect of miso on memory and naive T cells in spleen.

S4 Fig. Comparison of intestinal microflora analyzed from feces of mice fed control or 5% miso diets.

S5 Fig. Comparison of gene expression levels in spleen cells treated with the miso or other ingredients.

S6 Fig. Injection apparatus.

S1 Video. Intravital Ca2+ signaling by PBS or NaCl in the IECs of a mouse expressing YC3.60.

S2 Video. Intravital Ca2+ signaling by miso in the IECs of a mouse expressing YC3.60.

References

Effect of miso on Ca²⁺ signaling in the IECs of mice with ubiquitous YC3.60 expression in vivo

S1 Video. Intravital Ca²⁺ signaling by PBS or NaCl in the IECs of a mouse expressing YC3.60.

S2 Video. Intravital Ca²⁺ signaling by miso in the IECs of a mouse expressing YC3.60.