Abstract
Recently, we found a novel function of the lactic acid bacterium Tetragenococcus halophilus derived from miso, a fermented soy paste, that induces interleukin (IL)-22 production in B cells preferentially. IL-22 plays a critical role in barrier functions in the gut and skin. We further screened other bacteria species, namely, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Weissella, Pediococcus, and Bacillus, in addition to Tetragenococcus and found that some of them possessed robust IL-22-inducible function in B cells in vitro. This process resulted in the augmented expression of activation markers CD86 and CD69 on B and T cells, respectively. However, these observations were not correlated with IL-22 production. We isolated Bacillus coagulans sc-09 from miso and determined it to be the best strain to induce robust IL-22 production in B cells. Furthermore, feeding B. coagulans sc-09 to mice augmented the barrier function of the skin regardless of gut microbiota.
Keywords: food, IL-22, bacteria, B cell, skin barrier, miso
INTRODUCTION
Miso and soy sauce, which are traditional fermented foods in Japan, contain various microorganisms. In addition to a fungus (Aspergillus oryzae) and yeast, Tetragenococcus halophilus, a salt-tolerant lactic acid bacterium; other lactic acid bacteria; and Bacillus strains contribute to the fermentation processes of miso and soy sauce. Recently, the beneficial effects of these microorganisms and fermented foods on human health have been reported [1,2,3,4].
Recently, we isolated a strain of lactic acid bacteria, T. halophilus No. 1, which has immune regulatory functions, from miso, a fermented soy paste [5]. Administration of this strain augmented serum IgA and immune responses in mice. Notably, T. halophilus No. 1 induced interleukin (IL)-22 cytokine production in B cells. Thus, for the first time, we found that a subpopulation of B cells produce IL-22. Furthermore, T. halophilus induced production of interferon (IFN)-γ in B cells. We termed IL-22-producing and IFN-γ–producing B cell subpopulations as Bi22 and Big cells, respectively.
IL-22 is a member of the IL-10 family [6,7,8]. It was originally thought to be produced from T helper (Th)1 cells among CD4 T cells, and then subsequently it was found to be produced from Th17 and Th22 cells. Furthermore, γδT cells, NKT cells, and innate lymphoid cells are also known to produce IL-22. IL-22 has been identified in various tissues, such as the intestines, lung, liver, kidney, thymus, pancreas, and skin. It contributes to tissue regeneration and regulates host defense at barrier surfaces, such as the gut and skin. IL-22 is also involved in inflammatory tissue pathology. However, a comprehensive understanding of IL-22 remains elusive.
As IL-22 is a multifunctional cytokine, especially with respect to host defense functions, probiotics that induce IL-22 may be valuable to human health. Therefore, in this study, we investigated food-derived microorganisms that induce IL-22 production, identified IL-22-inducing bacteria, and assessed their in vivo functions.
MATERIALS AND METHODS
Ethics statement
C57BL/6 mice were maintained in our animal facility under specific pathogen free (SPF) conditions in accordance with guidelines of the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. Germ-free (GF) mice (C57BL/6NJcl) were obtained from CLEA Japan, Inc. All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (No. A2018-C3), and all experiments were carried out in accordance with the approved guidelines.
Bacteria
Bacteria were isolated from Japanese fermented foods, including miso, soy sauce, and amazake. Lactic acid bacteria were selected using MRS agar (Oxoid Ltd.) with CaCO3. Salt-tolerant lactic acid bacteria, such as T. halophilus, were separated in 10SG10N agar (10% soy sauce, 10% NaCl, 1% glucose, 1% yeast extract, 0.5% polypeptone, 0.2% sodium acetate trihydrate, 0.02% MgSO4·7H2O, 0.001% MnSO4·4H2O, 0.001% FeSO4·7H2O, 0.0025% Tween 80, and 1.5% agar; pH 6.8). Bacteria, such as Bacillus subtilis, were isolated in a standard methods agar (5.0 g/L pancreatic digest of casein, 2.5 g/L yeast extract, 1.0 g/L dextrose, 15.0 g/L agar; pH 7.0 ± 0.2). These bacteria were identified by microscopy and 16S rDNA analysis. Isolated bacteria were cultured, and cultures were sterilized by autoclaving at 121°C for 15 min. The bacteria were then collected by centrifugation, washed three times with water, and then lyophilized. These bacteria were directly used as a dietary supplement. Alternatively, these bacteria were suspended in PBS and used for in vitro immunological assay.
PCR amplification and bacterial 16S rDNA sequencing
Total bacterial DNA was extracted using a NucleoSpin Microbial DNA kit (Macherey-Nagel GmbH & Co. KG). Bacterial 16S rDNA was amplified by PCR using primers 10F (5ʹ-GTT TGA TCC TGG CTC A-3ʹ) and 1500R (5ʹ-TAC CTT GTT ACG ACT T-3ʹ). PCR products were purified using a FastGene Gel/PCR Extraction Kit (Nippon Genetics Co., Ltd). The purified PCR products were sequenced by FASMAC Co., Ltd., Japan, using an Applied Biosystems 3130 XL Genetic Analyzer (Applied Biosystems, Switzerland). To identify the bacterial species, the NCBI BLAST database was used for comparisons.
Cells and mice
The spleen cells of the C57BL/6 mice were prepared as described previously [9]. B220+ B cells were isolated from the spleen cells using a BDTM IMag Cell Separation System in accordance with the manufacturer’s instructions (Becton, Dickinson and Company). B220+ cells were recovered with a purity of >95%.
C57BL/6 mice (8 weeks old) were fed either a standard control diet (CE2, CLEA Japan, Inc.) or a diet supplemented with 1% Bacillus coagulans sc-09 for 3 weeks under SPF conditions. To investigate the effect of IL-22, recombinant mouse IL-22 (Tonbo) was administered to control mice by tail vein injection. IL-22 monoclonal antibodies (mAb; Thermo Fisher Scientific) were administered by tail vein injection to the mice fed the diet supplemented with 1% B. coagulans sc-09. The GF mice (C57BL/6NJcl; 8 weeks old) were either fed a standard control diet (CE2, CLEA Japan, Inc.) or a diet supplemented with 1% B. coagulans sc-09 for 4 weeks under an aseptic environment.
In vitro immunological assays
In vitro immunological assays were performed as described previously [5]. A total of 2 × 106 spleen cells were cultured in 1 mL of RPMI 1640 medium containing 10% FCS with or without 10 µg of bacterial cells for 2 days. Activation cell surface markers CD69 and CD86 on spleen cells were evaluated by flow cytometry. Viability was defined as the ration of viable cells to total cells and was determined as described previously [5]. The viability of total spleen cells in the control was 12.0% on average.
Cytokine assays
Spleen cells were cultured for 2 days at a concentration of 2 × 106 cells/mL in RPMI 1640 medium containing 10% FCS with or without 10 µg of bacterial cells. BD GolgiStopTM (in accordance with the manufacturer’s instructions; Becton, Dickinson and Company) was added to the medium at 6 hr before the end of the cultivation period. To measure intracellular cytokines, a BD Fixation/Permeabilization Solution Kit (Becton, Dickinson and Company) was used. Then, permeabilized cells were treated with phycoerythrin (PE)-labeled anti-IL-22 antibodies (clone 1H8PWSR, eBioscience). Cells were analyzed by flow cytometry. IL-22-positive cells in B220+ cells cultured without bacteria served as the control, and their number was defined as 100%. Based on this finding, the relative proportion of IL-22 positive cells cultured with bacteria was calculated as the relative IL-22 expression (%).
Flow cytometry
The cells were analyzed on a MACSQuant Flow Cytometer (Miltenyi Biotec) using the following antibodies: violetFluor™ 450-labeled anti-B220 antibodies (clone RA3-6B2) and APC-labeled anti-CD86 antibodies (clone GL-1) purchased from Tonbo Biosciences and Brilliant Violet 510TM anti-mouse CD4 antibodies (clone RM4-5) and PE-labeled anti-CD69 antibodies (clone H1.2F3) purchased from BioLegend. Dead cells were excluded using propidium iodide (PI) staining. Data analysis was conducted with FlowJo (FlowJo, LLC).
Evaluation of skin barrier function
Transepidermal water loss (TEWL) in mouse skin was measured using a DermaLab Combo system (Cortex Technology). TEWL measurements were recorded once the reading had stabilized at approximately 30 sec after the probe was placed on the skin.
Statistical analysis
Regarding the experimental date in Table 1, samples that had been measured one time and found to have increased were measured 1–6 more times, and the mean value and standard error (SE) were determined. Experimental data in Figs. 2, 3 are indicated as the mean ± SE. Experimental data in Figs. 4, 5 are indicated as the mean ± standard deviation (SD). Statistical significance was evaluated using a two-tailed Student’s t-test for unpaired data in Figs. 2, 3, 4b, and 5. The Tukey test was used for Fig. 4a. P values <0.05 were considered to be statistically significant.
Table 1. IL-22 production and CD86 expression in B cells caused by in vitro stimulation of bacteria.
| Strain | Relative IL-22 expression (%) | Relative CD86 expression (%) | n | ||||
|---|---|---|---|---|---|---|---|
| x– | SE | x– | SE | ||||
| Control | 100 | 100 | |||||
| Tetragenococcus halophilus | ta-01 | 100 | 255 | 1 | |||
| Tetragenococcus halophilus | ta-02 | 98 | 172 | 1 | |||
| Tetragenococcus halophilus | ta-03 | 97 | 172 | 1 | |||
| Tetragenococcus halophilus | ta-04 | 119 | 1 | 183 | 6 | 3 | |
| Tetragenococcus halophilus | ta-05 | 99 | 171 | 1 | |||
| Tetragenococcus halophilus | ta-06 | 100 | 192 | 1 | |||
| Tetragenococcus halophilus | ta-07 | 98 | 183 | 1 | |||
| Tetragenococcus halophilus | ta-08 | 109 | 191 | 1 | |||
| Tetragenococcus halophilus | ta-09 | 98 | 198 | 1 | |||
| Tetragenococcus halophilus | ta-10 | 96 | 170 | 1 | |||
| Tetragenococcus halophilus | ta-11 | 99 | 172 | 1 | |||
| Tetragenococcus halophilus | ta-12 | 102 | 178 | 1 | |||
| Tetragenococcus halophilus | ta-13 | 129 | 12 | 198 | 5 | 3 | |
| Tetragenococcus halophilus | ta-14 | 100 | 169 | 1 | |||
| Tetragenococcus halophilus | ta-15 | 95 | 166 | 1 | |||
| Tetragenococcus halophilus | ta-16 | 114 | 179 | 1 | |||
| Tetragenococcus halophilus | ta-17 | 99 | 172 | 1 | |||
| Tetragenococcus halophilus | ta-18 | 97 | 170 | 1 | |||
| Tetragenococcus halophilus | ta-19 | 112 | 158 | 1 | |||
| Tetragenococcus halophilus | ta-20 | 94 | 141 | 1 | |||
| Tetragenococcus halophilus | ta-21 | 138 | 13 | 162 | 12 | 3 | |
| Tetragenococcus halophilus | ta-22 | 95 | 137 | 1 | |||
| Tetragenococcus halophilus | ta-23 | 92 | 129 | 1 | |||
| Tetragenococcus halophilus | ta-24 | 94 | 144 | 1 | |||
| Tetragenococcus halophilus | ta-25 | 105 | 146 | 1 | |||
| Tetragenococcus halophilus | ta-26 | 100 | 153 | 1 | |||
| Tetragenococcus halophilus | ta-27 | 101 | 148 | 1 | |||
| Tetragenococcus halophilus | ta-28 | 100 | 105 | 1 | |||
| Tetragenococcus halophilus | ta-29 | 97 | 115 | 1 | |||
| Tetragenococcus halophilus | ta-30 | 91 | 104 | 1 | |||
| Tetragenococcus halophilus | ta-31 | 100 | 104 | 1 | |||
| Tetragenococcus halophilus | ta-32 | 97 | 106 | 1 | |||
| Tetragenococcus halophilus | ta-33 | 100 | 109 | 1 | |||
| Tetragenococcus halophilus | ta-34 | 111 | 155 | 1 | |||
| Tetragenococcus halophilus | ta-35 | 115 | 215 | 1 | |||
| Tetragenococcus halophilus | ta-36 | 106 | 179 | 1 | |||
| Tetragenococcus halophilus | ta-37 | 112 | 167 | 1 | |||
| Tetragenococcus halophilus | ta-38 | 123 | 1 | 168 | 15 | 3 | |
| Tetragenococcus halophilus | ta-39 | 107 | 156 | 1 | |||
| Tetragenococcus halophilus | ta-40 | 97 | 166 | 1 | |||
| Tetragenococcus halophilus | ta-41 | 99 | 169 | 1 | |||
| Tetragenococcus halophilus | ta-42 | 100 | 177 | 1 | |||
| Tetragenococcus halophilus | ta-43 | 97 | 162 | 1 | |||
| Tetragenococcus halophilus | ta-44 | 102 | 215 | 1 | |||
| Tetragenococcus halophilus | ta-45 | 97 | 138 | 1 | |||
| Tetragenococcus halophilus | ta-46 | 97 | 136 | 1 | |||
| Tetragenococcus halophilus | ta-47 | 100 | 189 | 1 | |||
| Tetragenococcus halophilus | ta-48 | 99 | 174 | 1 | |||
| Tetragenococcus halophilus | ta-49 | 137 | 3 | 235 | 8 | 3 | |
| Tetragenococcus halophilus | ta-50 | 118 | 111 | 1 | |||
| Tetragenococcus halophilus | ta-51 | 169 | 33 | 262 | 28 | 5 | |
| * | Tetragenococcus halophilus | ta-52 | 394 | 27 | 348 | 15 | 7 |
| Tetragenococcus halophilus | ta-53 | 91 | 256 | 1 | |||
| Tetragenococcus halophilus | ta-54 | 95 | 187 | 1 | |||
| Tetragenococcus halophilus | ta-55 | 96 | 158 | 1 | |||
| Tetragenococcus halophilus | ta-56 | 92 | 148 | 1 | |||
| Tetragenococcus halophilus | ta-57 | 90 | 221 | 1 | |||
| Tetragenococcus halophilus | ta-58 | 92 | 211 | 1 | |||
| Tetragenococcus halophilus | ta-59 | 93 | 169 | 1 | |||
| Tetragenococcus halophilus | ta-60 | 99 | 173 | 1 | |||
| Tetragenococcus halophilus | ta-61 | 94 | 165 | 1 | |||
| Tetragenococcus halophilus | ta-62 | 92 | 156 | 1 | |||
| Tetragenococcus halophilus | ta-63 | 91 | 187 | 1 | |||
| Tetragenococcus halophilus | ta-64 | 91 | 154 | 1 | |||
| Tetragenococcus halophilus | ta-65 | 90 | 171 | 1 | |||
| Tetragenococcus halophilus | ta-66 | 94 | 160 | 1 | |||
| Tetragenococcus halophilus | ta-67 | 94 | 142 | 1 | |||
| Tetragenococcus halophilus | ta-68 | 113 | 169 | 1 | |||
| Tetragenococcus halophilus | ta-69 | 90 | 143 | 1 | |||
| Tetragenococcus halophilus | ta-70 | 90 | 162 | 1 | |||
| Tetragenococcus halophilus | ta-71 | 92 | 156 | 1 | |||
| Tetragenococcus halophilus | ta-72 | 88 | 215 | 1 | |||
| Tetragenococcus halophilus | ta-73 | 92 | 149 | 1 | |||
| Tetragenococcus halophilus | ta-74 | 105 | 161 | 1 | |||
| Tetragenococcus halophilus | ta-75 | 93 | 186 | 1 | |||
| Tetragenococcus halophilus | ta-76 | 90 | 146 | 1 | |||
| Tetragenococcus halophilus | ta-77 | 90 | 167 | 1 | |||
| Tetragenococcus halophilus | ta-78 | 92 | 149 | 1 | |||
| Tetragenococcus halophilus | ta-79 | 90 | 143 | 1 | |||
| Tetragenococcus halophilus | ta-80 | 106 | 177 | 1 | |||
| Tetragenococcus halophilus | ta-81 | 94 | 147 | 1 | |||
| Tetragenococcus halophilus | ta-82 | 95 | 166 | 1 | |||
| Tetragenococcus halophilus | ta-83 | 108 | 199 | 1 | |||
| Tetragenococcus halophilus | ta-84 | 94 | 159 | 1 | |||
| Tetragenococcus halophilus | ta-85 | 93 | 240 | 1 | |||
| Tetragenococcus halophilus | ta-86 | 100 | 165 | 1 | |||
| Tetragenococcus halophilus | ta-87 | 92 | 160 | 1 | |||
| Tetragenococcus halophilus | ta-88 | 95 | 185 | 1 | |||
| Tetragenococcus halophilus | ta-89 | 98 | 148 | 1 | |||
| Tetragenococcus halophilus | ta-90 | 94 | 160 | 1 | |||
| Tetragenococcus halophilus | ta-91 | 94 | 162 | 1 | |||
| Tetragenococcus halophilus | ta-92 | 93 | 152 | 1 | |||
| Tetragenococcus halophilus | ta-93 | 99 | 166 | 1 | |||
| Tetragenococcus halophilus | ta-94 | 92 | 177 | 1 | |||
| Tetragenococcus halophilus | ta-95 | 92 | 178 | 1 | |||
| Enterococcus faecalis | fa-01 | 104 | 155 | 1 | |||
| Enterococcus faecalis | fa-02 | 123 | 3 | 155 | 7 | 3 | |
| Enterococcus faecalis | fa-03 | 110 | 153 | 1 | |||
| Enterococcus faecalis | fa-04 | 95 | 124 | 1 | |||
| Enterococcus faecalis | fa-05 | 100 | 121 | 1 | |||
| Enterococcus faecalis | fa-06 | 92 | 120 | 1 | |||
| Enterococcus faecalis | fa-07 | 102 | 107 | 1 | |||
| Enterococcus faecalis | fa-08 | 110 | 126 | 1 | |||
| Enterococcus faecalis | fa-09 | 113 | 137 | 1 | |||
| Enterococcus faecalis | fa-10 | 96 | 129 | 1 | |||
| Enterococcus faecalis | fa-11 | 112 | 139 | 1 | |||
| Enterococcus faecalis | fa-12 | 101 | 181 | 1 | |||
| Enterococcus faecium | fc-01 | 89 | 130 | 1 | |||
| Enterococcus faecium | fc-02 | 92 | 125 | 1 | |||
| Enterococcus faecium | fc-03 | 92 | 113 | 1 | |||
| Enterococcus faecium | fc-04 | 93 | 111 | 1 | |||
| Enterococcus faecium | fc-05 | 90 | 115 | 1 | |||
| Enterococcus faecium | fc-06 | 94 | 111 | 1 | |||
| Enterococcus faecium | fc-07 | 90 | 115 | 1 | |||
| Enterococcus faecium | fc-08 | 90 | 115 | 1 | |||
| Enterococcus faecium | fc-09 | 92 | 113 | 1 | |||
| Enterococcus faecium | fc-10 | 89 | 116 | 1 | |||
| Enterococcus faecium | fc-11 | 98 | 135 | 1 | |||
| Enterococcus faecium | fc-12 | 108 | 143 | 1 | |||
| Enterococcus faecium | fc-13 | 95 | 131 | 1 | |||
| Enterococcus faecium | fc-14 | 94 | 126 | 1 | |||
| Enterococcus faecium | fc-15 | 96 | 132 | 1 | |||
| Enterococcus faecium | fc-16 | 99 | 134 | 1 | |||
| Enterococcus faecium | fc-17 | 121 | 6 | 170 | 7 | 3 | |
| Enterococcus faecium | fc-18 | 99 | 160 | 1 | |||
| Enterococcus faecium | fc-19 | 134 | 4 | 188 | 12 | 3 | |
| Enterococcus faecium | fc-20 | 141 | 3 | 158 | 6 | 3 | |
| Enterococcus faecium | fc-21 | 99 | 138 | 1 | |||
| Enterococcus faecium | fc-22 | 98 | 131 | 1 | |||
| Enterococcus faecium | fc-23 | 119 | 142 | 1 | |||
| * | Enterococcus faecium | fc-24 | 215 | 19 | 197 | 9 | 7 |
| Enterococcus faecium | fc-25 | 111 | 146 | 1 | |||
| Enterococcus faecium | fc-26 | 106 | 155 | 1 | |||
| Enterococcus faecium | fc-27 | 95 | 136 | 1 | |||
| Lactobacillus acidipiscis | lb-01 | 218 | 30 | 343 | 102 | 3 | |
| Lactobacillus acidipiscis | lb-02 | 217 | 17 | 221 | 11 | 3 | |
| Lactobacillus acidipiscis | lb-03 | 243 | 48 | 392 | 54 | 3 | |
| Lactobacillus brevis | lb-04 | 190 | 53 | 218 | 18 | 3 | |
| Lactobacillus brevis | lb-05 | 115 | 225 | 1 | |||
| Lactobacillus brevis | lb-06 | 90 | 197 | 1 | |||
| Lactobacillus brevis | lb-07 | 100 | 218 | 1 | |||
| Lactobacillus brevis | lb-08 | 96 | 194 | 1 | |||
| Lactobacillus brevis | lb-09 | 88 | 169 | 1 | |||
| Lactobacillus brevis | lb-10 | 118 | 190 | 1 | |||
| Lactobacillus brevis | lb-11 | 209 | 39 | 166 | 17 | 3 | |
| Lactobacillus buchneri | lb-12 | 240 | 27 | 147 | 8 | 3 | |
| Lactobacillus buchneri | lb-13 | 89 | 177 | 1 | |||
| Lactobacillus casei | lb-14 | 92 | 206 | 1 | |||
| Lactobacillus casei | lb-15 | 93 | 316 | 1 | |||
| Lactobacillus casei | lb-16 | 89 | 205 | 1 | |||
| Lactobacillus casei | lb-17 | 97 | 157 | 1 | |||
| Lactobacillus curvatus | lb-18 | 96 | 198 | 1 | |||
| Lactobacillus fermentum | lb-19 | 100 | 215 | 1 | |||
| Lactobacillus fermentum | lb-20 | 102 | 223 | 1 | |||
| Lactobacillus fermentum | lb-21 | 94 | 193 | 1 | |||
| Lactobacillus fermentum | lb-22 | 96 | 208 | 1 | |||
| Lactobacillus fermentum | lb-23 | 95 | 193 | 1 | |||
| Lactobacillus fermentum | lb-24 | 96 | 197 | 1 | |||
| Lactobacillus fermentum | lb-25 | 156 | 39 | 228 | 53 | 2 | |
| Lactobacillus fermentum | lb-26 | 90 | 162 | 1 | |||
| Lactobacillus fructivorans | lb-27 | 89 | 190 | 1 | |||
| Lactobacillus fructivorans | lb-28 | 112 | 291 | 1 | |||
| Lactobacillus fructivorans | lb-29 | 104 | 192 | 1 | |||
| Lactobacillus fructivorans | lb-30 | 96 | 297 | 1 | |||
| Lactobacillus fructivorans | lb-31 | 95 | 312 | 1 | |||
| Lactobacillus fructivorans | lb-32 | 171 | 35 | 352 | 40 | 3 | |
| Lactobacillus helveticus | lb-33 | 91 | 122 | 1 | |||
| Lactobacillus helveticus | lb-34 | 210 | 9 | 184 | 38 | 3 | |
| Lactobacillus paracasei | lb-35 | 91 | 167 | 1 | |||
| Lactobacillus paracasei | lb-36 | 94 | 179 | 1 | |||
| Lactobacillus pentosus | lb-37 | 98 | 195 | 1 | |||
| Lactobacillus pentosus | lb-38 | 100 | 167 | 1 | |||
| Lactobacillus plantarum | lb-39 | 108 | 130 | 1 | |||
| Lactobacillus plantarum | lb-40 | 96 | 218 | 1 | |||
| Lactobacillus plantarum | lb-41 | 111 | 186 | 1 | |||
| Lactobacillus plantarum | lb-42 | 95 | 156 | 1 | |||
| Lactobacillus plantarum | lb-43 | 124 | 28 | 197 | 4 | 3 | |
| Lactobacillus plantarum | lb-44 | 102 | 212 | 1 | |||
| Lactobacillus plantarum | lb-45 | 122 | 218 | 1 | |||
| Lactobacillus plantarum | lb-46 | 112 | 182 | 1 | |||
| Lactobacillus plantarum | lb-47 | 96 | 185 | 1 | |||
| Lactobacillus plantarum | lb-48 | 92 | 266 | 1 | |||
| Lactobacillus plantarum | lb-49 | 92 | 175 | 1 | |||
| Lactobacillus plantarum | lb-50 | 94 | 181 | 1 | |||
| Lactobacillus plantarum | lb-51 | 114 | 121 | 1 | |||
| Lactobacillus plantarum | lb-52 | 95 | 163 | 1 | |||
| Lactobacillus plantarum | lb-53 | 91 | 174 | 1 | |||
| Lactobacillus rhamnosus | lb-56 | 102 | 262 | 1 | |||
| Lactobacillus sakei | lb-58 | 110 | 193 | 1 | |||
| Lactobacillus sakei | lb-59 | 92 | 193 | 1 | |||
| Lactobacillus sp. | lb-60 | 105 | 232 | 1 | |||
| Lactobacillus sp. | lb-61 | 155 | 6 | 166 | 35 | 3 | |
| Lactococcus lactis | lc-01 | 118 | 226 | 1 | |||
| Lactococcus lactis | lc-02 | 100 | 217 | 1 | |||
| Lactococcus lactis | lc-03 | 126 | 202 | 1 | |||
| Lactococcus lactis | lc-04 | 108 | 145 | 1 | |||
| Lactococcus lactis | lc-05 | 97 | 222 | 1 | |||
| Lactococcus lactis | lc-06 | 100 | 200 | 1 | |||
| Lactococcus lactis | lc-07 | 98 | 187 | 1 | |||
| Lactococcus plantarum | lc-08 | 138 | 12 | 282 | 101 | 3 | |
| Leuconostoc citreum | ls-01 | 98 | 231 | 1 | |||
| Leuconostoc citreum | ls-02 | 102 | 142 | 1 | |||
| Leuconostoc citreum | ls-03 | 94 | 205 | 1 | |||
| Leuconostoc citreum | ls-04 | 94 | 237 | 1 | |||
| Leuconostoc mesenteroides | ls-05 | 96 | 183 | 1 | |||
| Leuconostoc mesenteroides | ls-06 | 98 | 201 | 1 | |||
| Leuconostoc mesenteroides | ls-07 | 99 | 211 | 1 | |||
| Leuconostoc mesenteroides | ls-08 | 97 | 185 | 1 | |||
| L. pseudomesenteroides | ls-09 | 93 | 205 | 1 | |||
| L. pseudomesenteroides | ls-10 | 98 | 188 | 1 | |||
| Pediococcus acidilactici | pc-01 | 128 | 212 | 1 | |||
| Pediococcus acidilactici | pc-02 | 142 | 166 | 1 | |||
| Pediococcus acidilactici | pc-03 | 183 | 227 | 1 | |||
| Pediococcus acidilactici | pc-04 | 147 | 186 | 1 | |||
| Pediococcus acidilactici | pc-05 | 142 | 247 | 1 | |||
| Pediococcus acidilactici | pc-06 | 165 | 122 | 1 | |||
| Pediococcus acidilactici | pc-07 | 217 | 33 | 250 | 23 | 3 | |
| Pediococcus acidilactici | pc-08 | 128 | 242 | 1 | |||
| Pediococcus acidilactici | pc-09 | 146 | 200 | 1 | |||
| Pediococcus acidilactici | pc-10 | 122 | 279 | 1 | |||
| Pediococcus acidilactici | pc-11 | 167 | 321 | 1 | |||
| Pediococcus acidilactici | pc-12 | 128 | 296 | 1 | |||
| Pediococcus acidilactici | pc-13 | 110 | 167 | 1 | |||
| Pediococcus acidilactici | pc-14 | 146 | 204 | 1 | |||
| Pediococcus acidilactici | pc-15 | 171 | 234 | 1 | |||
| Pediococcus acidilactici | pc-16 | 151 | 208 | 1 | |||
| Pediococcus acidilactici | pc-17 | 286 | 34 | 237 | 52 | 3 | |
| Pediococcus acidilactici | pc-18 | 147 | 282 | 1 | |||
| * | Pediococcus acidilactici | pc-19 | 438 | 54 | 284 | 25 | 7 |
| Pediococcus acidilactici | pc-20 | 193 | 22 | 308 | 62 | 3 | |
| Pediococcus acidilactici | pc-21 | 149 | 370 | 1 | |||
| Pediococcus acidilactici | pc-22 | 94 | 230 | 1 | |||
| Pediococcus acidilactici | pc-23 | 105 | 263 | 1 | |||
| Pediococcus acidilactici | pc-24 | 104 | 244 | 1 | |||
| Pediococcus acidilactici | pc-25 | 245 | 7 | 299 | 90 | 3 | |
| Pediococcus acidilactici | pc-26 | 248 | 16 | 186 | 16 | 3 | |
| Pediococcus acidilactici | pc-27 | 107 | 222 | 1 | |||
| Pediococcus acidilactici | pc-28 | 97 | 253 | 1 | |||
| Pediococcus acidilactici | pc-29 | 166 | 253 | 1 | |||
| Pediococcus acidilactici | pc-30 | 110 | 276 | 1 | |||
| Pediococcus acidilactici | pc-31 | 114 | 232 | 1 | |||
| Pediococcus acidilactici | pc-32 | 95 | 253 | 1 | |||
| Pediococcus acidilactici | pc-33 | 103 | 300 | 1 | |||
| Pediococcus acidilactici | pc-34 | 93 | 201 | 1 | |||
| Pediococcus acidilactici | pc-35 | 97 | 209 | 1 | |||
| Pediococcus acidilactici | pc-36 | 99 | 265 | 1 | |||
| Pediococcus acidilactici | pc-37 | 87 | 173 | 1 | |||
| Pediococcus acidilactici | pc-38 | 225 | 28 | 447 | 99 | 3 | |
| Pediococcus acidilactici | pc-39 | 212 | 22 | 404 | 138 | 3 | |
| Pediococcus dextrinicus | pc-40 | 152 | 230 | 1 | |||
| Pediococcus pentosaceus | pc-41 | 119 | 171 | 1 | |||
| Pediococcus pentosaceus | pc-42 | 212 | 24 | 195 | 36 | 3 | |
| Pediococcus pentosaceus | pc-43 | 136 | 176 | 1 | |||
| Pediococcus pentosaceus | pc-44 | 139 | 239 | 1 | |||
| Pediococcus pentosaceus | pc-45 | 148 | 168 | 1 | |||
| Pediococcus pentosaceus | pc-46 | 104 | 175 | 1 | |||
| Pediococcus pentosaceus | pc-47 | 140 | 159 | 1 | |||
| Pediococcus pentosaceus | pc-48 | 165 | 195 | 1 | |||
| Pediococcus pentosaceus | pc-49 | 90 | 145 | 1 | |||
| Pediococcus pentosaceus | pc-50 | 134 | 112 | 1 | |||
| Pediococcus pentosaceus | pc-51 | 91 | 189 | 1 | |||
| Pediococcus pentosaceus | pc-52 | 197 | 11 | 158 | 25 | 3 | |
| Pediococcus pentosaceus | pc-53 | 141 | 95 | 1 | |||
| Pediococcus pentosaceus | pc-54 | 120 | 201 | 1 | |||
| Pediococcus pentosaceus | pc-55 | 99 | 163 | 1 | |||
| Pediococcus pentosaceus | pc-56 | 158 | 225 | 1 | |||
| Pediococcus pentosaceus | pc-57 | 103 | 190 | 1 | |||
| Pediococcus pentosaceus | pc-58 | 98 | 171 | 1 | |||
| Pediococcus pentosaceus | pc-59 | 119 | 215 | 1 | |||
| Pediococcus pentosaceus | pc-60 | 90 | 156 | 1 | |||
| Pediococcus pentosaceus | pc-61 | 151 | 223 | 1 | |||
| Pediococcus pentosaceus | pc-62 | 98 | 172 | 1 | |||
| Pediococcus pentosaceus | pc-63 | 116 | 211 | 1 | |||
| Pediococcus stilesii | pc-64 | 92 | 179 | 1 | |||
| Weissella cibaria | ws-01 | 111 | 177 | 1 | |||
| Weissella cibaria | ws-02 | 131 | 178 | 1 | |||
| Weissella cibaria | ws-03 | 138 | 235 | 1 | |||
| Weissella confusa | ws-04 | 107 | 241 | 1 | |||
| Weissella confusa | ws-05 | 135 | 210 | 1 | |||
| Weissella confusa | ws-06 | 161 | 7 | 322 | 67 | 3 | |
| Weissella halotolerans | ws-07 | 119 | 186 | 1 | |||
| Weissella hellenica | ws-08 | 102 | 197 | 1 | |||
| Weissella mesenteroides | ws-09 | 100 | 179 | 1 | |||
| Weissella paramesenteroides | ws-10 | 134 | 140 | 1 | |||
| Weissella paramesenteroides | ws-11 | 115 | 112 | 1 | |||
| Weissella paramesenteroides | ws-12 | 86 | 159 | 1 | |||
| Weissella paramesenteroides | ws-13 | 122 | 184 | 1 | |||
| Weissella paramesenteroides | ws-14 | 123 | 171 | 1 | |||
| Weissella paramesenteroides | ws-15 | 122 | 179 | 1 | |||
| Weissella paramesenteroides | ws-16 | 115 | 160 | 1 | |||
| Weissella paramesenteroides | ws-17 | 142 | 194 | 1 | |||
| Weissella paramesenteroides | ws-18 | 194 | 11 | 215 | 32 | 3 | |
| Weissella soli | ws-19 | 124 | 134 | 1 | |||
| Weissella viridescens | ws-20 | 134 | 178 | 1 | |||
| Weissella viridescens | ws-21 | 138 | 167 | 1 | |||
| Bacillus coagulans | sc-01 | 376 | 70 | 555 | 46 | 3 | |
| Bacillus coagulans | sc-02 | 168 | 20 | 262 | 58 | 3 | |
| Bacillus coagulans | sc-03 | 179 | 43 | 441 | 29 | 3 | |
| Bacillus coagulans | sc-04 | 243 | 22 | 688 | 48 | 3 | |
| Bacillus coagulans | sc-05 | 334 | 75 | 404 | 19 | 5 | |
| Bacillus coagulans | sc-06 | 423 | 54 | 414 | 17 | 5 | |
| Bacillus coagulans | sc-07 | 204 | 28 | 392 | 27 | 3 | |
| Bacillus coagulans | sc-08 | 444 | 104 | 385 | 17 | 5 | |
| * | Bacillus coagulans | sc-09 | 1,062 | 158 | 501 | 53 | 7 |
| Bacillus coagulans | sc-10 | 338 | 75 | 400 | 8 | 5 | |
| Bacillus coagulans | sc-11 | 253 | 16 | 417 | 27 | 3 | |
| Bacillus coagulans | sc-12 | 419 | 90 | 371 | 18 | 5 | |
| Bacillus coagulans | sc-13 | 143 | 19 | 364 | 27 | 3 | |
| Bacillus coagulans | sc-14 | 332 | 54 | 343 | 31 | 5 | |
| Bacillus coagulans | sc-15 | 309 | 41 | 353 | 17 | 5 | |
| Bacillus coagulans | sc-16 | 249 | 29 | 330 | 20 | 5 | |
| Bacillus coagulans | sc-17 | 131 | 10 | 182 | 58 | 3 | |
| Bacillus coagulans | sc-18 | 376 | 35 | 493 | 25 | 3 | |
| Bacillus coagulans | sc-19 | 349 | 31 | 474 | 28 | 3 | |
| Bacillus coagulans | sc-20 | 480 | 100 | 415 | 4 | 5 | |
| Bacillus subtilis | bs-01 | 282 | 525 | 1 | |||
| Bacillus subtilis | bs-02 | 355 | 474 | 1 | |||
| Bacillus subtilis | bs-03 | 236 | 485 | 1 | |||
| Bacillus subtilis | bs-04 | 176 | 561 | 1 | |||
| Bacillus subtilis | bs-05 | 457 | 37 | 460 | 90 | 4 | |
| Bacillus subtilis | bs-06 | 271 | 434 | 1 | |||
| Bacillus subtilis | bs-07 | 427 | 31 | 321 | 18 | 4 | |
| Bacillus subtilis | bs-08 | 353 | 453 | 1 | |||
| Bacillus subtilis | bs-09 | 230 | 333 | 1 | |||
| Bacillus subtilis | bs-10 | 245 | 215 | 1 | |||
| Bacillus subtilis | bs-11 | 154 | 248 | 1 | |||
| Bacillus subtilis | bs-12 | 332 | 471 | 1 | |||
| Bacillus subtilis | bs-13 | 218 | 553 | 1 | |||
| Bacillus subtilis | bs-14 | 135 | 206 | 1 | |||
| Bacillus subtilis | bs-15 | 262 | 453 | 1 | |||
| Bacillus subtilis | bs-16 | 252 | 622 | 1 | |||
| Bacillus subtilis | bs-17 | 245 | 475 | 1 | |||
| Bacillus subtilis | bs-18 | 262 | 526 | 1 | |||
| Bacillus subtilis | bs-19 | 298 | 311 | 1 | |||
| Bacillus subtilis | bs-20 | 193 | 424 | 1 | |||
| Bacillus subtilis | bs-21 | 133 | 366 | 1 | |||
| Bacillus subtilis | bs-22 | 178 | 535 | 1 | |||
| Bacillus subtilis | bs-23 | 145 | 307 | 1 | |||
| Bacillus subtilis | bs-24 | 223 | 378 | 1 | |||
| Bacillus subtilis | bs-25 | 372 | 74 | 437 | 51 | 4 | |
| Bacillus subtilis | bs-26 | 211 | 282 | 1 | |||
| Bacillus subtilis | bs-27 | 161 | 436 | 1 | |||
| Bacillus subtilis | bs-28 | 159 | 369 | 1 | |||
| Bacillus subtilis | bs-29 | 166 | 470 | 1 | |||
| * | Bacillus subtilis | bs-30 | 766 | 55 | 430 | 96 | 4 |
| Bacillus subtilis | bs-31 | 118 | 280 | 1 | |||
| Bacillus subtilis | bs-32 | 267 | 325 | 1 | |||
| Bacillus subtilis | bs-33 | 147 | 341 | 1 | |||
| * | Bacillus subtilis | bs-34 | 971 | 53 | 495 | 38 | 4 |
| Bacillus subtilis | bs-35 | 171 | 262 | 1 | |||
| Bacillus subtilis | bs-36 | 494 | 54 | 655 | 96 | 4 | |
| Bacillus subtilis | bs-37 | 176 | 561 | 1 | |||
| Bacillus subtilis | bs-38 | 275 | 449 | 1 | |||
| Bacillus subtilis | bs-39 | 513 | 68 | 294 | 44 | 4 | |
| Bacillus subtilis | bs-40 | 249 | 496 | 1 | |||
| Bacillus amyloliquefaciens | bi-01 | 369 | 499 | 1 | |||
| Bacillus amyloliquefaciens | bi-02 | 213 | 422 | 1 | |||
| Bacillus benzoevorans | bi-03 | 348 | 473 | 1 | |||
| Bacillus benzoevorans | bi-04 | 228 | 304 | 1 | |||
| Bacillus firmus | bi-05 | 161 | 189 | 1 | |||
| Bacillus megaterium | bi-06 | 148 | 209 | 1 | |||
| Bacillus megaterium | bi-07 | 176 | 412 | 1 | |||
| Bacillus megaterium | bi-08 | 267 | 434 | 1 | |||
| Bacillus megaterium | bi-09 | 264 | 270 | 1 | |||
| Bacillus novalis | bi-10 | 351 | 507 | 1 | |||
| Bacillus pumilus | bi-11 | 264 | 522 | 1 | |||
| Bacillus tequilensis | bi-12 | 219 | 378 | 1 | |||
x–: mean value; SE: standard error; n: number. The strains with high values are shown in bold and marked with an asterisk.
Fig. 2.
CD86 expression on B cells and CD69 expression on T cells cultured with bacterial strains.
The spleen cells from C57BL/6 mice were cultured with 10 µg of bacterial cells inducing high IL-22 production in 1 mL of RPMI 1640 medium containing 10% FCS for 2 days. The cells were collected and stained with anti-B220, anti-CD4, anti-CD69, and anti-CD86 mAb. Dead cells were stained with PI. The cells were analyzed by flow cytometry. (A–C) The viabilities of total spleen cells (A), B220+ cells (B), and CD4+ cells (C) cultured without bacterial cells, which served as controls, were defined as 100%. Based on this parameter, the relative viabilities of cells cultured with bacteria were calculated. Bars indicate the mean ± SE (n=6). (D, E) The CD86+ cells in B220+ cells and CD69+ cells in CD4+ cells cultured without bacteria served as controls, and their numbers were defined as 100%. Accordingly, the relative proportions of CD86+ cells and CD69+ cells, respectively, in B220+ cells (D) and CD4+ cells (E) cultured with bacteria were calculated. Bars indicate the mean ± SE (n=6). *p<0.05 vs. control by t-test. **p<0.01 vs. control by t-test. ***p<0.001 vs. control by t-test.
Fig. 3.
CD86 expression and IL-22 production in B cells cultured with bacterial strains.
Spleen B220+ cells prepared from C57BL/6 mice were cultured with 10 µg of bacterial cells that highly induced IL-22 production in 1 mL of RPMI 1640 medium containing 10% FCS for 2 days. The cells were collected and stained with anti-B220 and anti-CD86 mAb. Dead cells were stained with PI. The cells were analyzed by flow cytometry. Viability was assessed (A), and the viability of CD86+ cells in B220+ cells (B) cultured without bacterial cells, which served as control, was defined as 100%. On the basis of this parameter, the relative viability of cells and the relative CD86 expression of cells cultured with bacteria were calculated. Bars indicate the mean ± SE (n=4). (C) Cells cultured for 2 days were further incubated with GolgiStop and then collected and treated using a BD Fixation/Permeabilization Solution Kit. Subsequently, cells were stained and analyzed by flow cytometry. IL-22-positive cells in B220+ cells cultured without bacteria served as the control (0.08%), and their number was defined as 100%. Based on this parameter, the relative IL-22 expression of cells cultured with bacteria was calculated. Bars indicate the mean ± SE (n=4). *p<0.05 vs. control by t-test. **p<0.01 vs. control by t-test. ***p<0.001 vs. control by t-test.
Fig. 4.
Effect of B. coagulans sc-09 on murine skin barrier.
(A) C57BL/6 mice were divided into four groups (n=3 mice/group), with two groups fed a diet containing 1% B. coagulans sc-09 for 3 weeks and two groups fed a diet without B. coagulans. One group was specifically fed a diet containing 1% B. coagulans sc-09 and intravenously injected with the IL-22 antibody (20 µg/body) on the 14th and 17th days of feeding, respectively. The other group fed a diet without B. coagulans was intravenously injected with recombinant mouse IL-22 (2 µg/body) on the 14th and 17th days of feeding, respectively. On the 20th day of feeding, the backs of the mice were shaved, and on the 21st day, the TEWL of the skin on the back of the mice was measured (n=4). Bars indicate the mean ± SD of triplicate experiments. *p<0.05 vs. control by Tukey test. (B) Effect of B. coagulans sc-09 on skin barrier in GF mice. Diet containing 1% B. coagulans sc-09 was fed to GF mice. After feeding for 4 weeks in an aseptic environment, the TEWL of the back skin of the mice was measured. Just before the measurement, the hair on the backs of the mice was cut with clippers. Measurement of TEWL was performed four times each. Bars indicate the mean ± SD (n=5 mice). *p<0.05 vs. control by t-test.
Fig. 5.
Effect of B. coagulans sc-09 on IL-22 production in the Peyer’s patches and mesenteric lymph nodes in mice.
Diet containing 1% B. coagulans sc-09 was fed to C57BL/6 mice for 3 weeks (n=3 mice/group). Then, cells were collected from Peyer’s patches and mesenteric lymph nodes, and the percentages of IL-22-producing cells in B cells were analyzed by FACS. Mice fed without B. coagulans sc-09 were used as the control. Bars indicate the mean ± SD for Peyer’s patches (A) and mesenteric lymph nodes (B). The p values in A and B are 0.245 and 0.265, respectively.
RESULTS
Screening of IL-22-inducing bacteria in B cells
We isolated 367 bacteria from Japanese fermented foods, such as miso, soy sauce, and amazake. We collected 95 Tetragenococcus, 39 Enterococcus, 58 Lactobacillus, 8 Lactococcus, 10 Leuconostoc, 64 Pediococcus, 21 Weissella, and 72 Bacillus bacterial isolates. To evaluate the ability of these bacteria in inducing IL-22 production in immune cells, we established an in vitro immunological assay using mouse spleen cells [5]. The ability to induce IL-22 production was distinct for each bacterial species (Table 1 and Fig. 1). Most Tetragenococcus, Enterococcus, Lactococcus, Leuconostoc, and Weissella bacterial strains did not enhance the induction of IL-22 production. Lactobacillus and Pediococcus strains possessed higher abilities to induce IL-22 production than these lactic acid bacterial strains. Additionally, most of the Bacillus strains had higher abilities to induce IL-22 production than the lactic acid bacteria; B. coagulans sc-09, which was isolated from miso, had the highest ability to induce IL-22 production. B. subtilis bs-30 and bs-34 also possessed high IL-22-inducing ability. High IL-22-inducing bacterial strains also augmented activation marker CD86 on B cells. However, their abilities were not always proportional, suggesting that their inducing mechanisms were different.
Fig. 1.
Bacteria capable of inducing IL-22 and CD86.
The results in Table 1 are expressed in a column scatter plot. (A) Relative IL-22 expression in B cells. (B) Relative CD86 expression on B cells. The plots in the figure are divided according to category of bacteria, such as Tetragenococcus and Lactobacillus, and the relative value of each bacterium is plotted. The median value of each category is indicated by a bar.
Activation of B and T cells by IL-22-inducing bacterial strains
As shown in Table 1, the strains with high ability to induce IL-22 production also activated B cells. We assessed if six strains (T. halophilus ta-52, Enterococcus faecium fc-24, Pediococcus acidilactici pc-19, B. coagulans sc-09, B. subtilis bs-30, and B. subtilis bs-34) played a role in survival and activation of B and T cells based on activation markers, such as CD86 on B cells and CD69 on T cells, and determined their cell viability. All the strains augmented the viability of splenocytes, including B and T cells (Fig. 2A–C), and significantly increased CD86 expression on B cells and CD69 expression on CD4+ T cells (Fig. 2D and E). These results suggest that all tested strains activated B and CD4+ T cells and induced IL-22 in B cells.
Next, we examined whether the effect of these strains on IL-22 induction in B cells was direct or indirect. We isolated B cells, treated them with bacteria, and measured IL-22 production. As shown in Fig. 3A and B, these strains increased CD86 expression and B cell viability. In addition, IL-22 production similarly increased (Fig. 3C) in consistency with the results presented in Table 1. Among these strains, B. coagulans sc-09 most efficiently induced IL-22-producing B cells. This result indicates that these bacterial strains directly induce IL-22 production in B cells.
B. coagulans sc-09 augments skin barrier function independent of commensal bacteria
We examined the influence on skin barrier function by feeding mice B. coagulans sc-09. Specifically, we fed the mice 1% B. coagulans sc-09 for 3 weeks and measured TEWL. TEWL was significantly reduced in the skin of B. coagulans sc-09–fed mice as compared with that of the control mice (Fig. 4A). When the IL-22 mAb was administered to the B. coagulans sc-09–fed mice, TEWL increased and became significantly higher than that in the control mice. In contrast, TEWL significantly decreased in the control mice administered IL-22 by intravenous injection.
To determine whether this function is mediated by commensal bacteria, we utilized GF mice. We fed 1% B. coagulans sc-09 to GF mice for 4 weeks and measured TEWL. As shown in Fig. 4B, even in experiments with GF mice, TEWL significantly decreased in the skin of B. coagulans sc-09-fed mice as compared with that of the control mice. This decrease in TEWL in the B. coagulans sc-09-fed GF mice shows that skin barrier function is independent of commensal bacteria. These results indicate that B. coagulans sc-09 is effective in enhancing skin barrier function.
We examined the effect of B. coagulans sc-09 on IL-22 production in SPF mice. IL-22-producing B cells (Bi22 cells) in Peyer’s patches and mesenteric lymph nodes of B. coagulans sc-09-fed mice tended to be increased in comparison with those of control mice (Fig. 5), suggesting that B. coagulans sc-09-mediated IL-22 production contributed to the skin barrier function.
DISCUSSION
In this study, we screened bacteria from Japanese fermented foods for their ability to induce IL-22 production in B cells. We found that the ability to induce IL-22 production is dependent on the bacterial species, and Bacillus bacterial strains possessed high IL-22 induction potency. Among these strains, B. coagulans sc-09 was the highest IL-22 induction strain and had the ability to improve skin barrier function in vivo.
TEWL measurement is often used as an indicator for evaluating skin barrier function [10]. Because administration of IL-22 decreased TEWL and neutralization of IL-22 increased TEWL, the improvement of skin barrier function caused by B. coagulans sc-09 uptake may be attributed to IL-22. Although the significance of IL-22 produced by B cells is unknown, increased IL-22 may facilitate skin barrier function [7]. Our results suggest that IL-22-inducing bacteria have immunomodulatory abilities in addition to enhancement of skin barrier function.
Strains with high abilities to induce IL-22 production also possessed high abilities to activate B cells (Fig. 3); however, these capabilities were not directly proportional to each other (Table 1). Furthermore, only some subpopulations of activated B cells seemed to differentiate into IL-22-producing B cells (Bi22), as Bi22 cells are a minor population in B220+ B cells. This finding suggests that B cell activation and IL-22 induction are distinctly regulated. In our previous report [5], we showed that T. halophilus No. 1 induced multiple subsets in B cells similar to Th cells exist. Thus, some of the microorganisms harboring B cell activation ability may promote differentiation into a subset of B cells producing IL-22.
IL-22 is highly expressed in the skin and digestive and respiratory organs [7]. In the skin and intestines, IL-22 induces the production of antibacterial peptides and is considered to be involved in pathogen defense. Recently, reports have shown that Lactobacillus plantarum stimulation of NKs can enhance IL-22 production and defend against enterotoxigenic Escherichia coli-induced damage of the intestinal epithelial barrier [11]. Thus, IL-22-inducing bacteria including B. coagulans sc-09 may act on the barrier function of the intestinal tract, although IL-22 is produced in various types of immune cells.
Here, we found that B. coagulans sc-09 has a strong IL-22-inducing function in B cells. B. coagulans is a spore-forming bacterium that produces lactic acid. B. coagulans spores are probiotics and have beneficial effects in humans, such as amelioration of irritable bowel syndrome [12, 13], bacterial vaginosis [14], and intestinal disorders [15,16,17], and absorption of amino acids from proteins [18, 19]. In addition, their use in broilers and fish yields growth-promoting and disease-preventing effects [20, 21]. B. coagulans sc-09 isolated from miso appears to be a probiotic that improves skin barrier function and modulates immune function. Beneficial effects of B. coagulans on IL-22 induction in immune cells appear to contribute to human health when it is supplied as an ingredient in foods and supplements.
Acknowledgments
We are grateful to Ms. H. Iijima and Y. Mori for technical assistance. This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.A.) and by grants from the Canon Foundation (to T.A.).
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