The molecular mechanism for activating IgA production by Pediococcus acidilactici K15 and the clinical impact in a randomized trial

IgA secretion at mucosal sites is important for host defence against pathogens as well as maintaining the symbiosis with microorganisms present in the small intestine that affect IgA production. In the present study, we tested the ability of 5 strains of lactic acid bacteria stimulating IgA production, being Pediococcus acidilactici K15 selected as the most effective on inducing this protective immunoglobulin. We found that this response was mainly induced via IL-10, as efficiently as IL-6, secreted by K15-stimulated dendritic cells. Furthermore, bacterial RNA was largely responsible for the induction of these cytokines; double-stranded RNA was a major causative molecule for IL-6 production whereas single-stranded RNA was critical factor for IL-10 production. In a randomized, double-blind, placebo-controlled clinical trial, ingestion of K15 significantly increased the secretory IgA (sIgA) concentration in saliva compared with the basal level observed before this intervention. These results indicate that functional lactic acid bacteria induce IL-6 and IL-10 production by dendritic cells, which contribute to upregulating the sIgA concentration at mucosal sites in humans.


Results
IgA secretion by PBMCs in response to LAB via IL-6 and IL-10. We evaluated the IgA secretion by PBMCs from 7 donors in response to some strains of LAB (described in Table S1). Among tested strains of LAB, Pediococcus acidilactici K15 significantly induced IgA production in PBMCs (Fig. 1a). The other strains of LAB failed to enhance IgA production in this experimental system (Fig. 1a). B lymphocyte-induced maturation protein 1 (Blimp-1) is a master regulator for plasma cell differentiation 27 , and B cell activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) are both known to promote this class switching [28][29][30] . In K15-stimulated PBMCs, the gene expression encoding Blimp-1 and APRIL was upregulated, whereas that encoding BAFF did not change, in comparison with unstimulated PBMCs (Fig. 1b). Reportedly, IgA secretion by B cells is activated by IL-5 or IL-6 in mouse PBMCs 17,31 and by IL-6 and IL-10 in human PBMCs 32 . These cytokines promote the differentiation of IgA-producing plasma cells. We performed experiments in the presence of neutralizing Abs against IL-5, IL-6, and IL-10 to clarify which factor is involved in IgA production by human PBMCs in response to K15. Although a neutralizing Ab against IL-5 did not affect IgA secretion in response to K15 or to TLR ligands, both neutralizing Abs against IL-6 and IL-10 impaired IgA induction by their respective cytokines (Fig. 1c). As IL-5 was not involved in the induction of IgA secretion, the induction of IgA by LAB-stimulation likely occurs via a T cell-independent pathway. These results indicate that, in human PBMCs, the effects of LAB on activating IgA production are induced by IL-6 and IL-10, most likely secreted by DCs in response to LAB.
IgA production by B cells via the DC response to LAB. LAB contain many kinds of TLR ligands, such as bacterial cell walls, which are ligands for TLR2 and TLR4, dsRNA, which is a ligand of TLR3, and DNA, which is a ligand of TLR9 14,[33][34][35][36] . DCs are activated by these TLR ligands, and these cells, once activated, secrete many kinds of cytokines and IFNs. We isolated BDCA1 + DCs (mDC1s) and B cells from PBMCs and co-cultured them in the presence of neutralizing Abs against IL-5, IL-6, and IL-10. Following stimulation by K15, IgA production by B cells was slightly activated in the absence of mDC1s, whereas it was strongly activated in the presence of mDC1s (Fig. 2a). This indicates that DCs contribute to the activation of IgA secretion in response to LAB. When neutralizing Abs against IL-5, IL-6, and IL-10 were added to this co-culture system, the Abs against IL-6 and IL-10 significantly suppressed IgA production in response to LAB, and the combination of both of these Abs completely abolished IgA production (Fig. 2b). This result is in accordance with the observed IgA production by PBMCs described above (Fig. 1b). Together, these results strongly suggest that the IL-6 and IL-10 secreted by DCs in response to LAB activate IgA production by B cells. Previous reports identified IL-6 as a critical factor for IgA induction by LAB in mice 17 . However, our present research clearly demonstrates that IL-10 also makes a major contribution to this process in humans. IL-6 and IL-10 secretion by DCs in response to bacterial RNA. After finding that IL-6 and IL-10 are involved in the activation of IgA production that is induced by LAB, we investigated the levels of these cytokines secreted by mDC1s in response to several strains of LAB. K15 activated IL-6 and IL-10 production by DCs, and L. bulgaricus ATCC11842 T strongly induced IL-6 production (Fig. 3a). We previously reported that LAB contain a larger amount of dsRNA than pathogenic bacteria 14 . To test if RNA in LAB is responsible for the activation of IL-6 and IL-10 production by mDC1s, and thus in inducing IgA secretion, heat-killed K15 were treated with RNase A under 0 M NaCl to degrade both ssRNA and dsRNA or under 0.3 M NaCl to degrade only ssRNA 37 . IL-6 production by mDC1s in response to K15 was strongly impaired by the degradation of both ssRNA and dsRNA ( Fig. 3b) but not significantly by the degradation of ssRNA alone, indicating that bacterial dsRNA is the major component of LAB that induces IL-6 production. In contrast, IL-10 production was partially impaired by degradation under both conditions (Fig. 3b), indicating that ssRNA, but not dsRNA, contributes to IL-10 induction.
Activation of salivary sIgA production by K15 ingestion in a clinical trial. The effect of LAB on salivary IgA production was evaluated in a randomized, placebo-controlled trial using heat-killed K15. The secretion rate of salivary sIgA was significantly increased by ingestion of K15 for 8 and 12 weeks compared with baseline (p = 0.021 for 8 weeks, p = 0.002 for 12 weeks) and samples from participants who had ingested a placebo for 12 weeks (p = 0.036) ( Table 1). The salivary sIgA concentration was significantly enhanced only in the K15 group (p = 0.003) ( Table 1). These results indicate that K15 ingestion enhances salivary sIgA production, likely via IL-6 and IL-10 secreted by DCs, resulting in the activation of anti-viral or anti-bacterial immune responses. Any adverse reactions were not observed.  Table S1. The resulting IgA concentrations were measured by ELISA. Data are represented as mean ± SD of 7 donors. *p < 0.05 (vs medium alone, Student's t-test). (b) PBMCs were cultured in medium alone (−) or simulated with heat-killed K15. The resulting mRNA expressions of Blimp-1, April, and Baff were measured by qPCR. Data are represented as mean ± SD of duplicates and are representative of two independent experiments from different individuals. *p < 0.05, **p < 0.01 (Student's t-test). (c) PBMCs were cultured in medium alone (−) or stimulated with heatkilled K15, poly(I:C) (10 μg/mL), or LPS (10 μg/mL) for 5 days in the presence of control Ab (cont Ab), anti-IL-5 mAb (αIL-5 Ab), anti-IL-6 mAb (αIL-6 Ab), or anti-IL-10 mAb (αIL-10 Ab). The resulting IgA concentrations were measured by ELISA. Data are represented as the mean ± SD of triplicates and are representative of two independent experiments from different individuals. *p < 0.05, **p < 0.01 (Student's t-test).

Discussion
It has been reported that probiotic strains of LAB activate IgA production by B cells; this molecular mechanism was revealed in mice 17 . In the present study, we elucidated the mechanism for the effect of a functional LAB strain, Pediococcus acidilactici K15, on IgA production, focusing on the role of bacterial RNA in the interaction between DCs and B cells from human peripheral blood. In fact, K15, which activated IgA secretion from PBMCs in vitro, upregulated salivary sIgA concentration in healthy adults.
In mice, IL-6 secretion by PP DCs in response to some strains of Lactobacillus sp. promoted IgA + B cells to differentiate into IgA-producing plasma cells 17 . The present study found that in human PBMCs, IL-10 is another critical factor for activating IgA production from B cells. Moreover, we demonstrated via the use of a co-culture system composed of PBMC-derived mDC1s and B cells that the activation of IgA production by K15 is dependent on the function of DCs and their recognition of bacterial RNA.
A number of studies using animal models have revealed the beneficial effects of orally administered LAB, including Pedioccocus acidilactici 5,6,14,17,38 , suggesting that orally administered LAB are recognized in the intestine by antigen-presenting cells including DCs and macrophages. In our clinical trial, we confirmed that the salivary sIgA concentration was enhanced by K15 ingestion. Although we did not directly analyse the DCs in the guts of our human volunteers, our in vitro data and previous work on the immune events in the intestine suggest that the ingested K15 were probably recognized by DCs in PPs or the lamina propria, which then upregulated the IgA concentration in saliva. Arase et al. reported that a strong correlation is observed in humans between the levels of challenge-bacteria-specific IgA Ab in sublingual/submandibular secretions and those in gut lavages 39 . For augmentation of sIgA production at mucosal sites, an IgA class switch recombination to IgA + B cells induced by APRIL and BAFF is necessary [28][29][30] . In humans, it has been reported that APRIL, but not BAFF, is produced by monocyte-derived DCs following stimulation with CpG DNA or poly(I:C), although other TLR ligands were not able to induce either molecule 40 . In mice, all-trans-retinoic acid (RA) and IL-4 were shown to promote IgA class switching in cooperation with IL-5 41 , but we found that this mechanism was not involved in the IgA induction in our culture system. Here, we observed that the mRNA expression levels for APRIL, BAFF, and Blimp-1 were increased in PBMCs in the presence of LAB, which suggests that LAB may be sufficient to activate B cells for class switching and may also directly activate Tfh in the intestine. We also demonstrated in the present study that LAB RNA is the active component inducing the cytokine production that is critical for the enhancement of IgA production by B cells; dsRNA is a major causative molecule for IL-6 production whereas single-stranded RNA is critical factor for IL-10 production. Further investigations are needed to determine if these bacterial ligands to TLRs also contribute to IgA class switching and the promotion of IgA production via DC and Tfh functions.
Here, we used mDC1s isolated from PBMCs in a co-culture system with B cells. In addition to mDC1s, BDCA3 + DCs (mDC2s) and pDCs are also part of the composition of PBMCs, and, among these DC subsets,  mDC1s and pDCs are the more abundant cell populations 42,43 . mDC2s express a high level of TLR3, which recognizes the dsRNA in viruses and bacteria and secretes IFN-λ, a type III IFN 44 . pDCs express TLR7/8 and TLR9 and robustly secrete IFN-α during viral infections 16,[45][46][47] . mDC1s expressing many kinds of TLRs have been found to secrete IFN-β and many kinds of cytokines, including IL-12 and IL-27, in response to poly(I:C), R848, and LPS 43,[48][49][50] . Additionally, mDC1s secrete higher levels of IL-6 in response to Staphylococcus aureus, whose active ligand is TLR4 48 . Furthermore, IL-10 and IL-6 secretion levels in response to poly(I:C) were also reported to be higher in mDC1s than in mDC2s 51 . Thus, mDC1s seem to make a greater contribution to immunity than other DC subsets in the case of IgA induction via IL-6 and IL-10 production upon stimulation with bacteria. In addition to IgA induction, probiotic bacteria aid a variety of aspects relating to anti-infection immune functions. We previously reported that LAB contain large amounts of dsRNA, which induces IFN-β production via the TLR3 signalling pathway 14 . Type I IFNs, IFN-α and IFN-β, are well known to trigger a robust immune response against viral and bacterial infections 13,52 . LAB also activate IL-12 secretion from DCs or macrophages, which promotes Th1 cell differentiation, natural killer cell activation, and cytotoxic CD8 + T cell activation in concert with IL-27 and IL-18 production 49,53 . These subsets of cells protect against viral infection as well. Together, previous work and our current study suggest that functional strains of LAB, which has high safety for human and induce the production of IFN-β, IL-12, IL-6, and IL-10 by mDCs, protect the human body against viral and bacterial infection and maintain immune homeostasis by augmenting many of the pathways involved in protective immune responses.
Quantitative RT-PCR. Total RNA was extracted from the cells with a NucleoSpin RNA Kit (Takara, Kyoto, Japan) following the manufacturer's instructions. An equal amount of total RNA (300 ng) corresponding to each priming dose was reverse-transcribed using PrimeScript RT Reagent (Takara). The cDNA obtained after reverse transcription was amplified using specific primers for Blimp-1, APRIL, BAFF, and β-actin purchased from Takara and SYBR Premix Ex Taq II (Takara) following the protocols provided. The analysis of each gene expression was performed by normalizing to β-actin.
Preparation of clinical test foods. Test foods were prepared by Kikkoman Corporation. K15 was cultured in medium including soy peptone, yeast extract, and glucose. The cultured K15 were then heat-killed, washed with saline, concentrated, and spray-dried with dextrin. The test foods for the K15 and placebo groups were prepared using this K15 powder and a dextrin powder (Table S2). The subjects in the K15 group ingested 1 g of dextrin including 9.1 mg of heat-killed K15 (5 × 10 10 bacteria) every day for 12 weeks, while the subjects in the placebo group ingested 1 g of dextrin alone. Bacterial number in K15 samples was counted by using Nikon Eclipse 80i microscope (400×, Japna) and Micro Slide Glass (Matsunami Glass Ind., Ltd, Japan). The participants and the researchers were not able to discriminate between placebo and K15 samples.
Clinical study design. This study took place at Ueno Clinic, Aisei Hospital (Tokyo, Japan) between June 2016 and March 2017. A randomized, double-blinded, placebo-controlled trial was performed for 12 weeks. The trial was conducted by TTC Co. Ltd. funded by Kikkoman Corporation and approved by the Ethics Committee of Ueno Clinic, Aisei Hospital (Protocol No. 20160609-2) in compliance with the Declaration of Helsinki (2013). The trial was registered in the UMIN Clinical Trials Registry as UMIN000022880 (date of registration 01/04/2017). All participants provided written informed consent approved by the ethics committee. Data for body height, body weight, BMI, blood pressure, pulse, and biochemistry tests (BML Co. Ltd., Saitama, Japan) were obtained for all subjects before treatment and every 4 weeks after treatment. All subjects were followed up until the physician confirmed they had no important harms, and any important harms related to test foods were not observed. All subjects answered Profile of Mood States 2 Adult Short Form (POMS 2-A) and questionnaires about their weekly life events to confirm their fatigue and psychological stress. The salivary sIgA secretion rate was the primary outcome, and the salivary sIgA concentration and T scores in POMS 2-A were the secondary outcomes. We confirmed that Fatigue-Inertia (FI) T scores and Vigor-Activity (VA) T scores were significantly improved in both groups and were not different between two groups (Table S3).
Subjects for a clinical trial. The subjects were recruited from volunteers enrolled in Medical Art Laboratory Co. Ltd. (Tokyo, Japan), and 60 subjects were selected from 382 volunteers by the selection and exclusion criteria. The selection criteria were as follows: subjects who (a) were 20-64 years old and healthy; (b) had a lower than average salivary sIgA secretion rate; and (c) had a FI T score of more than 50 with a VA T score of less than 50 as assessed by POMS 2-A. The exclusion criteria were follows: subjects who (a) consumed food containing lactic acid bacteria, such as yogurt and other fermented foods; (b) had an allergic disease; (c) had mouth or teeth issues or were under treatment of them; (d) routinely engaged in vigorous exercises; (e) were pregnant or lactating; or (f) were inappropriate cases for the trial as defined by physicians. The researcher independent of this study (TTC Co. Ltd.) performed simple randomized allocation of the subjects by using a computer-generated list of random numbers. The allocation sequence was concealed from the researcher enrolling and assessing participants in the sealed envelope. This allocation sequence was opened by the independent researcher described above only after the enrolled participants completed all assessments.
Determination of salivary sIgA secretion rate and concentration. Saliva samples were collected for measurement of sIgA concentration and saliva flow rate before and every 4 weeks after the start of K15 or placebo ingestion. Salivary sIgA concentration was determined by ELISAs performed by Daiichi Kishimoto Clinical Laboratories, Inc. (Hokkaido, Japan). The salivary sIgA secretion rate was calculated from the saliva flow rate and salivary sIgA concentration.
Statistical analysis. Error bars on figures indicate the standard deviation (SD) of duplicate or triplicate samples for cell culture assay experiments. Statistical analysis was performed by PASW Statistics Version 18.0 (SPSS Inc, Chicago, IL, USA). The statistical significance was determined with a two-tailed Student's t-test for unpaired data, with p values of <0.05 considered significant (indicated on figures and tables as *p < 0.05, **p < 0.01). In the clinical trial, the consumption of test samples was monitored via diaries kept by the subjects, and these reports indicated that all subjects consumed more than 90% of total test samples. Two subjects (placebo group: n = 1, K15 group: n = 1) were excluded from the analysis due to meeting the exclusion criteria during the test period (Fig. S1). The subjects whose saliva flow rate strongly changed during the test period (CV > 1.5 SD) were also excluded from the analysis (placebo group: n = 4, K15 group: n = 2) (Fig. S1). Error bars on the figures presenting these data indicate standard error (SE). The statistical significance between two groups was determined with a two-tailed Student's t-test for unpaired data, with p values of <0.05 considered significant. The comparison between values from before and after the ingestion of test samples was performed by a paired t-test.