Abstract
We examined whether or not dietary fructooligosaccharides (FOS) in infancy can have a beneficial effect on the mucosal immune system. Newborn BALB/c mice, accompanied by their dams until 21 days of age, were fed either a control diet based on casein [FOS(–) diet group] or a FOS(−) diet supplemented with 5% (w/w) FOS [FOS(+) diet group]. Total IgA levels in tissue extracts from the intestines of mice in the FOS(+) diet group at 38 days of age were about twofold higher (P < 0·05) than those in the FOS(−) diet group in the jejunum, ileum and colon. Ileal and colonic polymeric immunoglobulin receptor (pIgR) expression in the FOS(+) diet group at 36 days of age was 1·5-fold higher than in the FOS(−) diet group (P < 0·05). Consistent with these results, the ileal IgA secretion rate of the FOS(+) diet group at 37 days of age was twofold higher than that of the FOS(−) diet group (P < 0·05). Moreover, the percentage of B220+IgA+ cells in Peyer's patches (PP) was significantly higher in the FOS(+) diet group than in the FOS(−) diet group (6·2% versus 4·3%, P < 0·05), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in the small intestine as well as the colon in infant mice.
Keywords: fructooligosaccharides, immunoglobulin A, polymeric immunoglobulin receptor, prebiotics
INTRODUCTION
Fructooligosaccharides (FOS) exist in a number of edible plants, such as onion and edible burdock, and are produced commercially by the action of fructosyltransferase on sucrose [1]. FOS are representative prebiotics: food products that escape digestion by pancreatic and small-intestinal enzymes in the human gut and therefore reach the colon, where they have beneficial effects. It has been reported that dietary FOS influences many aspects of intestinal function through fermentation [2].
Yamamoto et al. [3] and Juffrie [4] have reported that dietary FOS improve the faecal content and reduce the frequency of diarrhoea in infants, respectively. Numerous studies in adults or aged humans have shown that dietary FOS lead to an increase in faecal bifidobacteria numbers [1,5–7]. Because bifidobacteria, which are probiotics, are presumed to be antagonistic to pathogenic bacteria and to promote non-specific stimulation of the immune system, it is possible that the mechanisms by which dietary FOS confer beneficial effects on the host may include immunomodulatory effects on the intestinal immune system through the increase in bifidobacteria numbers.
There have been a few reports on the effect of dietary FOS on the mucosal immune system. Pierre et al. [8] demonstrated that dietary FOS enhanced the development of the gut-associated lymphoid tissue, and Hosono et al. [9] showed that dietary FOS up-regulated the faecal IgA content in the adult mouse. Furthermore, Swanson et al. [10] indicated that FOS plus mannanoligosaccharides influenced ileal IgA in the adult dog.
As the intestinal immune system in infants is immature compared to that in adults, it is of interest to investigate the effect of prebiotics on the intestinal immune system in infancy. Therefore, we have studied the effects of FOS ingestion on the mucosal IgA response and pIgR expression in the intestines of infant mice.
MATERIALS AND METHODS
Animals
Timed-pregnant BALB/c mice were purchased from Japan SLC (Shizuoka, Japan) and were maintained in individual plastic cages. The mice ate a non-purified diet (MF; Oriental Yeast, Tokyo, Japan) and drank water, both ad libitum. The day of birth is referred to as day 0. At 2 days of age, the litter sizes were adjusted to four to six pups on the basis of comparable mean body weight. Each dam and her pups were placed together in their own plastic cage. The dams were fed a control diet ad libitum based on casein [the FOS(−) diet] or on the FOS(−) diet supplemented with 5% (w/w) FOS [the FOS(+) diet] (Table 1). The added FOS (Meioligo-P®) were obtained from Meiji Seika Kaisya, Ltd (Tokyo, Japan). Pups were weaned at 21 days of age, and fed the same diets ad libitum until analysis. All procedures were approved by our institute's Committee for Research on Experimental Animals, and were conducted in accordance with the NRC Guide for the Care and Use of Laboratory Animals (1985).
Table 1. Composition of the FOS(−) diet (g/kg).
| Ingredient | FOS(−) diet |
|---|---|
| Casein | 220 |
| Sucrose | 50 |
| Starch | 600 |
| Cellulose | 30 |
| Soybean oil | 50 |
| Vitamins1 | 10 |
| Minerals2 | 40 |
The composition was as follows (IU/kg): vitamin A, 2 000 000; 7-dehydrocholesterol, 200 000; α-tocopheryl acetate (in g/kg): 10; menadione, 1; p-aminobenzoic acid, 10; inositol, 10; niacin, 4; calcium pantothenate, 4; choline chloride, 230·6; riboflavin, 0·8; thiamine HCl, 0·5; pyridoxine HCl, 0·5; folic acid, 0·2; d-biotin, 0·04; cyanocobalamine, 0·003.
The composition was as follows (in g/kg): NaCl, 139·3; KI, 0·79; KH2PO4, 389; MgCO3, 40·2; CaCO3, 381·4; Fe fumarate, 16·5; MnCO3, 3·05; ZnCO3, 0·58; CuSO4 5H2O, 0·6; CoCl2, 0·023.
Preparation of intestinal tissue samples and plasma membranes of the intestine
The procedure for the preparation of tissue samples from the small intestine and colon was a modification of a protocol described previously by our group [11]. Pups were killed under anaesthesia and the small intestine and colon were removed carefully from FOS(−) or FOS(+) diet-fed pups at 23, 30, 38 and 44 days of age. The small intestine from the pylorus to the ileocaecal junction was divided into two equal segments, defined as the jejunum and ileum. The luminal contents were flushed out with ice-cold phosphate buffered saline (PBS). Each segment was weighed after being washed. Then a 20-fold volume of PBS containing 1 mmol/l phenylmethylsulphonyl fluoride, 5 mmol/l EDTA, 100 µg/ml soybean trypsin inhibitor, 100 µg/ml leupeptin and 100 KIU/ml aprotinin in 50 mmol/l Tris-HCl (pH 6·8) was added, and the tissue was homogenized on ice using a Polytron homogenizer (Kinematica AG, Littau, Switzerland; setting 6; 30 s, three times). The suspension was centrifuged at 10 000 g for 15 min, and the supernatant obtained equalled the tissue extract used for the detection of intestinal IgA levels.
Intestinal plasma membranes were prepared from the suspension as described previously [12]. Briefly, intestinal suspensions were centrifuged (750 g for 10 min) to remove cells and nuclei. Membranes were pelleted from the supernatant by centrifugation (100 000 g for 30 min), resuspended and boiled (5 min) in Laemmli sample buffer for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [13].
Faecal sample preparation
Faecal samples were prepared as described by deVos and Dick, with modifications [14]. At 28, 36 and 42 days of age, pups were placed into stainless-steel wire-mesh cages without bedding materials for 24 h individually, and the faecal samples were collected. The material was lyophilized and dissolved in 50 volumes of PBS containing 0·5 mmol/l EDTA and 100 µg/ml soybean trypsin inhibitor. The sample was then homogenized on ice using a Polytron homogenizer (30 s, three times) and centrifuged at 1600 g for 15 min, and the supernatant equalled the extract used for the detection of faecal IgA levels.
Collection of ileal secretions
Ileal secretion was collected from isolated intestinal loops of anaesthetized mouse pups using a modified version of that described by Ahnen et al. [12]. At 37 days of age, pups (n = 6) were anaesthetized with sodium pentobarbital (100 mg/kg) injected intraperitoneally. Then, a 6–8-cm section of ileum beginning at the ileo–caecal junction was isolated. The loop was washed with PBS to remove luminal contents and ligated at both ends. After 30 min, the loops were excised and the luminal content was flushed out with PBS containing 10 mg/ml soybean trypsin inhibitor (5 ml × 3). The sample volume was increased to 20 ml by adding PBS, and then the sample was centrifuged at 750 g for 15 min. The supernatant was used for measuring the IgA concentration.
Cell culture
Peyer's patches (PP) from FOS(−) or FOS(+) diet-fed pups (n = 6) at 35 days of age were used for the cultures. A single-cell suspension of PP was prepared as described previously [15]. In brief, PP dissected from the small intestine were placed in 10 ml of RPMI-1640 medium (Gibco, Grand Island, NY, USA) in a glass dish, and then dissociated mechanically. The treated cells were washed twice with the RPMI-1640 medium. The PP cells (5 × 105 cells/0·2 ml/well) were cultured in triplicate in flat-bottomed, 96-well microtest plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) in RPMI-1640 medium containing 100 U/ml of penicillin (Gibco), 100 µg/ml of streptomycin (Gibco), 5 10−5 mol/l 2-mercaptoethanol and 10% heat-inactivated fetal bovine serum. The cells were incubated for 7 days at 37°C under 5% CO2 in air. The culture supernatants were collected and stored at −80°C until assayed.
Enzyme-linked immunosorbent assay (ELISA)
Total IgA concentration was measured by enzyme linked immunosorbent assay (ELISA). At 4°C overnight, 96-well microtitre plates (Nunc, Roskilde, Denmark) were coated with 100 µl of rat antimouse IgA antibodies (Pharmingen, San Diego, CA, USA) (1 µg/ml) dissolved in 0·15 m PBS. The unbound antibodies were removed by three washes with 125 µl of PBS containing 0·05% (w/v) Tween 20 (PBS-T). The plates were incubated with 125 µl of 1% (w/v) bovine serum albumin (BSA) (Intergen, Purchase, NY, USA) for 30 min at room temperature and washed with PBS-T three times. Intestinal tissue extracts, faecal samples, ileal secretions, culture supernatants or standard mouse IgA (mouse myeloma protein; ICN Biomedicals, Costa Mesa, CA, USA) were diluted with 0·01 mol/l PBS (pH 7·2) containing 0·5 mol/l NaCl and 0·1% Tween 20. The diluted samples or standards were added to the wells in triplicate. For each sample, an uncoated well blocked with 1% BSA was used as a control for non-specific binding. After incubation overnight at 4°C the plates were washed, and 100 µl of biotinylated rat antimouse IgA antibodies (Pharmingen) (50 ng/ml) were added. Following further incubation at room temperature for 2 h, the plates were washed and 100 µl of alkaline phosphatase-conjugated avidin (Organon Teknika, Durham, NC, USA) (1 µg/ml) were added to each well. Finally, 100 µl of p-nitrophenyl phosphate (1 mg/ml) dissolved in a diethanolamine buffer (pH 9·8) were added. After incubation at room temperature for 30 min, colour development was stopped by adding 50 µl of 5 mol/l NaOH, and then the absorbance at 405 nm of each well was measured. The total IgA content of each sample was calculated by means of a standard curve.
SDS-PAGE and antipolymeric immunoglobulin receptor immunoblotting
Antimouse polymeric immunoglobulin receptor (pIgR) antiserum was prepared in our laboratory. The procedure for preparing pIgR proteins followed the method described by Symersky et al. [16] with slight modifications. The BamH I-Sac I fragment of mouse pIgR cDNA was ligated with the pET32c vector. The mouse pIgR cDNA was kindly provided by Dr C. S. Kaetzel, University of Kentucky. The resultant plasmid was used to transform the BL21(DE3) strain of E. coli. After transformation the E. coli were incubated with shaking at 37°C until the OD 600 reached 0·6, then the bacteria were incubated with fresh TB medium containing 1 mm isopropyl-thio-β-D-galactopyranoside (IPTG) for 2 h at 37°C. The PITG-induced cells were collected by centrifugation, and crude fusion proteins were extracted in binding buffer (20 mm Tris-HCl pH 8·0, 5 mm imidazole, 500 mm NaCl) by sonication. The fusion proteins were purified by His binding column chromatography. Rabbits were immunized with the purified fusion proteins in Freund's complete adjuvant every 2 weeks for 3 months. Rabbit antimouse pIgR antiserum was obtained at 5 days after the last immunization.
Intestinal pIgR was measured by immunoblotting. The intestinal plasma membranes were subjected to 7·5% SDS-PAGE under reducing conditions [13]. The separated proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond C extra, Amersham International plc, Bucks, UK) and immunostained with the rabbit antimouse pIgR antiserum (×500) followed by horseradish peroxidase-conjugated goat antirabbit IgG antibodies (Zymed Laboratory, South San Francisco, CA, USA) (×10 000). Molecular weight markers (Bio-Rad Laboratories, Hercules, CA, USA) were used on each gel and the relative values of pIgR were estimated by densitometric scanning. The values were expressed relative to the average value in FOS(−) diet group that was normalized to 100.
Identification of PP cell phenotype by flow cytometry
PP cells from FOS(−) or FOS(+) diet-fed pups at 35 days of age (n = 5–6) were prepared as described above and used for two-colour FACS analysis. The antibodies used for flow cytometry were fluorescein isothiocyanate (FITC)-labelled antimouse IgA (C10-3; Pharmingen) (1 µg/ml), FITC-labelled antimouse IgM (1B4B1; eBioscience, San Diego, CA, USA) (1 µg/ml), and phycoerythrin (PE)-labelled antimouse B220 (RA3–6B2; Pharmingen) (40 ng/ml) antibodies. All incubations were performed in the dark. Cells were incubated for 30 min on ice with FITC-labelled and PE-labelled antibodies. The cells were then washed in PBS. Stained cells were analysed with an EPICS XL (Beckman Coulter Inc., Hialeah, FL, USA). The data were analysed with system ii software (Beckman Coulter Inc.).
Caecal short-chain fatty acids
Caecal short-chain fatty acids (SCFA; acetic, propionic and n-butyric acids) were measured using high performance liquid chromatography (HPLC) (Shimadu, Kyoto, Japan) by the internal standard method, as described previously [17]. Briefly, pups were sacrificed under anaesthesia and the caecum was removed carefully from FOS(−) or FOS(+) diet-fed pups at 36 days of age. Caecal contents were homogenized by ultrasonication (Sonifier 250, Branson, Danbury, CT, USA) with crotonic acid and then centrifuged (750 g). Fat-soluble substances in the supernatant were removed by extraction with chloroform. SCFA were separated using an ion exclusion column and detected using a post-column pH-buffered electroconductivity detection method. Caecal SCFA concentrations were expressed as mmol/kg caecal water.
Statistical analysis
The experimental data were expressed as means with their standard deviations for three to six pups per group. Differences were evaluated by the Student's t-test, the Mann–Whitney U-test or two-way anova with Tukey–Kramer's post hoc test using the StatView 4·0 program (Abacus Concept Inc., Berkeley, CA, USA). Differences were considered to be significant at P < 0·05.
RESULTS
Effect of dietary FOS on total IgA levels in intestinal tissue extracts
Throughout the experiments, the food intake and body weight did not differ between the FOS(−) and FOS(+) diet group (data not shown). The total IgA levels in the intestinal tissue extracts were higher (P < 0·05) in the FOS(+) compared with the FOS(−) diet group in the jejunum, ileum and colon (Table 2). In the jejunum, the IgA levels in the FOS(+) diet group were significantly higher at 30, 38 and 44 days of age than the respective levels in the FOS(−) diet group. This was especially noticeable at 38 days of age, when the IgA level of the FOS(+) diet group was about twofold higher than that of the FOS(−) diet group. Almost identical results were obtained for the ileum (38 days of age) and the colon (30 and 38 days of age).
Table 2. Effect of dietary FOS on the total IgA levels in intestinal tissue extracts1.
| FOS(−) | FOS(+) | ||||||
|---|---|---|---|---|---|---|---|
| Intestinal IgA (µg/g) | Age (days) | Mean | s.d. | n | Mean | s.d. | n |
| Jejunum | 23 | 46 | 28 | 6 | 42 | 14 | 4 |
| 30 | 96 | 29 | 6 | 167* | 71 | 6 | |
| 38 | 145 | 33 | 6 | 347* | 89 | 6 | |
| 44 | 307 | 48 | 6 | 384* | 49 | 6 | |
| Ileum | 23 | 9 | 5 | 6 | 10 | 5 | 4 |
| 30 | 38 | 22 | 6 | 69 | 46 | 6 | |
| 38 | 121 | 35 | 6 | 228* | 71 | 6 | |
| 44 | 252 | 38 | 6 | 281 | 36 | 6 | |
| Colon | 23 | 17 | 10 | 6 | 19 | 11 | 4 |
| 30 | 117 | 39 | 6 | 219* | 67 | 6 | |
| 38 | 202 | 31 | 6 | 399* | 47 | 6 | |
| 44 | 345 | 103 | 6 | 365 | 89 | 6 | |
FOS: fructooligosaccharides; s.d.: standard deviation.
The FOS(+) diet includes 5% fructooligosaccharides (Meiji Seika Kaisya, Ltd, Tokyo, Japan) + 95% (−) diet (for details of the FOS(−) diet, see Table 1.
Significant difference from the FOS(−) diet group (P < 0·05) by two-way anova with Tukey–Kramer's post hoc test.
Effect of dietary FOS on ileal IgA secretion and intestinal pIgR expression
The IgA content of dry matter faeces from the FOS(+) diet group at 36 days of age was significantly higher than that of the FOS(−) diet group (Fig. 1). This seemed to be due to an increase of intestinal IgA levels. However, it is accepted that some quantities of secretory IgA in the faeces are transported originally from the circulation into the bile in mice [18]. To eliminate the bilious secretory IgA contamination, we further examined the effect of dietary FOS on ileal IgA secretion by means of the intestinal loop method. The rate of ileal IgA secretion at 37 days of age was significantly higher in the FOS(+) compared with the FOS(−) diet group (Fig. 2). Moreover, ileal and colonic, but not jejunal, pIgR expression were significantly higher in the FOS(+) compared with the FOS(−) diet group (Fig. 3).
Fig. 1.
Effect of dietary FOS on faecal IgA levels in mouse pups. The faecal IgA levels were measured by ELISA for the FOS(−) (open circles) and FOS(+) diet (closed circles) groups. The results are expressed as the mean ± 1 s.d. (n = 6). *Significant difference from the FOS(−) diet group (P < 0·05) by two-way anova with Tukey–Kramer's post hoc test.
Fig. 2.
Effect of dietary FOS on ileal IgA secretion. The results are expressed as the mean + 1 s.d. (n = 6). *Significant difference from the FOS(−) diet group (P < 0·05) by the Student's t-test.
Fig. 3.
Effect of dietary FOS on pIgR expression in the jejunum, ileum and colon. The relative quantities of pIgR were estimated by densitometric scanning. The values were expressed relative to the average value in FOS(−) diet group that was normalized to 100. The results are expressed as the mean + 1 s.d. (n = 3). *Significant difference from FOS(−) diet group (P < 0·05) by the Mann–Whitney U-test.
Effect of dietary FOS on the concentration of short-chain fatty acids in the caecum contents
Short-chain fatty acids (mainly acetic, propionic and butyric acids) are formed in the caecum as a result of anaerobic bacterial fermentation of dietary FOS. Mice fed the FOS(+) diet had higher caecum acetate (P < 0·05), butyrate (P < 0·05) and propionate (P = 0·09) concentrations than mice fed the FOS(−) diet (Table 3). These results were consistent with those of previous reports [19].
Table 3. The caecum SCFA concentrations for mice fed FOS(−) or FOS(+) diets1.
| FOS(−) | FOS(+) | |||||
|---|---|---|---|---|---|---|
| Mean | s.d. | n | Mean | s.d. | n | |
| Acetate (mmol/kg caecum water) | 24·6 | 0·6 | 5 | 40·5* | 9·6 | 5 |
| Propionate (mmol/kg caecum water) | 5·1 | 0·5 | 5 | 6·4 | 1·4 | 5 |
| Butyrate (mmol/kg caecum water) | 2·8 | 0·6 | 5 | 7·6* | 2·3 | 5 |
SCFA: short-chain fatty acid; FOS: fructooligosaccharides; s.d.: standard deviation.
The FOS(+) diet includes 5% fructooligosaccharides (Meiji Seika Kaisya, Ltd, Tokyo, Japan) + 95% FOS(−) diet (for details of the FOS(−) diet, see Table 1.
Significant difference from the FOS(−) diet group (P < 0·05) by the Student's t-test.
Effect of dietary FOS on the IgA response and B cell subpopulations in PP
The culture period (7 days) established for PP cells was based on the results of a preliminary time–course study (data not shown). The IgA response of PP cells in the FOS(+) diet group was significantly higher than that in the FOS(−) diet group (89 ± 77 ng/ml versus 553 ± 160 ng/ml; P < 0·05). This result led us to analyse the percentages of PP cells identified as B220+, IgA+ and IgM+(Table 4). The diet supplemented with FOS did not have any effect on the percentages of B220+ cells in PP. However, the percentage of B220+IgA+ cells was significantly higher in the FOS(+) than the FOS(−) diet group (6·2% vs. 4·3%). The percentage of B220+IgM+ cells in PP was not significantly different between the two groups.
Table 4. Peyer's patch B cell subpopulations for mice fed FOS(−) or FOS(+) diets1.
| FOS(−) | FOS(+) | |||||
|---|---|---|---|---|---|---|
| Mean | s.d. | n | Mean | s.d. | n | |
| B220+ (%) | 77·1 | 2·4 | 5 | 77·4 | 2·9 | 6 |
| B220+IgM+ (%) | 72·1 | 2·0 | 5 | 71·3 | 3·4 | 6 |
| B220+IgA+ (%) | 4·0 | 1·1 | 5 | 6·5* | 0·8 | 6 |
FOS: fructooligosaccharides; s.d.: standard deviation.
The FOS(+) diet includes 5% fructooligosaccharides (Meiji Seika Kaisya, Ltd, Tokyo, Japan) + 95% FOS(−) diet (for details of the FOS(−) diet, see Table 1.
Significant difference from the FOS(−) diet group (P < 0·05) by the Student's t-test.
DISCUSSION
In the present study, we examined whether dietary FOS in infancy can have a beneficial effect on the mucosal immune system. Prebiotics are food products that are designed especially to benefit the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon [2,20]. It is assumed that prebiotics usually act in the colon. Most prebiotics resist hydrolysis, reach the colon intact and are fermented extensively in the colon by resident anaerobic bacteria. Thus, it is expected that the immunomodulatory effects of prebiotics would be observed mainly in the colon. Previous studies have shown that dietary FOS reduced the occurrence of colon tumours and trinitrobenzene sulphonic acid hapten-induced chronic colitis [21,22]. Furthermore, Buddington et al. [23] reported that diets supplemented with inulin and oligofructose lowered the incidence and growth of tumours in the colon after exposure to carcinogens. In agreement with these studies, our findings indicate that dietary FOS increase the total IgA levels in tissue extracts isolated from the colon and increase pIgR expression in the colon.
Similar to the colon, where solitary lymphoid follicles are present as gut-associated lymphoid tissue (GALT), the small intestine is another site where GALT, such as PP, is located and the mucosal IgA response is induced. In the human small intestine, it has been estimated that there are approximately 1010 immunoglobulin-producing cells per metre, most of which produce IgA, accounting for approximately 80% of all immunoglobulin-producing cells in the whole body [24]. Thus, we investigated whether dietary FOS showed a beneficial effect on the mucosal immune response in the small intestine. As demonstrated for the mucosal IgA response in the colon of infant mice, dietary FOS increased the total IgA levels in tissue extracts isolated from the jejunum and ileum (P < 0·05) (Table 2). Furthermore, pIgR expression in the ileum was significantly higher in the FOS(+) compared with the FOS(−) diet group. Thus, dietary FOS effect the mucosal immune system in the small intestine of infant mice as well as in the colon. However, the mechanism by which dietary FOS increases the IgA response and pIgR expression in the small intestine remains to be elucidated.
Dietary FOS has been shown to up-regulate the faecal IgA content in adult mice [9]. Our present findings in newborn mice are consistent with this observation. However, unlike humans, pIgR-mediated IgA transport occurs not only in the intestine but also in the liver in rodents [25]. Furthermore, it is possible that secretory IgA may be digested in the intestinal lumen, because some bacterial species have been shown to possess proteases capable of degrading IgA [26]. Therefore, faecal IgA levels may not reflect intestinal IgA secretion accurately, especially in mice. We found that ileal IgA secretion was enhanced significantly in the FOS(+) compared with that in the FOS(−) diet group. This may be due to the fact that both the total IgA level in tissue extracts and pIgR expression in the ileum was significantly higher in the FOS(+) diet group. In contrast, another group has shown that supplementation with FOS alone did not influence ileal IgA secretion in ileum-cannulated dogs [10]. The reason for this discrepancy is unclear. One possible reason, however, lies in the age differences between the experimental animals. In the latter study, the effects of feeding FOS were examined in approximately 3-year-old adult dogs, whose intestinal microflora would be less changeable than those in the newborn mice used in our study. In this regard, it has been reported that oral tolerance, which is considered to be a result of the suppressive mucosal immune response as well as the secretory IgA response, is restored in germ-free mice by reconstitution of the intestinal microflora at the neonatal, but not at a later, stage [27]. Therefore, we speculate that the prebiotic effect on the mucosal immune response can best be observed in newborn animals.
Faecal IgA production has been reported to decrease significantly in mice lacking PP caused by different types of gene targeting [28]. Furthermore, a similar result was obtained in a spontaneous mouse mutant whose PP were completely absent [29]. Therefore, it is likely that the mechanism underlying the prebiotic effect on the mucosal immune response includes the function of PP. We found that the in vitro IgA response of PP cells from the FOS(+) diet group was higher than that of the FOS(−) diet group. This is consistent with a previous study showing that dietary FOS up-regulates IgA secretion by PP cells [9]. However, it is possible that the PP B cells switch from IgM to IgA production during the in vitro culture period. We therefore performed phenotype analysis of PP lymphocytes by flow cytometry. Dietary FOS increased the percentage of B220+IgA+ cells (IgA-committed B cells) (P < 0·05 compared with the FOS(-) group), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. In mice, the peritoneal cavity is a major source of B cells [30]. However, a recent study has suggested that the contribution of B cells in the peritoneal cavity to total intestinal IgA may be limited [31]. Thus, our findings that dietary FOS increased the size of the IgA-committed B cell population in PP suggests that this cell population may be responsible, at least in part, for the observed increase in total IgA level in tissue extracts in the FOS(+) diet group.
It is known that intestinal pIgR plays a critical role in transepithelial transport of intestinal IgA onto mucosal surface. For example, Johansen et al. [32] reported a complete lack of active external IgA translocation in pIgR knockout mice. It is known that the expression of intestinal pIgR is strictly regulated during ontogeny in humans and rodents [33,34]. These findings [33,34] demonstrate that the production of pIgR protein in intestinal epithelial cells gradually increases after birth. Furthermore, protein–energy malnutrition depresses the production of pIgR protein in intestinal epithelial cells in the weanling mouse intestine [35,36]. Also, the concentration of pIgR is markedly depressed in the small intestine of iron-deprived newborn rats [37]. Therefore, nutritional factors are likely to affect the production of pIgR protein in intestinal epithelial cells in newborn animals. We found that the butyric acid concentration in the caecal contents was enhanced significantly in the FOS(+) compared with that in the FOS(−) diet group. Because butyric acid has been reported to up-regulate intestinal epithelial production of pIgR protein directly in vitro, it is possible that butyric acid production regulates intestinal pIgR expression via the fermentation of FOS in vivo[38].
Increasing evidence suggests a relationship between the intestinal microflora and allergy. It has been shown that the proportions of anaerobic bacteria in the intestinal flora are higher in non-allergic than allergic children [39]. In particular, non-allergic children harbour higher counts of bifidobacterium than allergic children [40,41]. Thus, there is a trend to supplement infant formula with substances with bifidogenic effects [42]. This seems to be reasonable, because human milk contains bioactive components that stimulate the growth of bifidobacteria in the intestine, such as oligosaccharides [43]. However, there are few studies on the effects of prebiotics on the mucosal immune system in infancy. Therefore, it is noteworthy that dietary FOS was shown to increase the IgA response and pIgR expression in the intestines of infant mice in this study.
In conclusion, we have demonstrated that dietary FOS increases the total IgA levels in tissue extracts from the jejunum, ileum, and colon in infant mice. Moreover, dietary FOS up-regulated ileal and colonic pIgR expression. Consistent with these results, the ileal IgA secretion rate in the FOS(+) diet group was increased. The percentage of B220+IgA+ cells in PP was significantly higher in the FOS(+) compared with the FOS(−) diet group, suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in both the small intestine and colon in infant mice.
Acknowledgments
We thank Meiji Seika Kaisha, Ltd for providing us with fructooligosaccharides (Meioligo-P®). We also thank Drs M. Totsuka, S. Hoshi and M. Yajima for their helpful suggestions.
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