Kazuhiro Hirayama

IgA production in the large intestine is modulated by a different mechanism than in the small intestine: Bacteroides acidifaciens promotes IgA production in the large intestine by inducing germinal center formation and increasing the number of IgA+ B cells

It has been demonstrated that intestinal commensal bacteria induce immunoglobulin (Ig) A production by promoting the development of gut-associated lymphoid tissues in the small intestine. However, the precise mechanism whereby these bacteria modulate IgA production in the large intestine, which harbors the majority of intestinal commensals, is poorly understood. In addition, it is not known which commensal bacteria induce IgA production in the small intestine and which induce production in the large intestine. To address these issues, we generated gnotobiotic mice mono-associated with different murine commensal bacteria by inoculating germ-free (GF) mice with Lactobacillus johnsonii or Bacteroides acidifaciens. In GF mice, IgA production was barely detectable in the small intestine and was not detected in the large intestine. Interestingly, total IgA secretion in the large intestinal mucosa of B. acidifaciens mono-associated (BA) mice was significantly greater than that of GF and L. johnsonii mono-associated (LJ) mice. However, there was no difference in total IgA production in the small intestine of GF, LJ and BA mice. In addition, in the large intestine of BA mice, the expression of IgA(+) cells and germinal center formation were more remarkable than in GF and LJ mice. Furthermore, B. acidifaciens-specific IgA was detected in the large intestine of BA mice. These results suggest that the production of IgA in the large intestine may be modulated by a different mechanism than that in the small intestine, and that B. acidifaciens is one of the predominant bacteria responsible for promoting IgA production in the large intestine.

Bacteroides Induce Higher IgA Production Than Lactobacillus by Increasing Activation-Induced Cytidine Deaminase Expression in B Cells in Murine Peyer's Patches

The gut mucosal immune system is crucial in host defense against infection by pathogenic microbacteria and viruses via the production of IgA. Previous studies have shown that intestinal commensal bacteria enhance mucosal IgA production. However, it is poorly understood how these bacteria induce IgA production and which genera of intestinal commensal bacteria induce IgA production effectively. In this study, we compared the immunomodulatory effects of Bacteroides and Lactobacillus on IgA production by Peyer’s patches lymphocytes. IgA production by Peyer’s patches lymphocytes co-cultured with Bacteroides was higher than with Lactobacillus. In addition, the expression of activation-induced cytidine deaminase increased in co-culture with Bacteroides but not with Lactobacillus. We found that intestinal commensal bacteria elicited IgA production. In particular, Bacteroides induced the differentiation of Peyer’s patches B cell into IgA+ B cells by increasing activation-induced cytidine deaminase expression.

Intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate the serum antibody responses induced by dietary antigen

Colonization of the gut by commensal bacteria modulates the induction of oral tolerance and allergy. However, how these intestinal bacteria modulate antigen-specific T cell responses induced by oral antigens remains unclear. In order to investigate this, we used germ-free (GF) ovalbumin (OVA)-specific T cell receptor transgenic (OVA23-3) mice. Conventional (CV) or GF mice were administered an OVA-containing diet. Cytokine production by CD4(+) cells from spleen (SP), mesenteric lymph nodes (MLN) and Peyers patches (PP) was evaluated by ELISA, as was the peripheral antibody titer. T cell phenotype was assessed by flow cytometry. CD4(+) cells from the SP and MLN of CV and GF mice fed an OVA diet for 3 weeks produced significantly less IL-2 than the corresponding cells from mice receiving a control diet, suggesting that oral tolerance could be induced at the T cell level in the systemic and intestinal immune systems of both bacterial condition of mice. However, we also observed that the T cell hyporesponsiveness induced by dietary antigen was delayed in the systemic immune tissues and was weaker in the intestinal immune tissues of the GF mice. Intestinal MLN and PP CD4(+) T cells from these animals also produced lower levels of IL-10, had less activated/memory type CD45RB(low) cells, and expressed lower levels of CTLA-4 but not Foxp3 compared to their CV counterparts. Furthermore, GF mice produced higher serum levels of OVA-specific antibodies than CV animals. CD40L expression by SP CD4(+) cells from GF mice fed OVA was higher than that of CV mice. These results suggest that intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate serum antibody responses induced by dietary antigens through modulation of the intestinal and systemic T cell phenotype.

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota.

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.

Effects of soy protein-isoflavone diet on plasma isoflavone and intestinal microflora in adult mice

Effects of supplementing soy protein or casein diet with isoflavones on intestinal microflora and plasma concentrations of lipids and isoflavone metabolites were studied. Male mice were fed a soy protein or casein diet supplemented with isoflavones for four weeks, and feces and plasma samples were collected. Animals were also fed the soy protein or casein diet and feces were collected to investigate the capacity to produce equol from daidzein in vitro. The number of fusiform-shaped bacteria was significantly lower in the soy-isoflavone diet group than in the casein-isoflavone diet group, whereas the number of lactobacilli was significantly higher. No significant difference was observed in the plasma lipid concentration between the soy-isoflavone diet group and casein-isoflavone diet group. Plasma equol concentration was significantly higher in the soy-isoflavone diet group than in the casein-isoflavone diet group. After incubation of daidzein in vitro with the feces from the mice fed the soy protein and casein diets, the production of equol from daidzein was significantly more in the soy protein diet group. The present study indicates that the soy protein diet supplemented with isoflavone has an impact on the composition and metabolism of intestinal microflora and suggests that soy protein plays some roles in the effect of dietary isoflavones on the host through their effects on the intestinal microflora.

Metabolism of Isoflavones Found in the Pueraria thomsonii Flower by Human Intestinal Microbiota

Isoflavones contained in the root and flower of Kudzu (Pueraria lobata and related species) are suggested to be the critical component for its effects. Although metabolism of soy isoflavones has been well studied, the composition of isoflavones found in Kudzu is completely different from that of soy isoflavones. In the present study, we investigated whether isoflavones found in the flower of Pueraria thomsonii, a species of Kudzu, were metabolized by human fecal microbiota and murine small intestinal enzymes. Among 5 glycosidic isoflavones of the Pueraria thomsonii flower, tectorigenin 7-O-xylosylglucoside, tectoridin, genistin and glycitin were completely hydrolyzed by a homogenate of germfree mouse small intestine without contribution of bacteria. Released aglycones were not further metabolized, except that up to half of glycitein disappeared. Mouse small intestinal enzymes did not metabolize 6-hydroxygenistein 6,7-di-O-glucoside. Isoflavone aglycones as well as 6-hydroxygenistein 6,7-di-O-glucoside were highly metabolized by most of the human fecal suspensions. Metabolites were not detected with the present analytical methods in most cases. Although further investigations of the pharmacokinetics of Pueraria thomsonii flower isoflavones are needed, the results of the present study indicate active metabolism of Pueraria thomsonii flower isoflavones in the human intestine.

Effects of Guar Gum and Cellulose on Cecal Enzyme Activity and Cecal Short-Chain Fatty Acids in Young and Aged Mice

The effects of cellulose or guar gum on cecal enzyme activity and cecal short-chain fatty acids (SCFAs) in young and aged mice were studied. Male Crj:CD-1 (ICR) mice were fed an MF diet for 4 (young mice) or 23 months (aged mice). The MF diet was then replaced with a semisynthetic diet supplemented with 5% guar gum or 5% cellulose. The mice were fed the guar gum or cellulose diet for 3 weeks. There was no significant difference in cecal content between the two diet groups. There were no significant differences in total short-chain fatty acid production between the young mice fed the cellulose and those fed the guar gum diet, and between the aged mice fed the cellulose and guar gum diet. There were significant differences in cecal enzyme activity between the young mice fed the cellulose and those fed the guar gum diet. β-Glucuronidase activity was significantly higher in the young mice fed the guar gum diet than in those fed the cellulose diet. There were also significant differences in cecal enzyme activity between the aged mice fed the cellulose diet and those fed the guar gum diet. β-Glucuronidase activity was significantly higher in the aged mice fed the guar gum diet than in these fed the cellulose diet. β-Glucosidase activity was significantly lower in the aged mice fed the guar gum diet than in those fed the cellulose diet. The effect of cellulose on the microflora between the young and aged mice might be different from the effect of guar gum. The degree of adaptation to the diet of microflora in young and aged mice fed the cellulose diet might differ from that in those fed the guar gum diet. The higher enzyme activities of microflora in aged animals compared to young animals, might have some relation with the incidence of colon cancer in aged animals.

Intestinal Bifidobacterium association in germ-free T cell receptor transgenic mice down-regulates dietary antigen-specific immune responses of the small intestine but enhances those of the large intestine

Bifidobacterium is a dominant bacterial species among commensals in the human intestine and is thought to have probiotic immunomodulatory effects. In this study, we investigated the effect of the association with Bifidobacterium pseudocatenulatum JCM 7041 (Bp) on dietary ovalbumin (OVA)-specific immune responses using germ-free OVA-specific T cell receptor transgenic mice (OVA23-3 mice). We established germ-free OVA23-3 mice, and then associated with Bp (BIF group) or without (CONT group) and additionally associated with segmented filamentous bacteria (SFB) and clostridia in both groups. BIF and CONT mice were fed an egg-white diet containing OVA for 1 week. Cytokine production in response to OVA by cells of Peyers patches (PPs) and lamina propria (LP) from the small and large intestine was measured. Interferon (IFN)-gamma and interleukin (IL)-6 production by PP cells from BIF group mice was lower than that of the CONT group. The proportion of PP cells expressing CD4+CD62L(low), an activated/memory T cell phenotype, was higher in BIF group mice than the CONT group. Furthermore, LP cells from the small intestine in Bp-associated mice showed a tendency to produce slightly lower IFN-gamma and IL-6, while the cells from large intestine produced markedly higher IFN-gamma, IL-5 and IL-6 than those in the CONT group. The pattern of cytokine production by PP in BIF animals was similar to those isolated from conventional mice. These results suggest that intestinal association with Bp might down-regulate excessive immune responses to dietary antigens of the small intestine but enhance those of the large intestine.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

Establishment and Analysis of Germfree T Cell Receptor Transgenic Mice

Recent studies have shown that intestinal bacteria affect the intestinal immune system. In order to elucidate the effects of intestinal microflora on T-cell mediated immune responses of the intestine, we established germfree (GF) T cell receptor transgenic (TCR-Tg) mice. GF ovalbumin (OVA)-specific TCR-Tg mice were obtained from conventional (CV) TCR-Tg mice by hysterectomy. Cells from spleen, Peyer’s patch (PP) and lamina propria (LP) were isolated from GF TCR-Tg mice, and the ratio of CD4+ T cells was assessed by flow cytometry and the production of cytokines, in response to in vitro OVA stimulation, was measured by ELISA. The numbers of PPs were significantly lower in GF TCR-Tg mice compared with CV TCR-Tg mice and the ratios of CD4+ T cells in PP and LP cells from GF TCR-Tg mice were decreased. When stimulated with OVA, PP cells from GF TCR-Tg mice secreted higher levels of interferon (IFN)-γ, interleukin (IL)-5 and IL-6, and LP cells from GF mice secreted higher levels of IFN-γ and IL-6 compared with CV mice. These results suggested that although the gut-associated lymphoid tissue is poorly developed in germfree mice, intestinal T cells developing under these conditions possessed an enhanced ability to secrete cytokines in response to antigenic stimulation. Our TCR-Tg system should be an informative system to evaluate the effect of intestinal microflora on antigen-specific T cell responses.