Yuji Aiba

Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice

Indigenous microbiota have several beneficial effects on host physiological functions; however, little is known about whether or not postnatal microbial colonization can affect the development of brain plasticity and a subsequent physiological system response. To test the idea that such microbes may affect the development of neural systems that govern the endocrine response to stress, we investigated hypothalamic–pituitary–adrenal (HPA) reaction to stress by comparing germfree (GF), specific pathogen free (SPF) and gnotobiotic mice. Plasma ACTH and corticosterone elevation in response to restraint stress was substantially higher in GF mice than in SPF mice, but not in response to stimulation with ether. Moreover, GF mice also exhibited reduced brain‐derived neurotrophic factor expression levels in the cortex and hippocampus relative to SPF mice. The exaggerated HPA stress response by GF mice was reversed by reconstitution with Bifidobacterium infantis. In contrast, monoassociation with enteropathogenic Escherichia coli, but not with its mutant strain devoid of the translocated intimin receptor gene, enhanced the response to stress. Importantly, the enhanced HPA response of GF mice was partly corrected by reconstitution with SPF faeces at an early stage, but not by any reconstitution exerted at a later stage, which therefore indicates that exposure to microbes at an early developmental stage is required for the HPA system to become fully susceptible to inhibitory neural regulation. These results suggest that commensal microbiota can affect the postnatal development of the HPA stress response in mice.

Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model

Objectives:We examined whether or not the lactobacilli administered to treat Helicobacter pylori (H. pylori) infection can suppress the colonization of H. pylori, and we also sought to elucidate the mechanism of such suppression.Methods:We used an in vitro culture system and an H. pylori-infected gnotobiotic murine model.Results:Among the lactobacillus species examined in vitro, Lactobacillus salivarius (L. salivarius) but not L. casei or L. acidophilus proved to be capable of producing a high amount of lactic acid and thus completely inhibiting the growth of H. pylori in a mixed culture. The validity of L. salivarius as a probiotic to suppress H. pylori and thus reduce the inflammatory response was again confirmed in vivo by using an H. pylori-infected gnotobiotic murine model.Conclusion:Based on our findings, L. salivarius was found to be a potentially effective probiotic against H. pylori.

Inhibition of the Accumulation of Uremic Toxins in the Blood and Their Precursors in the Feces after Oral Administration of Lebenin®, a Lactic Acid Bacteria Preparation, to Uremic Patients Undergoing Hemodialysis

The plasma levels of phenol, p-cresol, and indican are markedly increased in uremic patients, and cannot be efficiently reduced by hemodialysis. Such uremic toxins, which are produced in the intestine as bacterial putrefactive metabolites, accumulate to a great degree in the feces of hemodialysis patients. Oral administration of Lebenin, a preparation consisting of antibiotic-resistant lactic acid bacteria, reduced the levels of fecal putrefactive metabolites to levels comparable with those of healthy subjects. Moreover, the plasma level of indican also significantly decreased in these Lebenin-treated patients. An analysis of the fecal microflora revealed that a disturbed composition of the microflora characterized by an overgrowth of aerobic bacteria is restored to normal by oral administration of Lebenin in hemodialysis patients. These results thus demonstrate that oral administration of lactic acid bacteria in uremic patients is effective in reducing the levels of uremic toxins, especially that of indican, in the blood by inhibiting bacterial production by means of correcting the intestinal microflora.

Critical role of gut microbiota in the production of biologically active, free catecholamines in the gut lumen of mice

There is increasing interest in the bidirectional communication between the mammalian host and prokaryotic cells. Catecholamines (CA), candidate molecules for such communication, are presumed to play an important role in the gut lumen; however, available evidence is limited because of the lack of actual data about luminal CA. This study evaluated luminal CA levels in the gastrointestinal tract and elucidated the involvement of gut microbiota in the generation of luminal CA by comparing the findings among specific pathogen-free mice (SPF-M), germ-free mice (GF-M), and gnotobiotic mice. Substantial levels of free dopamine and norepinephrine were identified in the gut lumen of SPF-M. The free CA levels in the gut lumen were lower in GF-M than in SPF-M. The majority of CA was a biologically active, free form in SPF-M, whereas it was a biologically inactive, conjugated form in GF-M. The association of GF-M with either Clostridium species or SPF fecal flora, both of which have abundant β-glucuronidase activity, resulted in the drastic elevation of free CA. The inoculation of E. coli strain into GF-M induced a substantial amount of free CA, but the inoculation of its mutant strain deficient in the β-glucuronidase gene did not. The intraluminal administration of DA increased colonic water absorption in an in vivo ligated loop model of SPF-M, thus suggesting that luminal DA plays a role as a proabsorptive modulator of water transport in the colon. These results indicate that gut microbiota play a critical role in the generation of free CA in the gut lumen.

Impact of Intestinal Microbiota on Intestinal Luminal Metabolome

Low–molecular-weight metabolites produced by intestinal microbiota play a direct role in health and disease. In this study, we analyzed the colonic luminal metabolome using capillary electrophoresis mass spectrometry with time-of-flight (CE-TOFMS) —a novel technique for analyzing and differentially displaying metabolic profiles— in order to clarify the metabolite profiles in the intestinal lumen. CE-TOFMS identified 179 metabolites from the colonic luminal metabolome and 48 metabolites were present in significantly higher concentrations and/or incidence in the germ-free (GF) mice than in the Ex-GF mice (p < 0.05), 77 metabolites were present in significantly lower concentrations and/or incidence in the GF mice than in the Ex-GF mice (p < 0.05), and 56 metabolites showed no differences in the concentration or incidence between GF and Ex-GF mice. These indicate that intestinal microbiota highly influenced the colonic luminal metabolome and a comprehensive understanding of intestinal luminal metabolome is critical for clarifying host-intestinal bacterial interactions.

Cd1d-dependent regulation of bacterial colonization in the intestine of mice

The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen-free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I-like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with alpha-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d-/- mice in comparison to WT mice under specific pathogen-free conditions. Germ-free Cd1d-/- mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.

An oral introduction of intestinal bacteria prevents the development of a long-term Th2-skewed immunological memory induced by neonatal antibiotic treatment in mice

Background Recent epidemiological studies indicate that antibiotic use in infancy may be associated with an increased risk of developing atopy. Our previous work on animals demonstrated that kanamycin use during infancy promotes a shift in the Th1/Th2 balance towards a Th2‐dominant immunity.

Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells

When oral tolerance was induced in either specific pathogen‐free (SPF) or germ‐free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA‐specific immunoglobulin G1 (IgG1) and IgE titres in OVA‐fed GF mice were comparable to those in phosphate‐buffered saline‐fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25+ CD4+/CD4+ cells in the mesenteric lymph node (MLN) and the absolute number of CD25+ CD4+ cells in the Peyers patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25+ CD4+ cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose‐dependent manner than those from the GF mice. In addition, the CD25+ CD4+ regulatory T (Treg) cells from the naive SPF mice produced higher amounts of interleukin (IL)‐10 and transforming growth factor (TGF)‐β than those from the GF mice. When anti‐TGF‐β neutralizing antibody, but not anti‐IL‐10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25+ CD4+ Treg cells from the SPF mice was attenuated to the same level as that of the CD25+ CD4+ cells from the GF mice. In conclusion, the TGF‐β‐producing CD25+ CD4+ Treg cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25+ CD4+ cells from the naive GF mice was much lower than that of the CD25+ CD4+ Treg cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25+ CD4+ Treg cells.

Commensal microbiota modulate murine behaviors in a strictly contamination-free environment confirmed by culture-based methods

There is increasing evidence suggesting the existence of an interaction between commensal microbiota, the gut and the brain. The aim of this study was to examine the influence of commensal microbiota on the host behaviors in a contamination‐free environment, which was verified by culture‐based methods.

The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyers patch (PP) cells bearing PNA (peanut agglutinin)+/anti‐κ+ phenotype, which represents plasma cell‐like B cells. Moreover, treatment of those B. vulgatus‐implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti‐κ+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.