Probiotic Bifidobacterium breve Prevents Memory Impairment Through the Reduction of Both Amyloid-ß Production and Microglia Activation in APP Knock-In Mouse.

BACKGROUND: Probiotic supplementation reestablishes microbiome diversity and improves brain function in Alzheimer's disease (AD); their molecular mechanisms, however, have not yet been fully illustrated. OBJECTIVE: We investigated the effects of orally supplemented Bifidobacterium breve MCC1274 on cognitive function and AD-like pathologies in AppNL-G-F mice. METHODS: Three-month-old AppNL-G-F mice were orally supplemented with B. breve MCC1274 for four months. The short-term memory function was evaluated using a novel object recognition test. Amyloid plaques, amyloid-ß (Aß) levels, Aß fibril, amyloid-ß protein precursor and its processing enzymes, its metabolic products, glial activity, and cell proliferation in the subgranular zone of the dentate gyrus were evaluated by immunohistochemistry, Aß ELISA, western blotting, and immunofluorescence staining. The mRNA expression levels of pro- and anti-inflammatory cytokines were determined by qRT-PCR analysis. RESULTS: We found that the oral B. breve MCC1 274 supplementation prevented memory impairment in AppNL-G-F mice and decreased hippocampal Aß levels through the enhancement of the a-disintegrin and metalloproteinase 10 (ADAM10) level. Moreover, administration of the probiotic activated the ERK/HIF-1α signaling pathway responsible for increasing the ADAM10 level and also attenuated microglial activation, which in turn led to reduction in the mRNA expression levels of pro-inflammatory cytokines in the brain. In addition, B. breve MCC1274 supplementation increased the level of synaptic proteins in the hippocampus. CONCLUSION: Our findings support the possibility that oral B. breve MCC1274 supplementation might be used as a potential preventive therapy for AD progression.

Oral Administration of Probiotic Bifidobacterium breve Improves Facilitation of Hippocampal Memory Extinction via Restoration of Aberrant Higher Induction of Neuropsin in an MPTP-Induced Mouse Model of Parkinson's Disease.

We previously reported that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease (PD) model mice (PD mice) facilitate hippocampal memory extinction, which may be the cause of cognitive impairment in PD. Recent studies on the consumption of probiotics have reported a variety of beneficial effects on the central nervous system via the microbiota-gut-brain axis. In this study, we investigated the effects of oral administration of Bifidobacterium breve strain A1 [MCC1274] (B. breve A1) on the facilitation of hippocampal memory extinction observed in PD mice. We found that four-day consecutive oral administration of B. breve A1 restored facilitation of contextual fear extinction in PD mice. Hippocampal mRNA expression levels of postsynaptic density protein-95 and synaptophysin significantly decreased in the PD mice, but mRNA and protein expression levels of neuropsin increased. Furthermore, CA1 apical spine density was significantly reduced in PD mice. On the other hand, administration of B. breve A1 to PD mice recovered all these expression levels and the CA1 spine density to control levels. These results suggest that increased induction of neuropsin is involved in abnormal changes in hippocampal synaptic plasticity, and that B. breve A1 imposes reins on its expression, resulting in the restoration of abnormal hippocampal synaptic plasticity and the facilitation of fear extinction in PD mice.

Potential Effects of Indole-3-Lactic Acid, a Metabolite of Human Bifidobacteria, on NGF-induced Neurite Outgrowth in PC12 Cells.

Gut microbiota-derived tryptophan metabolites such as indole derivatives are an integral part of host metabolome that could mediate gut-brain communication and contribute to host homeostasis. We previously reported that infant-type Human-Residential Bifidobacteria (HRB) produced higher levels of indole-3-lactic acid (ILA), suggesting the former might play a specific role in microbiota-host crosstalk by producing ILA in human infants. Nonetheless, the biological meaning of bifidobacteria-derived ILA in infant health development remains obscure. Here, we sought to explore the potential role of ILA in neuronal differentiation. We examined the neurite outgrowth and acetylcholinesterase (AchE) activity of PC12 cells following exposure to ILA and NGF induction. We found that ILA substantially enhanced NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner, and had the most prominent effect at 100 nM. Significant increases in the expression of TrkA receptor, ERK1/2 and CREB were observed in ILA-treated PC12 cells, suggesting ILA potentiated NGF-induced neurite outgrowth through the Ras/ERK pathway. Additionally, ILA was found to act as the aryl hydrocarbon receptor (AhR) agonist and evoked NGF-induced neurite outgrowth in an AhR-mediated manner. These new findings provide clues into the potential involvement of ILA as the mediator in bifidobacterial host-microbiota crosstalk and neuronal developmental processes.

Heat-Killed Bifidobacterium breve B-3 Enhances Muscle Functions: Possible Involvement of Increases in Muscle Mass and Mitochondrial Biogenesis.

A previous clinical study on pre-obesity subjects revealed that Bifidobacterium breve B-3 shows anti-obesity effects and possibly increases muscle mass. Here, we investigated the effects of B-3 on muscle function, such as muscle strength and metabolism, and some signaling pathways in skeletal muscle. Male rodents were orally administered live B-3 (B-3L) or heat-killed B-3 (B-3HK) for 4 weeks. We found that administration of B-3 to rats tended to increase muscle mass and affect muscle metabolism, with stronger effects in the B-3HK group than in the B-3L group. B-3HK significantly increased muscle mass and activated Akt in the rat soleus. With regard to muscle metabolism, B-3HK significantly increased phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and cytochrome c oxidase (CCO) gene expression in the rat soleus, suggesting an effect on the AMPK-PGC1α-mitochondrial biogenesis pathway. Furthermore, B-3HK promoted oxidative muscle fiber composition in the gastrocnemius. We also observed a significantly higher level of murine grip strength in the B-3HK group than in the control group. These findings suggest the potential of heat-killed B-3 in promoting muscle hypertrophy and modifying metabolic functions, possibly through the Akt and AMPK pathways, respectively.

Neonatal oral fluid as a transmission route for bifidobacteria to the infant gut immediately after birth.

Bifidobacteria are one of the most abundant bacterial groups in the infant gut microbiota and are closely associated with infant health and can potentially affect health in later life. However, the details regarding the source of bifidobacteria have yet to be completely elucidated. This study aimed to assess neonatal oral fluid (OF) as a transmission route for bifidobacteria to the infant gut during delivery. Neonatal OF and infant feces (IF) were collected immediately and one month after birth from 15 healthy vaginally delivered newborns. Bifidobacterium strains were isolated from OF and IF samples, and the similarity of strains between the OF-IF pairs was evaluated based on the average nucleotide identity (ANI) value. The 16S rRNA gene sequencing results revealed the presence of Bifidobacteriaceae at >1% relative abundance in all OF samples. Bifidobacterium strains were isolated from OF (9/15) and IF (11/15) samples, and those sharing high genomic homology (ANI values >99.5%) between the neonatal OF and IF samples were present in one-third of the OF-IF pairs. The results of this study indicate that viable bifidobacteria are present in neonatal OF and that OF at birth is a possible transmission route of bifidobacteria to the infant gut.

Heat-killed Lactobacillus helveticus strain MCC1848 confers resilience to anxiety or depression-like symptoms caused by subchronic social defeat stress in mice.

The gut microbiota is involved in the pathogenesis of stress-related disorders. Probiotics can benefit the central nervous system via the microbiota-gut-brain axis, which raises the possibility that probiotics are effective in managing depression. In the present study, we examined the effects of heat-killed Lactobacillus helveticus strain MCC1848 in subchronic and mild social defeat stress (sCSDS) model mice (a widely used animal model of depression). MCC1848 supplementation significantly enhanced the interaction time in the social interaction test and sucrose preference ratio in the sucrose preference test, suggesting that MCC1848 improved anxiety- or depressive-like behaviors in sCSDS mice. The gene expression profile analysis of the nucleus accumbens, which plays an important role in stress resilience, indicated that MCC1848 ameliorated sCSDS-induced gene expression alterations in signal transduction or nervous system development. These findings suggest that MCC1848 supplementation is useful as a preventive strategy for chronic-stress-induced depression.

Therapeutic potential of Bifidobacterium breve strain A1 for preventing cognitive impairment in Alzheimer's disease.

It has previously been shown that the consumption of probiotics may have beneficial effects not only on peripheral tissues but also on the central nervous system and behavior via the microbiota-gut-brain axis, raising the possibility that treatment with probiotics could be an effective therapeutic strategy for managing neurodegenerative disorders. In this study, we investigated the effects of oral administration of Bifidobacterium breve strain A1 (B. breve A1) on behavior and physiological processes in Alzheimer's disease (AD) model mice. We found that administration of B. breve A1 to AD mice reversed the impairment of alternation behavior in a Y maze test and the reduced latency time in a passive avoidance test, indicating that it prevented cognitive dysfunction. We also demonstrated that non-viable components of the bacterium or its metabolite acetate partially ameliorated the cognitive decline observed in AD mice. Gene profiling analysis revealed that the consumption of B. breve A1 suppressed the hippocampal expressions of inflammation and immune-reactive genes that are induced by amyloid-ß. Together, these findings suggest that B. breve A1 has therapeutic potential for preventing cognitive impairment in AD.

Bovine lactoferrin ingestion protects against inflammation via IL-11 induction in the small intestine of mice with hepatitis.

Accumulating evidence suggests that orally ingested lactoferrin protects against inflammation. To assess the efficacy of orally administered bovine lactoferrin (bLF) against hepatitis and to identify the underlying mechanism, in the present study, we used four mouse models of hepatitis induced by d-galactosamine (GalN), carbon tetrachloride (CCl4), GalN plus lipopolysaccharide (LPS) and zymosan plus LPS. Intraperitoneal (i.p.) injection of GalN (500 mg/kg body weight) in mice treated with bovine serum albumin (BSA) for 14 d significantly increased serum aspartate aminotransferase (AST) concentrations compared with the untreated mice. However, orally administered bLF reduced AST concentrations compared with BSA treatment. In mice that received a single injection (0·4 ml/kg) and twice-weekly injections (0·08 ml/kg) of CCl4 for 24 weeks and pretreated with bLF for 14 d and 24 weeks, respectively, significantly suppressed alanine aminotransferase and AST concentrations were observed compared with the BSA-treated control. Oral administration of bLF for 14 d before i.p. injection of LPS (5 mg/kg) plus GalN (1 g/kg) significantly improved the survival rate. In mice that received intravenous injection of zymosan (25 mg/kg) and LPS (15 µg/kg) at 7 d intervals, bLF reduced the elevation of AST concentrations and enhanced the production of IL-11 and bone morphogenetic protein 2 in the small intestine compared with the BSA-treated control. To evaluate the effects of IL-11, we used IL-11 receptor α-null mice treated with GalN, CCl4 and zymosan plus LPS. In this group, the activity of bLF was not significantly different from that of BSA. These data indicate that orally ingested bLF enhances the expression of IL-11 in the small intestine and up-regulates protective activity in mice with hepatitis.

A lactoferrin-receptor, intelectin 1, affects uptake, sub-cellular localization and release of immunochemically detectable lactoferrin by intestinal epithelial Caco-2 cells.

Intelectin 1 (IntL) is known as a lectin expressed in intestinal epithelia and also as a receptor for an iron-binding protein, lactoferrin (LF). Uptake of LF with bound iron by enterocytes via receptor-mediated endocytosis has been well investigated, whereas subsequent fate of endocytized LF and LF/IntL complexes remains largely unknown. In the present study, we examined contribution of IntL to the uptake, sub-cellular localization and subsequent release of LF by intestinal Caco-2 IntL-transfectants using two-site ELISA and fluorescence confocal microscopy. LF taken up by IntL-transfectants was immunochemically detected mostly as intact protein in the cell lysates, and it was a little larger in amount than that of the mock-transfectants. In the IntL-transfectants cultured on porous membrane, LF taken up from the apical side was detected immunochemically as punctate signals in the apical-side cytoplasmic region near nucleus. The LF signals were co-localized with IntL and, in a time-dependent manner, partially with early endosome antigen 1 (EEA1), but not with alkaline phosphatase. LF taken up, retained and subsequently released by the IntL-transfectants was larger in amount than that of mock-transfectants. Moreover, uptake of LF altered sub-cellular localization of IntL and markedly enhanced the IntL signals within the cells.

Bovine lactoferrin induces interleukin-11 production in a hepatitis mouse model and human intestinal myofibroblasts.

PURPOSE: Orally administered bovine lactoferrin (bLF) exerts an anti-inflammatory effect on hepatitis and colitis animal models. To investigate the mechanism underlying the action of bLF, we explored the expression of inflammation-related factors in the intestine of a hepatitis mouse model after the oral administration of bLF and in several human intestinal cell lines treated with bLF. METHODS: The effects of bLF on the expression of interleukin-11 (IL-11) and bone morphogenetic protein 2 (BMP2) in the intestinal mucosa of a hepatitis mouse model as well as in cell cultures of human intestinal epithelial cells, myofibroblasts, and monocytes were examined using the real-time reverse transcription polymerase chain reaction. Epithelial cells and myofibroblasts were also cocultured using transwells. bLF transport, and IL-11 and BMP2 induction, as well as the interactions between the two cell types, were then analyzed after bLF treatment. RESULTS: In vivo, oral bLF administration increased the production of IL-11 and BMP2 in intestinal specimens. In vitro, bLF only stimulated the production of IL-11 in human intestinal myofibroblasts; i.e., it had no effect on BMP2 production in any cell type. In the transwell cocultures, bLF passed through the epithelium and directly stimulated IL-11 production in the myofibroblasts on the basolateral side. The IL-11 produced in the myofibroblasts subsequently acted protectively on the epithelial cells of the coculture. CONCLUSIONS: bLF upregulated the activity of anti-inflammatory factors, such as IL-11, in the intestine of a hepatitis mouse model and human intestinal myofibroblasts.

Oral administration of lactoferrin increases NK cell activity in mice via increased production of IL-18 and type I IFN in the small intestine.

Evidence that lactoferrin (LF) influences various immune functions is now accumulating. Recent reports have shown that bovine LF (BoLF) enhances antimicrobial, antiviral, and antitumor immune activities when orally administered. Here, we report that orally administered BoLF increases natural killer (NK) cell populations in peripheral blood and spleen in a dose-dependent manner and enhances interferon-gamma (IFN-gamma) production by NK cells. Using intraperitoneal (i.p.) injection of poly(I:C) to induce NK cell trafficking into the peritoneum, oral BoLF increased NK cell migration. Oral BoLF also produced an immediate increase in the levels of interleukin-18 (IL-18) in the portal circulation. In IL-18 knockout (KO) mice, BoLF did not increase the numbers of NK cells, although NK cell cytotoxic activity and poly(I:C)-induced trafficking activity were both enhanced by oral BoLF, even in IL-18 KO mice. Furthermore, oral BoLF increased the expression of IFN-alpha and IFN-beta in Peyer's patches (PP) and mesenteric lymph nodes (MLN). Oral administration of 2- chloroadenosine selectively depleted the PP cells and blocked the ability of oral BoLF to increase NK cell accumulation in the peritoneum following poly(I:C) i.p. injection. Collectively, these results demonstrate that orally administered BoLF stimulates intestine-associated immune functions, including the production of IL- 18 and type I IFNs and increased NK cell activity.