Bifidobacterium Longum: Protection against Inflammatory Bowel Disease.

The prevalence of inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), increases gradually worldwide in the past decades. IBD is generally associated with the change of the immune system and gut microbiota, and the conventional treatments usually result in some side effects. Bifidobacterium longum, as colonizing bacteria in the intestine, has been demonstrated to be capable of relieving colitis in mice and can be employed as an alternative or auxiliary way for treating IBD. Here, the mechanisms of the Bifidobacterium longum in the treatment of IBD were summarized based on previous cell and animal studies and clinical trials testing bacterial therapies. This review will be served as a basis for future research on IBD treatment.

Gene Networks Underlying the Resistance of Bifidobacterium longum to Inflammatory Factors.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host's immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter's growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro. By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

BACKGROUND: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections. METHODS: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified. FINDINGS: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain. INTERPRETATION: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung. FUNDING: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE).

Maturation of Gut Microbiota and Circulating Regulatory T Cells and Development of IgE Sensitization in Early Life.

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium. These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed.

Modulation of T Regulatory and Dendritic Cell Phenotypes Following Ingestion of Bifidobacterium longum, AHCC® and Azithromycin in Healthy Individuals.

The probiotic Bifidus BB536 (BB536), which contains Bifidobacterium longum, has been shown to have enhanced probiotic effects when given together with a standardized extract of cultured Lentinula edodes mycelia (AHCC®, Amino Up Co. Ltd., Sapporo, Japan). BB536 and AHCC® may modulate T cell and dendritic cell (DC) phenotypes, and cytokine profiles to favour anti-inflammatory responses following antibiotic ingestion. We tested the hypothesis that orally administered BB536 and/or AHCC®, results in modulation of immune effector cells with polarisation towards anti-inflammatory responses following antibiotic usage. Forty healthy male volunteers divided into 4 equal groups were randomised to receive either placebo, BB536, AHCC® or a combination for 12 days in a double-blind manner. After 7 days volunteers also received 250 mg azithromycin for 5 days. Cytokine profiles from purified CD3+ T cells stimulated with PDB-ionomycin were assessed. CD4+ CD25+ forkhead box P3 (Foxp3) expression and peripheral blood DC subsets were assessed prior to treatment and subsequently at 7 and 13 days. There was no difference in cytokine secretion from stimulated CD3+ T cells between treatment groups. Compared with baseline, Foxp3 expression (0.45 ± 0.1 vs. 1.3 ± 0.4; p = 0.002) and interferon-gamma/interleukin-4 (IFN-γ/IL-4) ratios were increased post-treatment in volunteers receiving BB536 (p = 0.031), although differences between groups were not significant. For volunteers receiving combination BB536 and AHCC®, there was an increase in myeloid dendritic cells (mDC) compared with plasmacytoid DC (pDC) counts (80% vs. 61%; p = 0.006) at post treatment time points. mDC2 phenotypes were more prevalent, compared with baseline, following combination treatment (0.16% vs. 0.05%; p = 0.002). Oral intake of AHCC® and BB536 may modulate T regulatory and DC phenotypes to favour anti-inflammatory responses following antibiotic usage.

Immunomodulatory Effects by Bifidobacterium longum KACC 91563 in Mouse Splenocytes and Macrophages.

The present study evaluates the immunomodulatory effect of Bifidobacterium longum KACC 91563 in murine primary splenocytes and macrophages. B. longum KACC 91563 regulated Tand B-cell proliferation and inhibited the Th1 (IL-2, IFN-γ)/Th2 (IL-4, IL-10) cytokine imbalance and immune cytokine production. Moreover, immunoglobulin E (IgE) levels were significantly lower after treatment with B. longum KACC 91563. These findings suggest that B. longum KACC 91563 could modulate the systemic immune system toward both IgE production and regulation of the Th1/Th2 balance.

Probiotic Supplementation During Human Pregnancy Affects the Gut Microbiota and Immune Status.

The consumption of probiotics and fermented foods has been very popular in recent decades. The primary aim of our study was to evaluate the effect of probiotics on the gut microbiota and the changes in inflammatory cytokines after an average of 6.7 weeks of probiotic administration among normal pregnant women. Thirty-two healthy pregnant women at 32 weeks of gestation were recruited and divided into two groups. The probiotic group ingested combined probiotics until after birth. The base characteristics of the probiotics and control groups showed no significant differences. The structure of the fecal microbiota at the genus level varied during the third trimester, and administration of probiotics had no influence on the composition of the fecal microbiota however, many highly abundant taxa and core microbiota at the genus level changed in the probiotic group when compared to the control group. The analysis of cytokines showed that IL-5, IL-6, TNF-α, and GM-CSF had equal levels between the baseline and control groups but were significantly increased after probiotic administration (baseline = control < probiotics). Additionally, levels of IL-1ß, IL-2, IL-12, and IFN-γ significantly increased among the three groups (baseline < control < probiotics). This result demonstrated that probiotics helped to shift the anti-inflammatory state to a pro-inflammatory state. The correlation analysis outcome suggested that the relationship between the microbiota and the cytokines was not strain-dependent. The gut microbiota varied during the third trimester. The probiotics demonstrated immunomodulation effects that helped to switch over to a pro-inflammatory immune state in the third trimester, which was important for labor.

Influence of monostrain and multistrain probiotics on immunity, intestinal ultrastructure and microbiota in experimental dysbiosis.

The biological effects of three probiotic strains Lactobacillus rhamnosus K32, Bifidobacterium longum GT15, Enterococcus faecium L3 and their mixture were studied using a model of dysbiosis induced in rats by antibiotics. It was found that after taking different probiotics intestinal microbiota changed in a strain-specific manner. The maximal activity against pathogens was revealed after the administration of a mixture of bacterial strains under study or a single strain of enterococci. The strain E. faecium L3 showed the most activity against both Klebsiella spp. and Bacteroides fragilis. It helped to restore the original content of Faecalibacterium prausnitzii. The number of Klebsiella spp. was the same in the group receiving L. rhamnosus K32 and the group of animals, which was not consuming probiotics. Different probiotic strains included in the composition had various immunological effects. Probiotic bifidobacteria, enterococci and the mixture of three probiotics stimulated of mRNA expression of interleukin (IL)-10 in mesenteric lymph nodes. The changes in microbiota after consuming an enterococcal probiotic correlated with an increase in transforming growth factor (TGF)-ß and IL-10 content in blood serum and an increase of the intestinal mucus layer. Consumption of L. rhamnosus K32 led to the stimulation of IL-8 expression in mesenteric lymph nodes. Control group not receiving probiotics was characterised by expression of pro-inflammatory cytokines, damage of epithelial cells and the destruction of their tight junctions. The damage to the ultrastructure of the mucosa was prevented in all the groups taking probiotics.

Targeting the gut microbiome to treat the osteoarthritis of obesity.

Obesity is a risk factor for osteoarthritis (OA), the greatest cause of disability in the US. The impact of obesity on OA is driven by systemic inflammation, and increased systemic inflammation is now understood to be caused by gut microbiome dysbiosis. Oligofructose, a nondigestible prebiotic fiber, can restore a lean gut microbial community profile in the context of obesity, suggesting a potentially novel approach to treat the OA of obesity. Here, we report that - compared with the lean murine gut - obesity is associated with loss of beneficial Bifidobacteria, while key proinflammatory species gain in abundance. A downstream systemic inflammatory signature culminates with macrophage migration to the synovium and accelerated knee OA. Oligofructose supplementation restores the lean gut microbiome in obese mice, in part, by supporting key commensal microflora, particularly Bifidobacterium pseudolongum. This is associated with reduced inflammation in the colon, circulation, and knee and protection from OA. This observation of a gut microbiome-OA connection sets the stage for discovery of potentially new OA therapeutics involving strategic manipulation of specific microbial species inhabiting the intestinal space.

The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.

Anti-PD-1-based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients' microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.

Impact of ageing and a synbiotic on the immune response to seasonal influenza vaccination; a randomised controlled trial.

BACKGROUND & AIMS: Ageing increases risk of respiratory infections and impairs the response to influenza vaccination. Pre- and pro-biotics offer an opportunity to modulate anti-viral defenses and the response to vaccination via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486, combined with a prebiotic, gluco-oligosaccharide, on the B and T cell response to seasonal influenza vaccination in young and older subjects . METHODS: In a double-blind, randomized controlled trial, 58 young (18-35 y) and 54 older (60-85 y) subjects were supplemented with the synbiotic for 8 weeks. At 4 weeks they were administered with a seasonal influenza vaccine. B and T cell phenotype and responsiveness to in vitro re-stimulation with the vaccine were assessed at baseline, 4, 6 and 8 weeks. RESULTS: B and T cell profiles differed markedly between young and older subjects. Vaccination increased numbers of memory, IgA+ memory, IgG+ memory and total IgG+ B cells in young subjects, but failed to do so in older subjects and did not significantly alter T cell subsets. Seroconversion to the H1N1 subunit in the older subjects was associated with higher post-vaccination numbers of plasma B cells, but seroconversion was less consistently associated with T cell phenotype. B and T cell subsets from both young and older subjects demonstrated a strong antigen-specific recall challenge, and although not influenced by age, responsiveness to the recall challenge was associated with seroconversion. In older subjects, CMV seropositivity was associated with a significantly lower recall response to the vaccine, but the synbiotic did not affect the responsiveness of B or T cells to re-stimulation with influenza vaccine. CONCLUSIONS: Antigen-specific B and T cell activation following an in vitro recall challenge with the influenza vaccine was influenced by CMV seropositivity, but not by a synbiotic. Registered under ClinicalTrials.gov Identifier no. NCT01066377.

Antitumor effect of oral cancer vaccine with Bifidobacterium delivering WT1 protein to gut immune system is superior to WT1 peptide vaccine.

Despite the revolutionary progress of immune checkpoint inhibitors (CPIs) for cancer immunotherapy, CPIs are effective only in a subset of patients. Combining CPIs and cancer vaccines to achieve better clinical outcomes is a reasonable approach since CPI enhances cancer vaccine-induced tumor-associated antigen (TAA) specific CTL. Among the various TAAs so far identified, WT1 protein is one of the most promising TAAs as a cancer vaccine target. Until now clinical trials of WT1 vaccine have demonstrated only modest clinical efficacy. These WT1 vaccines were based on peptides or dendritic cells (DCs), and there was no oral cancer vaccine. Recently, we developed a WT1 oral cancer vaccine using a recombinant Bifidobacterium displaying WT1 protein, which can efficiently deliver WT1 protein to the gut immune system, and we demonstrated that this oral cancer vaccine had a significant anti-tumor effect in a C1498-WT1 murine leukemia syngeneic tumor model. The WT1 protein displayed in this vaccine consists of about 70% of the WT1 amino acid sequence including multiple known CD4 and CD8 T-cell epitopes of WT1. In this commentary, we introduce our recent data indicating the superior anti-tumor effect of a WT1 oral cancer vaccine delivering WT1 protein to the gut immune system compared to a peptide vaccine.

Long-term safety and efficacy of perinatal probiotic intervention: Evidence from a follow-up study of four randomized, double-blind, placebo-controlled trials.

BACKGROUND: Societies worldwide are faced with a progressive increase in immune-mediated health problems such as allergic, autoimmune, and inflammatory diseases, as well as obesity. Perinatal administration of specific probiotic bacteria is an attractive approach in reducing the risk of these conditions, but long-term efficacy and safety data are lacking. The aim here was to evaluate the clinical benefit and long-term safety of specific probiotics administered during the perinatal period. METHODS: The probiotic strains used were Lactobacillus rhamnosus GG, Bifidobacterium lactis Bb-12, Lactobacillus paracasei ST11, and Bifidobacterium longum BL999. The children involved have subsequently undergone prospective long-term follow-up. In addition to physical examination, data were collected by structured questionnaires on non-communicable diseases and continued probiotic use, and growth data from welfare clinics and school nurses. RESULTS: Altogether 303 mother-infant pairs were included in the analysis. Seventy-six of 163 (47%) children receiving perinatal probiotics had developed allergic disease compared with 79 of 140 (56%) receiving placebo (OR 0.67, 95% confidence intervals [CI] 0.43-1.06, p = 0.09). Fifty-nine of 133 (44%) children receiving L. rhamnosus GG perinatally had developed allergic disease, OR 0.62, 95% CI 0.38-0.99, p = 0.047, as compared to placebo. We found no differences in growth or non-communicable disease prevalence between children receiving perinatally probiotics or placebo. CONCLUSIONS: Perinatal probiotic administration is safe in long-term follow-up. Children receiving L. rhamnosus GG perinatally tended to have decreased allergy prevalence.

The Surface-Associated Exopolysaccharide of Bifidobacterium longum 35624 Plays an Essential Role in Dampening Host Proinflammatory Responses and Repressing Local TH17 Responses.

The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immune-regulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPSneg) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPSneg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPSneg did not protect against the development of colitis, with associated enhanced recruitment of IL-17+ lymphocytes to the gut. Moreover, intranasal administration of sEPSneg also resulted in enhanced recruitment of IL-17+ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local TH17 responses. IMPORTANCE: Particular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensal-host interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surface-associated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses.

Bifidobacterium bifidum R0071 decreases stress-associated diarrhoea-related symptoms and self-reported stress: a secondary analysis of a randomised trial.

Psychological stress is associated with gastrointestinal (GI) distress. This secondary analysis from a randomised, double-blind, placebo-controlled study examined whether three different probiotics could normalise self-reported stress-associated GI discomfort and reduce overall self-reported stress. Undergraduate students (n=581) received Lactobacillus helveticus R0052, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium bifidum R0071, or placebo. Participants self-reported 2 outcomes for a 6-week period, which included final academic exams: daily level of stress (0=no stress to 10=extremely stressed) and weekly three diarrhoea-related symptoms (DS, 1=no discomfort to 7=severe discomfort) using the GI Symptom Rating Scale. Self-reported stress was positively related to DS (P=0.0068). Mean DS scores were lower with B. bifidum versus placebo at week 2 at the average level of stress and the average body mass index (BMI). DS scores were lower with B. bifidum at week 5 versus week 0 and 1 and with B. infantis R0033 at week 6 versus week 0. DS scores were higher when antibiotics were used in the prior week with placebo (P=0.0092). DS were not different with or without antibiotic use with the probiotics. Only B. bifidum had an effect on self-reported stress scores (P=0.0086). The self-reported stress score was also dependent on hours of sleep per day where it decreased by 0.13 for each additional hour of sleep. During a stressful period, B. bifidum R0071 decreases DS and self-reported stress scores. This trial was registered at clinicaltrials.gov as NCT01709825.

Role of NLRP3 inflammasome in Bifidobacterium longum-regulated visceral hypersensitivity of postinfectious irritable bowel syndrome.

We aimed to explore the role of NLRP3 inflammasome in Bifidobacterium longum-regulated visceral hypersensitivity of postinfectious irritable bowel syndrome (PI-IBS). Fifty NIH mice were divided into five groups (n = 10). The visceral sensitivities of PI-2W and PI-8W groups significantly exceeded than those of normal control groups (P < 0.05), which was significantly decreased in PI-B group (P < 0.05). Significantly more IL-18 and IL-1ß were expressed in PI-2W and PI-8W groups than those in normal control groups (P < 0.05), which was significantly decreased in PI-B group (P < 0.05). Bifidobacterium longum may down-regulate IL-18 and IL-1ß expressions by inhibiting NLRP3 inflammasome and thus reduce the visceral hypersensitivity of PI-IBS.