Treg-inducing capacity of genomic DNA of Bifidobacterium longum subsp. infantis.

Background: Allergic and autoimmune diseases comprise a group of inflammatory disorders caused by aberrant immune responses in which CD25+ forkhead box P3-positive regulatory T cells (Treg) cells that normally suppress inflammatory events are often poorly functioning. This has stimulated an intensive investigative effort to find ways of increasing Tregs as a method of therapy for these conditions. Commensal microbiota known to have health benefits in humans include the lactic acid-producing, probiotic bacteria B. longum subsp. infantis and Lactobacillus rhamnosus. Mechanistically, several mechanisms have been proposed to explain how probiotics may favorably affect host immunity, including the induction of Tregs. Analysis of emerging data from several laboratories, including our own, suggest that DNA methylation may be an important determinant of immune reactivity responsible for Treg induction. Although methylated CpG moieties in normal mammalian DNA are both noninflammatory and lack immunogenicity, unmethylated CpGs, found largely in microbial DNA, are immunostimulatory and display proinflammatory properties. Objective: We hypothesize that microbiota with more DNA methylation may potentiate Treg induction to a greater degree than microbiota with a lower content of methylation. The purpose of the present study was to test this hypothesis by studying the methylation status of whole genomic DNA (gDNA) and the Treg-inducing capacity of purified gDNA in each of the probiotic bacteria B. longum subsp. infantis and L. rhamnosus, and a pathogenic Escherichia coli strain B. Results: We showed that gDNA from B. longum subsp. infantis is a potent Treg inducer that displays a dose-dependent response pattern at a dose threshold of 20 µg of gDNA. No similar Treg-inducing responses were observed with the gDNA from L. rhamnosus or E. coli. We identified a unique CpG methylated motif in the gDNA sequencing of B. longum subsp. infantis which was not found in L. rhamnosus or E. coli strain B. Conclusion: Although the literature indicates that both B. longum subsp. infantis and L. rhamnosus strains contribute to health, our data suggest that they do so by different mechanisms. Further, because of its small molecular size, low cost, ease of synthesis, and unique Treg-inducing feature, this methylated CpG oligodeoxynucleotide (ODN) from B. longum would offer many attractive features for an ideal novel therapeutic vaccine candidate for the treatment of immunologic diseases, such as the allergic and autoimmune disorders, in which Treg populations are diminished.

Pre-treatment with Bifidobacterium infantis and its specific antibodies enhance targeted radiosensitization in a murine model for lung cancer.

PURPOSE: The hypoxic microenvironments of solid tumours are complex and reduce the susceptibility of cancer cells to chemo- and radiotherapy. Conventional radiosensitisers have poor specificity, unsatisfactory therapeutic effects, and significant side effects. Anaerobic bacteria colonise and destroy hypoxic areas of the tumour and consequently enhance the effects of radiation. METHODS: In this study, we treated a Lewis lung carcinoma transplant mouse model with Bifidobacterium infantis (Bi) combined with its specific monoclonal antibody (mAb) and radiotherapy (RT) to investigate its ability to radiosensitise the tumour. The tumour metabolism and hypoxia in the tumour tissue were monitored by micro-18F-FDG and 18F-FMISO PET/CT imaging. Immunohistochemistry was used to detect phosphorylated histone (γ-H2AX), proliferation (Ki-67), platelet endothelial cell adhesion molecules (CD31), tumour necrosis factor-α (TNF-α), hypoxia-inducible factor-1α (HIF-1α), and glucose transporter 1 (Glut-1) levels. RESULTS: Tumour growth was slowed and survival time was markedly prolonged in mice subjected to the combination of B. infantis, specific antibody, and radiotherapy. Levels of HIF-1α, Glut-1, Ki-67, and CD31 expression, as well as uptake of FDG and FMISO, were the lowest in the combination-treated mice. In contrast, γ-H2AX and TNF-α expression levels were elevated and hypoxia in tumour tissue was reduced compared with controls. CONCLUSION: In conclusion, our data indicated that the curative effect of radiotherapy for lung cancer was enhanced by pre-treating mice with a combination of B. infantis and its specific monoclonal antibody.

Mechanism of the Immunomodulatory Effect of the Combination of Live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus on Immunocompromised Rats.

Immunodeficiency is a very common condition in suboptimal health status and during the development or treatment of many diseases. Recently, probiotics have become an important means for immune regulation. The present study aimed to investigate the mechanism of the immunomodulatory effect of a combination of live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus (CBLEB), which is a drug used by approximately 10 million patients every year, on cyclophosphamide-immunosuppressed rats. Cyclophosphamide (40 mg/kg) was intraperitoneally injected to induce immunosuppression in a rat model on days 1, 2, 3, and 10. Starting from day 4, the rats were continuously gavaged with CBLEB solution for 15 days. The samples were collected to determine routine blood test parameters, liver and kidney functions, serum cytokine levels, gut microbiota, fecal and serum metabolomes, transcriptomes, and histopathological features. The results indicated that CBLEB treatment reduced cyclophosphamide-induced death, weight loss, and damage to the gut, liver, spleen, and lungs and eliminated a cyclophosphamide-induced increase in the mean hemoglobin content and GGT, M-CSF, and MIP-3α levels and a decrease in the red blood cell distribution width and total protein and creatinine levels in the blood. Additionally, CBLEB corrected cyclophosphamide-induced dysbiosis of the gut microbiota and eliminated all cyclophosphamide-induced alterations at the phylum level in rat feces, including the enrichment in Proteobacteria, Fusobacteriota, and Actinobacteriota and depletion of Spirochaetota and Cyanobacteria. Furthermore, CBLEB treatment alleviated cyclophosphamide-induced alterations in the whole fecal metabolome profile, including enrichment in 1-heptadecanol, succinic acid, hexadecane-1,2-diol, nonadecanoic acid, and pentadecanoic acid and depletion of benzenepropanoic acid and hexane. CBLEB treatment also alleviated cyclophosphamide-induced enrichment in serum D-lyxose and depletion of serum succinic acid, D-galactose, L-5-oxoproline, L-alanine, and malic acid. The results of transcriptome analysis indicated that the mechanism of the effect of CBLEB was related to the induction of recovery of cyclophosphamide-altered carbohydrate metabolism and signal transduction. In conclusion, the present study provides an experimental basis and comprehensive analysis of application of CBLEB for the treatment of immunodeficiency.

Treatment of Guillain-Barré syndrome with Bifidobacterium infantis through regulation of T helper cells subsets.

BACKGROUND: Guillain-Barré syndrome (GBS) is a rare, autoimmune-mediated disease. The use of Bifidobacterium is reportedly effective in alleviating GBS since they act by regulating T helper (Th) cells. OBJECTIVES: In this study, we explored the differentiation of T helper cell subsets in patients with GBS. We also evaluated the effect of GBS on Bifidobacterium levels in patients and the likely protective influence of this bacterium in alleviating the disease in an animal model. MATERIALS AND METHODS: We used flow cytometry, and real-time polymerase chain reaction (PCR) to determine the T cell subsets differentiation among 30 GBS patients and 20 healthy controls (HC). The concentration of Bifidobacterium was assayed by real-time PCR. Experimental autoimmune neuritis (EAN) animal model was established to support the protective role of Bifidobacterium in GBS. RESULTS: The expression of Th cells, Th2 and Th17 in the patients was significantly higher than that in the HC, while Treg cells decreased substantially. Moreover, the levels of Bifidobacterium in the GBS patients were considerably lower than those in the HC, the concentration of Bifidobacterium correlating with Th2 and Th17 subsets negatively. Treatment with Bifidobacterium significantly reduced the levels of Th2 and Th17 and promoted the levels of Treg cells. CONCLUSIONS: We concluded from this study that Bifidobacterium alleviated GBS by regulating Th cells, although in-depth studies might be required to fully understand the mechanism of action.

Probiotics SOD inhibited food allergy via downregulation of STAT6-TIM4 signaling on DCs.

Bacterial probiotics are of increasing use against intestinal disorders such as food allergy. However, the detailed molecular mechanism underlying probiotics-mediated anti-allergic effect remains unknown. In the present study, we orally treated OVA-sensitized mice with Bifidobacterium infantis (BB) for two weeks. It was found that OVA specific-IgE and-IgG levels in serum were significantly decreased after BB administration. BB treatment also significantly reduced the release of IL-4, -5, -13 in splenocytes. Besides, after challenge with OVA, the occurrence of temperature drop and diarrhea was dramatically reduced in BB group. Moreover, the protective effect of BB on allergic mice was correlated with its antioxidative enzyme, superoxide dismutase (SOD). The antioxidative effect of BB on Dendritic cells (DCs0 was further demonstrated to be mediated by cAMP/PKA signaling. We also found that the mRNA and protein expression levels of TIM4 were attenuated in BB group. Finally, ChIP-qPCR assay studies indicate that BB reduced the binding of STAT6 to its response elements in the TIM4 promoter. In conclusion, orally administration of BB protected allergic mice via attenuation of oxidative stress, which further reduced TIM4 expression by inhibiting its transcription factor STAT6.