663 A Secreted Polysaccharide from Bifidobacterium Infantis 35624 Promotes Development of FOXP3+ T Regulatory Cells in Gnotobiotic Mice In Vivo.

Background& Aim: The enteric microbiota condition and prime the host immune response. We and others have previously demonstrated that selected microbial strains, such as Bifidobacterium infantis 35624, drive the development of immune-regulatory networks involving the induction of regulatoryT cells. While the exact mechanisms underpinning this immunological effect are unclear, recent reports suggest that commensal polysaccharides modulate host immune maturation. Thus, the aim of our study was to determine if polysaccharides from Bifidobacterium infantis 35624 could stimulate T regulatory cells In Vivo. Methods: Cell-free supernatants from Bifidobacterium infantis 35624 were generated and a high molecular weight extra-cellular polysaccharide (EPS)-rich fraction was isolated using standard biochemical techniques. THP-1 dendritic cells were exposed to EPS and NF-?B activation measured using the secreted embryonic alkaline phosphatase (SEAP) reporter system. Cell viability was determined using the alamar blue assay while cytokine production was measured using MSD kits. EPS was co-incubated with LPS to assess if EPS could inhibit LPS-induced cellular activation. In addition, EPS was administered orally (20mg/kg) to Swiss Webster germ-free animals and induction of Foxp3+ T regulatory cells monitored using RT-PCR and flow cytometry. Cytokine production was measured using RT-PCR and MSD kits. Results: The EPS preparation was confirmed to be DNA-free and LPS-free with minimal protein contamination. EPS did not induce NF-?B activation or cytokine secretion by THP-1 cells and was non-toxic over a wide range of concentrations tested. A modest attenuation of LPS-induced NF-?B activation (approx. 40% reduction) was observed with =250pg/ml EPS. Administration of the EPS fraction to germ-free mice resulted in the increased expression of Foxp3 gene expression within the ileum. Flow cytometric assessment of splenic CD4+/CD25+/ Foxp3+ T cell subsets revealed a significant increase in regulatory T cell numbers in EPS-fed germ-free animals (3.4+/-0.3%, n=15) compared to saline-treated controls (1.6+/-0.2%, n=12). In contrast, no differences in splenic cytokine secretion was observed in the EPS-treated animals. Conclusions:Apolysaccharide enriched fraction fromBifidobacterium infantis 35624 enhances the expression of Foxp3+ T regulatory cells In Vivo and is not pro-inflammatory In Vitro. The results provide further support for the distinct nature and beneficial aspects of this strain of Bifidobacterium and underscore the importance of strain-specificity in the context of probiotic function.