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 immuneregulatory 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 sEPS neg ) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPS neg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPS neg did not protect against the development of colitis, with associated enhanced recruitment of IL-17 ؉ lymphocytes to the gut. Moreover, intranasal administration of sEPS neg 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 T H 17 responses. IMPORTANCEParticular 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 commensalhost 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 surfaceassociated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses. T he gut microbiota contributes significantly to host health via multiple mechanisms, including the digestion of foods, competitive exclusion of pathogens, enhancement of epithelial cell differentiation, and promotion of mucosa-associated lymphoid tissue proliferation (1, 2). Furthermore, accumulating evidence suggests that the composition and metabolic activity of the gut microbiota has profound effects on proinflammatory activity and the induction of immune tolerance within mucosal tissue (3-5). Certain microbes induce regulatory responses, while others induce effector responses, resulting in the case of healthy individuals in a balanced homeostatic...

Adhesion and anti-inflammatory properties of eight strains of bifidobacteria were tested using the intestinal epithelial cell lines Caco-2, T84, and HT29. Two strains were selected for further assessment of their antiinflammatory capacity in two murine models of colitis. In vivo results confirmed the high anti-inflammatory capacity of a Bifidobacterium bifidum strain.Bifidobacteria are an important group of the intestinal microbiota (21). Several beneficial health effects have been claimed to be based on the presence of bifidobacteria in the colon (reviewed in reference 16), and thus, they become increasingly interesting for probiotic applications in pharmaceutical and dairy products. A promising application is their use in probiotic intervention in inflammatory bowel disease (IBD). Probiotics containing bifidobacteria have been shown to be effective in reducing the severity of inflammation in several rodent models of IBD and in patients with IBD (3,[6][7][8][9]12,19). Several studies have reported an upregulation of the receptors for bacterial lipopolysaccharide (LPS), CD14, and TLR4 in the intestinal epithelium in murine models of IBD and in patients with IBD (4, 13, 15), suggesting a contribution of abnormal LPS stimulation to chronic intestinal inflammation. In a previous study, we were able to show that different strains of bifidobacteria are able to inhibit LPS-induced inflammatory events in intestinal epithelial cells (IECs) (18). Besides the manufacturing criteria, shelf life and gut transit, two main characteristics are crucial for selecting potential probiotic strains: (i) the desired probiotic property, in the case of IBD, an anti-inflammatory effect, and (ii) high adhesion to the intestinal mucosa (reviewed in reference 10).Adhesion to IEC lines. Here, we present our data from an in vitro analysis of eight strains of bifidobacteria covering the major species isolated from human fecal samples. All of the strains were tested for adhesion and anti-inflammatory effects using Caco-2, T84, and HT-29 IECs. For adhesion experiments, Caco-2, T84, and HT-29 cells were grown in 24-well tissue culture plates as described previously (17,18). Bifidobacterium adolescentis NCC251, B. lactis NCC362, B. longum NCC2705, B. bifidum NCC189, S16, and S17, B. longum/infantis E18, and B. breve MB226 (all described previously [18]) were grown to stationary phase at 37°C in MRS medium supplemented with 0.5 g cysteine/liter (MRSC) in anaerobic jars using Anaerocult A (Merck). Bacteria were washed three times with phosphate-buffered saline (PBS) and resuspended at 1 ϫ 10 8 CFU/ml RPMI medium (Gibco) supplemented with 1% nonessential amino acids (Gibco). One-milliliter aliquots were incubated with the IECs, i.e., a bacterium-to-cell ratio of 100:1, for 1 h. After three washings with PBS to remove nonadherent bacteria, IEC monolayers were lysed and bacterial adhesion was quantified in serial dilutions of the lysates by plate counts on MRSC agar. Adhesion was expressed as percent adherent bacteria relative to the initially added C...

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.