Evidence for the Modulation of the Immune Response in Peripheral Blood Mononuclear Cells after Stimulation with a High Molecular Weight β-glucan Isolated fromLactobacillus fermentumLf2

Abstract

27Lactobacillus fermentum Lf 2 produces large amounts of exopolysaccharides under 28 optimized conditions (~2 g/L, EPS) which have been shown to possess 29 immunomodulatory activity. In this study, the crude EPS was fractionated to give a 30 high molecular weight (HMw) homoglycan and a mixture of medium molecular 31 weight heteroglycans. The HMw EPS was isolated and identified as a -glucan. 32 Peripheral blood mononuclear cells (PBMC) were pre-treated with purified 33 polysaccharide to determine if the HMw -glucan is responsible for the 34 immunomodulatory activity. Cells were also stimulated with either lipopolysaccharide 35 (LPS) or phytohemagglutinin (PHA) and their effects, both with and without -glucan 36 pre-treatment, compared. 37Exposure of the cells to -glucan increased their metabolic activity and whilst a small 38 but statistically significant drop in CD14 expression was observed at Day 1, the 39 levels were significantly elevated at Day 2. High levels of CD14 expression were 40 observed in cells initially exposed to the -glucan and subsequently stimulated with 41 either LPS or PHA. In contrast, reduced levels of TLR-2 expression were observed 42 for cells initially exposed to the -glucan and subsequently stimulated with LPS. 43TNF-α levels were elevated in -glucan treated cells (Day1) with the levels dropping 44 back once the -glucan had been removed (Day 2). The stimulants LPS and PHA 45 both induced significant rises in TNF- levels, however, this induction was 46 completely (LPS) or partially blocked (PHA) in -glucan pre-treated cells. 47 IMPORTANCE 50The use of foods containing probiotics (i.e. live microorganisms providing health 51 benefits) is growing in popularity. Whilst there is empirical evidence identifying 52 specific microorganisms as having beneficial effects, the precise mechanisms by 53 which these strains exert influence is not well understood. One of the problems is 54 that there are a large number of structures presented at the surface of 55 microorganisms which can interact with the host and which can initiate a host 56 response. In the current work, we addressed this issue by choosing a potential 57 probiotic strain of bacteria associated with cheese production, L. fermentum Lf2, 58 which produces large quantities of polysaccharides at its surface. From this 59 polysaccharide mixture, we have isolated, purified and characterised a HMw -60 glucan and demonstrated that it has immunomodulatory activity. This study 61 demonstrates that the -glucan produced by L. fermentum Lf2 can modify the 62 immune response of PBMC cells when subjected to a bacterial endotoxin. 63 64 4 1. Introduction 65 Species of Lactobacillus fermentum are obligate heterofermentative Gram-positive 66bacteria that can be isolated from a wide variety of sources and include members 67 that are normal inhabitants of the human microflora (1; 2). A significant number of 68 strains are closely associated with the production of fermented foods (3-7) and a 69 number have been exploited for their probioti...

Exopolysaccharides Produced by Lactic Acid Bacteria: From Biosynthesis to Health-Promoting Properties

“…This has been confirmed by studies showing that phosphate is the molecule that triggers the immune response, as chemical dephosphorylation of these HePS leads to a decrease in the stimulatory effect [ 168 , 171 , 190 , 194 , 195 ]. However, immunomodulatory activity has also been associated with high molecular weight HePS, which appears to suppress the immune response [ 168 , 171 , 185 , 190 , 196 , 197 , 198 ]. In addition, the monosaccharide composition of EPS has been shown to influence its anti-inflammatory properties, possibly by association with the recognition of receptors on the surface of immune cells [ 199 , 200 ].…”

Unravelling the Immunotoxicity of Polycaprolactone Nanoparticles—Effects of Polymer Molecular Weight, Hydrolysis, and Blends

“…The results from both cytokine and NO production inhibition, showed an anti-inflammatory activity of PCL 2 /Glu NPs which contrasts with all other PCL-based NPs. As β-glucans are widely reported in the literature to induce TNF-α release in PBMCs, − the fact that PCL 2 /Glu NPs did not induce TNF-α production, and even inhibited its release when stimulated by LPS was a surprising result. However, this phenomenon has been previously reported.…”

Structural characterisation of two medium molecular mass exopolysaccharides produced by the bacterium Lactobacillus fermentum Lf2

“…composition of the EPS and in characterising the polysaccharides present. In an earlier study, we reported that the crude EPS contains a mixture of both high and medium molecular mass polysaccharides and identified the high molecular mass polysaccharide (HMMP) as a glucan, which presented biological activity [22]. In this paper, we report the characterisation of the medium molecular mass polysaccharides (MMMP).…”

“…The sample eluted as two main peaks: HMMP eluted in the early fractions and MMMP eluted in tubes 25-30. The characterisation of the structure of the HMMP has already been reported [22]. USA) was used for the data analysis.…”

“…Determination of the purity of the MMMP EPS fraction and its weight average molecular mass.Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALLS-Wyatt technology, Santa Barbara, CA, USA) was used to determine the purity of the fractions; the procedures employed have been reported in a previous paper[22]. Briefly, EPS(1 mg.mL -1 ) were prepared in aq.…”