Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function

Abstract

Objectives: Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in selected bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of selected bifidobacteria on HMO stimulates the intestinal epithelium.

Methods: Caco-2 and HT-29 cells were incubated with lactose (LAC)- or HMO-grown Bifidobacterium longum subsp infantis (B infantis) or B bifidum. Bacterial adhesion and translocation were measured by real-time quantitative polymerase chain reaction. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real-time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy.

Results: We showed that HMO-grown B infantis had a significantly higher rate of adhesion to HT-29 cells compared with B bifidum. B infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand-1. Both B infantis and B bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin-10 compared with LAC-grown bacteria in Caco-2 cells. B bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cells and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation.

Conclusions: The study provides evidence for the specific relation between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction.

Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1

Adhesion of B. bifidum and B. infantis grown on LAC or HMO to the Caco-2 (a) or HT-29 (b) cells. Bifidobacteria were incubated with fully differentiated epithelial cells for 2h at 37°C in anaerobic conditions. Quantification of the bacterial genomes was based on the presence of 16s rRNA. The results are presented as the percentage of bacteria recovered vs. applied on monolayers. Values are the mean ± SD and * indicates statistical significance (p < 0.05).

Figure 2

Analysis of the effects of HMO- grown bifidobacteria on tight junction protein mRNA expression in Caco-2 (a) or HT-29 (b) cells. Cells were incubated for 2h, then the expression was measured by real-time PCR, normalized to β-actin and presented as fold induction relative to LAC-grown bifidobacteria. The data are the means ± SD where * indicates statistical significance between LAC and HMO (p

Figure 3

Effects of HMO-grown B. bifidum and B. infantis on mRNA expression of SELPLG and SERPING in Caco-2 cells was measured by real-time PCR, normalized to β-actin and presented as fold induction relative to LAC-grown bifidobacteria. The data are the means ± SD where * indicates statistical significance between LAC and HMO (p < 0.05).

Figure 4

Cytokine mRNA expression in Caco-2 (a) and HT-29 (b) cells incubated with bifidobacterial strains grown on HMO was measured by real-time PCR, normalized to β-actin and presented as fold induction relative to LAC-grown bifidobacteria. * indicates statistical significance between LAC and HMO (p

Figure 5

Translocation of bifidobacteria in Caco-2 (a) and HT-29 (b) monolayers. Cells were incubated with bacteria apically for 2h, and then basolateral translocation was measured. Data are means ± SD. Significance was at p