Immunomodulatory and Prebiotic Effects of 2'-Fucosyllactose in Suckling Rats

Abstract

Human milk oligosaccharides are unconjugated complex glycans present in high concentration in human milk that serve as pre-biotics and immunomodulators. They are not primarily absorbed or metabolized by the infant and reach the lower part of the intestinal tract unaltered. One of the main oligosaccharides found in human milk is 2'-fucosyllactose (2'-FL). This study aimed to investigate the effects of daily oral administration of 2'-FL in healthy suckling rats. From days 2 to 16 of life, rats were daily given the oligosaccharide (2'-FL) or vehicle (REF), weighed and their stool characteristics were assessed. On days 8 and 16 of life the morphometry, intestinal architecture, and cytokine release, mesenteric lymph nodes cell composition, plasma immunoglobulin concentrations, fecal microbiota composition, cecal short-chain fatty acids content, and the urinary metabolic profile were assessed. Animals given 2'-FL showed higher plasma IgG and IgA and more T cell subsets in the mesenteric lymph nodes on day 16. Moreover, at intestinal level, villus heights, and areas were increased on day 8. Cecal samples displayed a higher Lactobacillus proportion and a different urinary metabolic profile was observed on day 8, and a higher proportion of butyrate on day 16. In conclusion, supplementation of 2'-FL in early life has a pre-biotic and intestinal trophic effect and promotes maturation of the immune system.

Keywords: 2′-FL; HMO; immunoglobulins; metabolomics; microbiota.

Figure 1

Effect of 2'-FL on CD8 surface expression in the mesenteric lymph node lymphocytes. (A) Percentage of lymphocytes expressing CD8. (B) Percentage of CD8+ lymphocytes expressing CD8αα or CD8αβ. (C) Ratio between the percentage of CD8αα and CD8αβ subsets. Results are expressed as mean ± S.E.M. (n = 4/group). *p < 0.05 compared to REF group (by Student T-test). REF, Reference (REF); 2′-FL, 2′-fucosyllactose.

Figure 2

Effect of 2′-FL on the intestinal gene expression and production of cytokines. (A) The intestinal function was assessed on day 8 by the relative quantification of IgA secretion, Toll-like receptors, cytokines, epithelial barrier, and maturation by real-time PCR. The relative gene expression in the experimental groups was calculated with respect to REF, which corresponded to 100% of transcription (indicated with a horizontal dotted line). A collection of cytokines (IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α) were quantified in the intestinal wash of animals on (B) day 8 and (C) day 16 of life. Results are expressed as mean ± S.E.M. (n = 8/group in (A), n = 6/group in (B,C) *p < 0.05 compared to REF group (by Mann-Whitney U-test). pIgR, Polymeric immunoglobulin receptor; IgA, immunoglobulin A; TLR, Toll-like receptor; TNF, tumor necrosis alpha; IL, interleukin; IFN, interferon; TGF-β, tumor growth factor-beta; MUC2, mucin 2; Ocln, occludin; Cldn2, claudin 2; FcRn, neonatal constant fragment receptor; Blimp-1, B-lymphocyte-induced maturation-protein-1; REF, reference; 2′-FL, 2′-fucosyllactose.

Figure 3

Effect of 2′-FL on intestinal histomorphometric variables on day 8. (A) Representative images of distal jejunum sections stained with hematoxylin and eosin, 100×. (B) Villi height, villi width, villi area, crypts depth, villi height/crypts depth ratio, and perimeter of the jejunum of suckling rats. Results are expressed as mean ± S.E.M. (n = 8/group). *p < 0.05 compared to REF group (by Student T-test). REF, Reference; 2′-FL, 2′-fucosyllactose.

Figure 4

Effect of 2′-FL on the fecal microbiota composition on day 8. The sequencing of the amplicon targeting the V3–V4 region of the 16S rRNA was performed following the 16S Metagenomic Sequencing Library Illumina 15044223 B protocol. The relative proportion of the bacteria was calculated for (A) phylum, family, genus, and species. (B) PCA of the proportion of bacteria at the level of family. (C) Venn Diagrams showing the number of families and genera in the REF and 2′-FL groups. Results are expressed as the mean value (n = 3/group, corresponding to one random animal/litter). *p < 0.05 compared to REF group (by Mann–Whitney U-test). REF, Reference; 2′-FL, 2′-fucosyllactose.

Figure 5

Effect of 2′-FL on the cecal SCFA production on day 16. Acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid production was quantified by HS-GC-MS. Results are expressed as the mean relative percentage ± S.E.M. (n = 8/group). *p < 0.05 compared to REF group (by Mann-Whitney U-test). REF, Reference; 2′-FL; 2′-fucosyllactose.

Figure 6

Effect of 2′-FL on the urinary metabolic profile on day 16. (A) NMR spectra was analyzed by natural clustering by PCA. (B) OPLS-DA coefficient plot showing the most significant metabolites. In this plot, positive peaks represent metabolites excreted at higher concentrations in urines of rats which received 2′-FL supplementation compared to the REF group, whereas negative peaks indicate metabolites excreted in higher concentration in the REF group (n = 4–5/group). REF, Reference; 2′-FL, 2′-fucosyllactose.