Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4 - PubMed (original) (raw)

Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4

Ekaterina Kurakevich et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10(-/-) mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10(-/-) mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10(-/-);St3gal4(-/-) mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10(-/-) mice to lactating St3gal4(-/-) mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c(+) dendritic cells and induced production of cytokines required for expansion of TH1 and TH17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c(+) cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.

Keywords: carbohydrate; glycan; mouse innate immunity; prebiotics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

St3gal4 deficiency attenuates spontaneous intestinal inflammation in Il10 −/− mice. (A) Microscopic analysis of colon sections from 6- and 14-wk-old _Il10_−/− and S3/I10−/− mice, stained with H&E. Representative images from three independent experiments. (Scale bar, 200 μm.) (B) Histological score based on evaluation of morphological changes of epithelium and immune cell infiltration. Six- and 14-wk-old Il10 −/− and S3/I10−/− mice (n = 8–12) were analyzed. (C) Frequency of rectal prolapse in _Il10_−/− and S3/I10−/− mice. Il10 −/− mice (n = 46) and S3/I10−/− mice (n = 63) were monitored over a period of 26 wk. The graph represents percentage of mice with rectal prolapse from the total number mice at the age of 10–12 wk and 24–26 wk, respectively. WT, wild-type mice; S3/I10−/−, St3g4 −/− ; Il10 −/− mice; 6W, 6-wk-old mice; 14W, 14-wk-old mice.

Fig. 2.

Fig. 2.

Decreased leukocyte infiltration and inflammation in colons of St3gal4 −/− /Il10 −/− mice. (A) Flow cytometry analysis of lamina propria leukocytes (LPLs) isolated from a distal part of the colon of 6- and 14-wk-old Il10 −/− and S3/I10−/− mice (n = 5–7) or 6- to 8-wk-old WT controls. Data are presented as percentage of CD45+, CD4+, CD4+FoxP3+ (Treg), CD11c+, Ly6Chi, and Ly6G+ cells from all isolated cells. (B) Expression levels of cytokines in colons of 6- and 14-wk-old Il10 −/−, S3/I10−/− mice (n = 6–8) and WT control mice (n = 5–6) were determined by real-time PCR and normalized to GAPDH.

Fig. 3.

Fig. 3.

3SL supplementation aggravates colonic inflammation. S3/I10−/− mice were fed daily from birth until weaning (21 d) with 25 mM 3SL or lactose; control mice were fed with water. Mice were analyzed at the age of 6 wk (day 48). (A) Schematic representation of 3SL and lactose supplementation. (B) Representative microscopic images (n = 9) of colon sections from 6-wk-old _Il10_−/− and S3/I10−/− mice either with or without 3SL supplementation stained with hematoxylin and eosin. (Scale bar, 200 μm.) (C) Histological score based on evaluation of morphological changes of epithelium and immune cell infiltration from WT (n = 4), Il10 −/−, and S3/I10−/− mice (n = 8–9). WT, wild-type mice; S3/I10−/−, St3g4 −/− ; Il10 −/− mice; 3SL, sialyl(α2,3)lactose.

Fig. 4.

Fig. 4.

3SL directly stimulates dendritic cells (DCs). DCs were isolated from mesenteric lymph nodes of 6-wk-old WT, St3gal4 −/−, and Il10 −/− mice and purified with CD11c MicroBeads. (A) CD11c+ cells were stimulated for 14 h with 625 μM of 3SL, 6SL, or lactose (Lac). Stimulations with LPS (500 ng/mL) or PBS (buff.) were used as controls. Cell surface expression of CD40, CD80, and CD86 was analyzed by flow cytometry. (B) Measurement of secreted cytokines in the medium of stimulated CD11c+ cells. (C) Increase in Ly-6C+/CD11c+ cells in Il10 −/− mice during inflammation. Data are presented as percentage of CD11b+, CD103+, Ly-6C+, or CD8α+ cells from CD11c+ cells (n = 6). MFI, mean fluorescence intensity; 3SL, sialyl(α2,3)lactose; 6SL, sialyl(α2,6)lactose.

Fig. 5.

Fig. 5.

3SL feeding induces colon inflammation and 3SL is sensed by TLR4. Six-week-old Il10 −/− mice were treated with 3SL, lactose (Lac), or control (null) for 4 d and analyzed at day 5 (n = 6 per group). (A) Microscopic analysis of colon sections. (Scale bar, 200 μm.) (B) Histological score based on evaluation of morphological changes of epithelium and immune cell infiltration. (C) Lamina propria CD45+ cells are presented as percentage of all isolated cells. (D) Expression levels of cytokines in colons of mice fed with 3SL and lactose (Lac) for 4 d were determined by real-time PCR and normalized to GAPDH (n = 5). (E) CD11c+ cells from MLN of WT, Myd88 −/−, Tlr2 −/−, and Tlr4 −/− mice were stimulated with 625 μM of 3SL or 6SL for 14 h. Stimulation with LPS (500 ng/mL) or PBS (buffered) was used as controls. Cell surface expression of CD80, CD86, and CD40 on CD11c+/MHCII+ cells was analyzed by flow cytometry and quantified in two independent experiments (n = 4). MFI, mean fluorescence intensity; 3SL, sialyl(α2,3)lactose; 6SL, sialyl(α2,6)lactose; Unstim, nonstimulated cells.

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