Human Milk Blocks DC-SIGN-Pathogen Interaction via MUC1

Abstract

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.

Keywords: DC-SIGN; human milk; immune modulation; intestine; mucin.

Figure 1

(A) Binding of monocyte-derived dendritic cells to skimmed human, formula, bovine, or camel milk. (B) Binding of skimmed milk to DC-SIGN-Fc or MGL-Fc. (C) Binding of monocyte-derived dendritic cells to skimmed human milk in different dilutions in the presence of EGTA, DC-SIGN blocking antibody AZN-D1, or MGL blocking antibody 1G6.6.Three independent experiments were performed. Data are indicated as mean ± SD.

Figure 2

(A) Binding of milk proteins to DC-SIGN. MUC1, MUC4, κ-casein, and α-lactalbumin were captured from human milk using antibodies, whereas lactoferrin was used in purified form. One representative experiment out of three is presented. (B) Correlation of MUC1 levels with DC-SIGN binding in fractionated human milk. Human milk was fractionated into 30 fractions, based on protein size. MUC1 levels and DC-SIGN binding was tested in ELISA. (C) Correlation of Lewis x levels on MUC1 in 40 milk donors with the capacity of captured MUC1 binding to DC-SIGN. (D) Correlation of Lewis y levels on MUC1 in 40 milk donors with the capacity of captured MUC1 binding to DC-SIGN. Typical experiment out of two is presented.

Figure 3

(A) IL-10 expression in human monocyte-derived dendritic cells incubated in human milk with and without the blocking DC-SIGN antibody AZN-D1. Expression is normalized to GAPDH. (B) Cell surface expression of the maturation markers CD83, CD86, and HLA-DR on human monocyte-derived dendritic cells following incubation with human milk and after LPS stimulation. Data are indicated as mean ± SD of three experiments performed.

Figure 4

(A) Binding of N. gonorrhoeae to human monocyte-derived dendritic cells in the presence of the DC-SIGN blocking antibody AZN-D1, MGL blocking antibody 1g6.6, human milk or formula milk. Three independent experiments were performed. (B) Binding of N. gonorrhoeae to monocyte-derived dendritic cells in the presence of fractionated milk samples. (C) DC-SIGN binding capacity of fractionated milk samples in ELISA. (D) MUC1 levels in fractionated milk samples detected by ELISA. (E) Lewis x expression on MUC1 captured from fractionated milk samples in ELISA. (F) Correlation of MUC1 levels in fractionated milk samples and the capacity of these samples to inhibit N. gonorrhoeae binding to monocyte-derived dendritic cells. (G) Binding of phase variants J223.3 and J223.8 from H. pylori to DC-SIGN-Fc in ELISA. (H) Binding of H. pylori phase variants to DC-SIGN-Fc in the presence of the calcium chelator EGTA, the DC-SIGN ligand mannan and human milk. (I) Binding of H. pylori phase variant J223.3 to monocyte-derived dendritic cells in the presence of anti- DC-SIGN or anti-MGL antibody, human or formula milk. (J) Correlation of MUC1 levels in fractionated milk samples and the capacity of these samples to inhibit H. pylori J223.3 binding to monocyte-derived dendritic cells. Data are indicated as mean ± SD of three experiments performed.

Figure 5

DC-SIGN expression in (A) stomach, (B) duodenum, (C,D) ileum, and (E,F) colon of young infants. Arrows indicate DC-SIGN positive cells in the lamina propria and arrowheads point to positive cells in the submucosa. Nuclei are stained in blue. Magnification ×20.