Human Milk Oligosaccharide 2'-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells

Abstract

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2'-fucosyllactose (2'FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2'FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4⁺ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNβ, IFNλ1 and CCL5 secretion. Pre-incubation with 2'FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2'FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2'FL. These results provide insights regarding the protective capacity of human milk components in early life.

Keywords: 2′-fucosyllactose; T-cell; host-defense; moDC; poly I:C; viral infection.

Figure 1

Cytokine and chemokine secretion from IEC upon stimulation with pIC. IEC were grown in 48-well plates until confluency and pre-incubated with 2′FL (0.5% w/v). After 24 h incubation, IEC were stimulated with naked or LV-pIC for 20 h after which the supernatant was collected (A) and IL-8 (B), GROα (C), CCL20 (D), CXCL10 (E), IFNβ (F) IFNλ1 (G) and CCL5 (H) were measured. Results are represented as mean ± SEM of n = 6 independent experiments, except for CXCL10 n = 4 (# p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Cytokine and chemokine secretion upon stimulation with LV-pIC. IEC were grown in 12-well transwell inserts until confluency and pre-incubated with 2′FL (0.5% w/v). After 24 h pre-incubation, IEC were stimulated with LV-pIC and fresh 2′FL, and co-cultured with immature moDC for 48 h. Alternatively, moDC alone were stimulated with LV-pIC alone in the presence or absence of 2′FL for 48 h. After incubation, the basolateral supernatant was collected (A) and IL-8 (B), GROα (C), CCL20 (D), CXCL10 (E), IFNβ (F), IFNλ1 (G) and CCL5 (H) were measured. Results are represented as mean ± SEM of n = 6 independent moDC donors except for CCL20 n = 4 (# p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Galectin secretion upon stimulation with LV-pIC. IEC were grown in 12-well transwell inserts until confluency and pre-incubated with 2′FL (0.5% w/v). After 24 h pre-incubation, IEC were stimulated with LV-pIC and fresh 2′FL, and co-cultured with immature moDC for 48 h. Alternatively, moDC alone were stimulated with LV-pIC in the presence or absence of 2′FL for 48 h (A). After incubation, the basolateral supernatant was collected and galectin-3 (B), -4 (C) and -9 (D) were measured. Results are represented as mean ± SEM of n = 6 independent moDC donors (# p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Cytokine secretion upon stimulation with LV-pIC. IEC were grown in 12-well transwell inserts until confluency and pre-incubated with 2′FL (0.5% w/v). After 24 h pre-incubation, IEC were stimulated with LV-pIC and fresh 2′FL, and co-cultured with immature moDC for 48 h. Alternatively, moDC alone were stimulated with LV-pIC in the presence or absence of 2′FL for 48 h (A). After incubation, the basolateral supernatant was collected and IL-12p70 (B), IL-15 (C) and IL-6 (D) concentrations were measured. Results are represented as mean ± SEM of n = 6 independent moDC donors (# p < 0.1, * p < 0.05).

Figure 5

Cytokine secretion in DC/T-cell assay. Conditioned moDC (ccDC) previously exposed to LV-pIC activated IEC or to LV-complexed pIC directly, were incubated with naïve T-cells for 5 days (A) After incubation, the basolateral supernatant was collected and IFNγ (B), IL-22 (C) and IL-13 (D) were measured. Results are represented as mean ± SEM of n = 6 independent moDC donors (# p < 0.1, * p < 0.05, ** p < 0.01).