Mustard seed major allergen Sin a1 activates intestinal epithelial cells and also dendritic cells that drive type 2 immune responses.

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization.

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

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 IFNß, 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.

Modulation of the Epithelial-Immune Cell Crosstalk and Related Galectin Secretion by DP3-5 Galacto-Oligosaccharides and ß-3'Galactosyllactose.

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized ß-3'galactosyllactose (ß-3'GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or ß-3'GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized ß-3'GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by ß-3'GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized ß-3'GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.