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.

Specific Human Milk Oligosaccharides Differentially Promote Th1 and Regulatory Responses in a CpG-Activated Epithelial/Immune Cell Coculture.

Proper early life immune development creates a basis for a healthy and resilient immune system, which balances immune tolerance and activation. Deviations in neonatal immune maturation can have life-long effects, such as development of allergic diseases. Evidence suggests that human milk oligosaccharides (HMOS) possess immunomodulatory properties essential for neonatal immune maturation. To understand the immunomodulatory properties of enzymatic or bacterial produced HMOS, the effects of five HMOS (2'FL, 3FL, 3'SL, 6'SL and LNnT), present in human milk have been studied. A PBMC immune model, the IEC barrier model and IEC/PBMC transwell coculture models were used, representing critical steps in mucosal immune development. HMOS were applied to IEC cocultured with activated PBMC. In the presence of CpG, 2'FL and 3FL enhanced IFNγ (p < 0.01), IL10 (p < 0.0001) and galectin-9 (p < 0.001) secretion when added to IEC; 2'FL and 3FL decreased Th2 cell development while 3FL enhanced Treg polarization (p < 0.05). IEC were required for this 3FL mediated Treg polarization, which was not explained by epithelial-derived galectin-9, TGFß nor retinoic acid secretion. The most pronounced immunomodulatory effects, linking to enhanced type 1 and regulatory mediator secretion, were observed for 2'FL and 3FL. Future studies are needed to further understand the complex interplay between HMO and early life mucosal immune development.

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.

Epithelial-derived galectin-9 containing exosomes contribute to the immunomodulatory effects promoted by 2'-fucosyllactose and short-chain galacto- and long-chain fructo-oligosaccharides.

Introduction: Early life exposure to non-digestible oligosaccharides (NDO) or microbial components is known to affect immune development. NDO in combination with a TLR9 agonist mimicking bacterial triggers (CpG) promoted the secretion of galectins through unknown pathways. We aimed to study the contribution of exosomes in epithelial galectin secretion and subsequent immunoregulation upon exposure to a mixture of NDO by inhibiting exosome biogenesis. Methods: Human intestinal epithelial cells (IEC) (FHs 74 Int or HT-29) were apically exposed to 2'-fucosyllactose (2'FL) and short-chain galacto- and long-chain fructo-oligosaccharides (GF), alone or with CpG. Basolaterally, non-activated or αCD3/CD28-activated peripheral blood mononuclear cells (PBMC) were added. After 24 h incubation, IEC were washed and incubated in fresh medium to analyze epithelial-derived galectin secretion. Additionally, before exposure to NDO and CpG, IEC were exposed to GW4869 to inhibit exosome biogenesis. After 24 h of incubation, IEC were washed and incubated for additional 24 h in the presence of GW4869, after which epithelial-derived galectin secretion was studied. Also, epithelial-derived exosomes were isolated to study the presence of galectins within the exosomes. Results: Compared to CpG alone, exposure to 2'FL/GF mixture and CpG, significantly enhanced Th1-type IFNγ, and regulatory IEC-derived galectin-9 secretion in the HT-29/PBMC model. Similarly, in the FHs 74 Int/PBMC co-culture, 2'FL/GF induced immunomodulatory effects in the absence of CpG. Interestingly, galectin-9 and -4 were present in CD63-expressing exosomes isolated from HT-29 supernatants after IEC/PBMC co-culture. Exposure to GW4869 suppressed 2'FL/GF and CpG induced epithelial-derived galectin-9 secretion, which subsequently prevented the rise in IL-10 and reduction in IL-13 secretion observed in the HT-29/PBMC co-culture model upon exposure to 2'FL/GF and CpG. Discussion: Exposure to 2'FL/GF and CpG or 2'FL/GF promoted Th1-type regulatory effects in HT-29/PBMC or FHs 74 Int/PBMC co-culture respectively, while Th2-type IL-13 was reduced in association with increased galectin-9 release. Galectin-9 and -4 were present in exosomes from HT-29 and the inhibition of exosome biogenesis inhibited epithelial-derived galectin secretion. This, also affected immunomodulatory effects in IEC/PBMC co-culture suggesting a key role of galectin expressing IEC-derived exosomes in the mucosal immune regulation induced by NDO.

A Fermented Milk Matrix Containing Postbiotics Supports Th1- and Th17-Type Immunity In Vitro and Modulates the Influenza-Specific Vaccination Response In Vivo in Association with Altered Serum Galectin Ratios.

During a specific milk fermentation process with Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LactofidusTM), postbiotics with possible immunomodulatory properties are produced. We investigated the effects of this fermentation product (FP) in vitro using a model that allows crosstalk between intestinal epithelial (IEC) and immune cells. IECs were exposed to FP and αCD3/CD28-activated peripheral blood mononuclear cells after which the mediator secretion was measured. Additionally, using a murine influenza vaccination model, immune development was assessed. Mice were fed an AIN93G diet containing FP or lactose as control. Vaccine-specific immunity was measured as delayed-type hypersensitivity (DTH) and correlated to intestinal and systemic immunomodulation levels. In vitro, exposure to FP enhanced IFNγ, TNFα and IL-17A concentrations. Moreover, IEC-derived galectin-3/galectin-9 and galectin-4/galectin-9 ratios were increased. In vivo, dietary intervention with FP increased vaccine-specific DTH responses as compared to the lactose-receiving group. Although no effects on humoral immunity and vaccine-specific T-cell responses were detected, an enhanced systemic serum galectin-3/galectin-9 and galectin-4/galectin-9 ratio correlated with a shift in RORγ (Th17) mRNA expression over regulatory TGFß1 in the ileum. This was also positively correlated with the increased DTH response. These results indicate that FP can enhance epithelial galectin-3 and -4 over galectin-9 release, and boost adaptive immunity by promoting Th1- and Th17-type cytokines under inflammatory conditions in vitro. Similar variations in galectin and immune balance were observed in the vaccination model, where FP improved the influenza-specific DTH response.

Exposure of Intestinal Epithelial Cells to 2'-Fucosyllactose and CpG Enhances Galectin Release and Instructs Dendritic Cells to Drive Th1 and Regulatory-Type Immune Development.

Intestinal epithelial cells (IEC) release immunomodulatory galectins upon exposure to CpG DNA (mimicking bacterial triggers) and short-chain galacto- and long-chain fructo-oligosaccharides (GF). This study aims to investigate the immunomodulatory properties of 2'-fucosyllactose (2'-FL), a non-digestible oligosaccharide (NDO) abundantly present in human milk, using a co-culture model developed to study the crosstalk between IEC and innate and adaptive immune cells. IECs, co-cultured with αCD3/CD28-activated peripheral blood mononuclear cells (PBMC), were apically exposed to NDOs and CpG, washed and co-cultured with immature monocyte-derived dendritic cells (moDC). Subsequently, moDC were co-cultured with naïve CD4+ T-cells. In the presence of CpG, both 2'-FL or GF-exposed IEC enhanced Th1-type IFNγ and regulatory IL-10 secretion of PBMCs, compared to CpG alone, while Th2-type IL-13 was reduced. Both NDOs increased IEC-derived galectin-3, -4, -9 and TGF-ß1 of CpG-exposed IEC. Only galectin-9 correlated with all modified immune parameters and TGF-ß1 secretion. MoDCs exposed to 2'-FL and CpG-conditioned IEC instructed IFNγ and IL-10 secretion by CD4+ T-cells, suggesting the development of a regulatory Th1 response. These results reveal that 2'-FL and GF could contribute to the mucosal immune development by supporting the effect of microbial CpG DNA associated with the modulation of epithelial galectin and TGF-ß1 secretion.

Raw Cow's Milk Reduces Allergic Symptoms in a Murine Model for Food Allergy-A Potential Role For Epigenetic Modifications.

Epidemiological studies identified raw cow's milk consumption as an important environmental exposure that prevents allergic diseases. In the present study, we investigated whether raw cow's milk has the capacity to induce tolerance to an unrelated, non-milk, food allergen. Histone acetylation of T cell genes was investigated to assess potential epigenetic regulation. Female C3H/HeOuJ mice were sensitized and challenged to ovalbumin. Prior to sensitization, the mice were treated with raw milk, processed milk, or phosphate-buffered saline for eight days. Allergic symptoms were assessed after challenge and histone modifications in T cell-related genes of splenocyte-derived CD4+ T cells and the mesenteric lymph nodes were analyzed after milk exposure and after challenge. Unlike processed milk, raw milk decreased allergic symptoms. After raw milk exposure, histone acetylation of Th1-, Th2-, and regulatory T cell-related genes of splenocyte-derived CD4+ T cells was higher than after processed milk exposure. After allergy induction, this general immune stimulation was resolved and histone acetylation of Th2 genes was lower when compared to processed milk. Raw milk reduces allergic symptoms to an unrelated, non-milk, food allergen in a murine model for food allergy. The activation of T cell-related genes could be responsible for the observed tolerance induction, which suggested that epigenetic modifications contribute to the allergy-protective effect of raw milk.

Diversity of Human Milk Oligosaccharides and Effects on Early Life Immune Development.

One of the well-known features of human milk, is the capacity to protect against the risk and impact of neonatal infections, as well as to influence the onset of allergic and metabolic disease manifestations. The major objective of this review is to provide a detailed overview regarding the role of human milk, more specifically the diversity in human milk oligosaccharides (HMOS), on early life immune development. Novel insights in immune modulatory effects of HMOS obtained by in vitro as well as in vivo studies, adds to the understanding on how early life nutrition may impact immune development. Extensive description and analysis of single HMOS contributing to the diversity within the composition provided during breastfeeding will be discussed with specific emphasis on immune development and the susceptibility to neonatal and childhood infections.

Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner.

Intestinal epithelial cells (IEC) drive regulatory T cell (Treg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103+ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC.