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

Modulation of the Epithelial-Immune Cell Crosstalk and Related Galectin Secretion by DP3-5 Galacto-Oligosaccharides and β-3′galactosyllactose

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligode-oxynucleotides (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 ca-pacities of DP3-sized β-3′galactosyllactose (β-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 β-3′GL in the presence of CpG, and basolater-ally 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 β-3′GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galec-tin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3,-4, and-9 secretion was increased by β-3′GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized β-3′GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity

Simulation in industrial statistics

We provide a brief introduction to simulation methods and tools within Industrial Statistics. We illustrate both Monte Carlo and discrete event simulation, within a common framework, through several motiovating examples showing the relevance of simulation. We then describe random number and variate generation, and how to obtain and analyse output data from a simulation model. A case study in modelling a workflow line within the digitalisation industry guides our discussion. Relevant pointers to references and url's are provided

Dietary, nondigestible oligosaccharides and Bifidobacterium breve M-16V suppress allergic inflammation in intestine via targeting dendritic cell maturation

Dietary intervention with short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and Bifidobacterium breve M-16V (Bb) (GF/Bb) suppresses food allergic symptoms in mice, potentially via intestinal epithelial cell (IEC)-derived galectin-9. Furthermore, in vitro studies showed galacto- and fructo-oligosaccharides (GF) to enhance the immunomodulatory capacity of a TLR9 ligand representing bacterial CpG DNA when exposed to IEC. In this study, we investigated whether GF/Bb modulates dendritic cells (DCs) and subsequent Th2 and regulatory T cell (Treg) frequency in the small intestinal lamina propria (SI-LP). BALB/c mice were fed GF/Bb during oral OVA sensitization. DC and T cell phenotype were determined in SI-LP mononuclear cells using flow cytometry. Murine bone marrow-derived DCs (BMDCs) were exposed to recombinant galectin-9 or human monocyte-derived DCs (moDCs) and were cultured in IEC-conditioned medium from GF and TLR9 ligand-exposed HT-29 cells. GF/Bb reduced allergic symptoms and enhanced serum galectin-9 levels, while suppressing activation, restoring phagocytic capacity, and normalizing CD103 expression of SI-LP DCs of OVA-allergic mice. In vitro, galectin-9 suppressed LPS-induced activation markers and cytokine secretion by BMDCs, and IEC-conditioned medium suppressed moDC activation in a galectin-9-dependent manner. Besides suppression of SI-LP DC activation, dietary GF/Bb also lowered the frequency of activated Th2 cells, while enhancing Treg in the SI-LP of OVA-allergic mice compared to the control diet. Dietary intervention with GF/Bb enhances galectin-9 and suppresses allergic symptoms of OVA-allergic mice in association with reduced intestinal DC and Th2 activation and increased Treg frequency in these mice

DataSheet_1_Epithelial-derived galectin-9 containing exosomes contribute to the immunomodulatory effects promoted by 2’-fucosyllactose and short-chain galacto- and long-chain fructo-oligosaccharides.pdf

IntroductionEarly 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.MethodsHuman 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.ResultsCompared 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.DiscussionExposure 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.</p

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

Dietary, nondigestible oligosaccharides and Bifidobacterium breve M-16V suppress allergic inflammation in intestine via targeting dendritic cell maturation

Dietary intervention with short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and Bifidobacterium breve M-16V (Bb) (GF/Bb) suppresses food allergic symptoms in mice, potentially via intestinal epithelial cell (IEC)-derived galectin-9. Furthermore, in vitro studies showed galacto- and fructo-oligosaccharides (GF) to enhance the immunomodulatory capacity of a TLR9 ligand representing bacterial CpG DNA when exposed to IEC. In this study, we investigated whether GF/Bb modulates dendritic cells (DCs) and subsequent Th2 and regulatory T cell (Treg) frequency in the small intestinal lamina propria (SI-LP). BALB/c mice were fed GF/Bb during oral OVA sensitization. DC and T cell phenotype were determined in SI-LP mononuclear cells using flow cytometry. Murine bone marrow-derived DCs (BMDCs) were exposed to recombinant galectin-9 or human monocyte-derived DCs (moDCs) and were cultured in IEC-conditioned medium from GF and TLR9 ligand-exposed HT-29 cells. GF/Bb reduced allergic symptoms and enhanced serum galectin-9 levels, while suppressing activation, restoring phagocytic capacity, and normalizing CD103 expression of SI-LP DCs of OVA-allergic mice. In vitro, galectin-9 suppressed LPS-induced activation markers and cytokine secretion by BMDCs, and IEC-conditioned medium suppressed moDC activation in a galectin-9-dependent manner. Besides suppression of SI-LP DC activation, dietary GF/Bb also lowered the frequency of activated Th2 cells, while enhancing Treg in the SI-LP of OVA-allergic mice compared to the control diet. Dietary intervention with GF/Bb enhances galectin-9 and suppresses allergic symptoms of OVA-allergic mice in association with reduced intestinal DC and Th2 activation and increased Treg frequency in these mice

Expression Patterns of the <i>Drosophila</i> Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain

<div><p>The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, <i>in situ</i> hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly <i>Drosophila melanogaster</i>. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.</p> </div

Cartoon of our proposed mechanism for the hormonal action of CCHamide-2 in third instar larvae.

<p>This hypothetical model is based on the sensory nature of the CCHamide-2 immunoreactive endocrine cells in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g001" target="_blank">Figure 1</a>) and on our findings that there is abundant expression of the CCHamide-2 receptor gene in the brain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D</a>, a), while this expression is virtually absent in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D, b</a>), combined with the high CCHamide-2 peptide gene expression in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9C, b</a>). This model predicts that the quality of the food in the gut lumen is sensed by the CCHamide-2 immunoreactive endocrine cells in the gut wall. These cells release the CCHamide-2 peptides into the circulation, after which they reach the brain and bind to the CCHamide-2 receptors. This binding starts a cascade that leads to an altered (adapted) feeding behavior of the animal. In addition to this long-distance hormonal CCHamide-2 signaling, there is a local (synaptic or paracrine) CCHamide-2 signaling in the larval CNS, possibly also associated with feeding.</p