Pro-inflammatory T cells-derived cytokines enhance the maturation of the human fetal intestinal epithelial barrier.

Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development.

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development.

Mildly Pasteurized Whey Protein Promotes Gut Tolerance in Immature Piglets Compared with Extensively Heated Whey Protein.

Human milk is the optimal diet for infant development, but infant milk formula (IMF) must be available as an alternative. To develop high-quality IMF, bovine milk processing is required to ensure microbial safety and to obtain a protein composition that mimics human milk. However, processing can impact the quality of milk proteins, which can influence gastro-intestinal (GI) tolerance by changing digestion, transit time and/or absorption. The aim of this study was to evaluate the impact of structural changes of proteins due to thermal processing on gastro-intestinal tolerance in the immature GI tract. Preterm and near-term piglets received enteral nutrition based on whey protein concentrate (WPC) either mildly pasteurized (MP-WPC) or extensively heated (EH-WPC). Clinical symptoms, transit time and gastric residuals were evaluated. In addition, protein coagulation and protein composition of coagulates formed during in vitro digestion were analyzed in more detail. Characterization of MP-WPC and EH-WPC revealed that mild pasteurization maintained protein nativity and reduced aggregation of ß-lactoglobulin and α-lactalbumin, relative to EH-WPC. Mild pasteurization reduced the formation of coagulates during digestion, resulting in reduced gastric residual volume and increased intestinal tract content. In addition, preterm piglets receiving MP-WPC showed reduced mucosal bacterial adherence in the proximal small intestine. Finally, in vitro digestion studies revealed less protein coagulation and lower levels of ß-lactoglobulin and α-lactalbumin in the coagulates of MP-WPC compared with EH-WPC. In conclusion, minimal heat treatment of WPC compared with extensive heating promoted GI tolerance in immature piglets, implying that minimal heated WPC could improve the GI tolerance of milk formulas in infants.

A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine.

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

Beneficial Effect of Mildly Pasteurized Whey Protein on Intestinal Integrity and Innate Defense in Preterm and Near-Term Piglets.

BACKGROUND: The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1-2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition. METHODS: WPC was obtained by cold filtration and mildly pasteurized (73 °C, 30 s) or extensively heat treated (73 °C, 30 s + 80 °C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis. RESULTS: A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-ß, IL-8 and TNF-α expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC. CONCLUSIONS: Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivoIn vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice.

The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy.

Lactobacillus reuteri and Staphylococcus aureus differentially influence the generation of monocyte-derived dendritic cells and subsequent autologous T cell responses.

INTRODUCTION: In early-life, the immature mucosal barrier allows contact between the gut microbiota and the developing immune system. Due to their strategic location and their ability to sample luminal antigen, dendritic cells (DC) play a central role in the interaction of microbes and immune cells in the gut. Here, we investigated how two bacteria associated with opposite immune profiles in children, that is, Lactobacillus (L.) reuteri and Staphylococcus (S.) aureus, influenced the differentiation of monocytes in vitro as well how the generated DC impacted T cell responses. METHODS: We exposed monocyte cultures to cell-free supernatants (CFS) from these bacteria during their differentiation to DC. RESULTS: The presence of L. reuteri-CFS during DC differentiation resulted in DC with a more mature phenotype, in terms of up-regulated surface markers (HLA-DR, CD86, CD83, CCR7) and enhanced cytokine production (IL6, IL10, and IL23), but had a reduced phagocytic capacity compared with non-treated monocyte-derived DC (Mo-DC). However, upon LPS activation, L. reuteri-CFS-generated DC displayed a more regulated phenotype than control Mo-DC with notable reduction of cytokine responses both at mRNA and protein levels. In contrast, S. aureus-CFS-generated DC were more similar to control Mo-DC both without and after LPS stimulation, but they were still able to induce responses in autologous T cells, in the absence of further T cell stimulation. CONCLUSIONS: We show that bacterial signals during DC differentiation have a profound impact on DC function and possibly also for shaping the T cell pool.

Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

Postbiotic Modulation of Retinoic Acid Imprinted Mucosal-like Dendritic Cells by Probiotic Lactobacillus reuteri 17938 In Vitro.

Lactobacilli are widely used as probiotics with beneficial effects on infection-associated diarrhea, but also used in clinical trials of e.g., necrotizing enterocolitis and inflammatory bowel diseases. The possibility of using probiotic metabolic products, so-called postbiotics, is desirable as it could prevent possible side effects of live bacteria in individuals with a disturbed gut epithelial barrier. Here, we studied how Lactobacillus reuteri DSM 17938 cell-free supernatant (L. reuteri-CFS) influenced retinoic acid (RA)-driven mucosal-like dendritic cells (DC) and their subsequent effect on T regulatory cells (Treg) in vitro. RA clearly imprinted a mucosal-like DC phenotype with higher IL10 production, increased CD103 and CD1d expression, and a downregulated mRNA expression of several inflammatory-associated genes (NFκB1, RELB, and TNF). Treatment with L. reuteri-CFS further influenced the tolerogenic phenotype of RA-DC by downregulating most genes involved in antigen uptake, antigen presentation, and signal transduction as well as several chemokine receptors, while upregulating IL10 production. L. reuteri-CFS also augmented CCR7 expression on RA-DC. In cocultures, RA-DC increased IL10 and FOXP3 expression in Treg, but pre-treatment with L. reuteri-CFS did not further influence the Treg phenotype. In conclusion, L. reuteri-CFS modulates the phenotype and function of mucosal-like DC, implicating its potential application as postbiotic.

Corrigendum: Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

[This corrects the article on p. 437 in vol. 7, PMID: 27065990.].