Intestinal Epithelial Cell Monolayers Research Articles

Soybean polysaccharide fermentation products regulate the air-liquid interface in co-cultured Caco-2 cells by increasing short chain fatty acids transport

Soybean polysaccharides have a large molecular weight and complex structure, which is not conducive to body absorption and exerting their biological activities. After the in vitro hydrolysate digestion of soybean polysaccharides, their interactions with intestinal epithelial cell monolayers during soybean polysaccharide-derived short chain fatty acids (SCFAs) uptake and transport were determined by co-culturing soybean polysaccharide hydrolysate products with Caco-2 cells. Based on prepared soybean polysaccharide hydrolysates, physicochemical indices and hydrolysate components were explored and the interface characteristics between SCFAs and Caco-2 cells were characterized using interfacial rheology methods for the first time. Transwell chambers were used to explore relationships between SCFAs transport and the air–liquid interface in Caco-2 cells. We showed that physicochemical properties, cell proliferation rates, and the interfacial tension of soybean polysaccharide hydrolysis products were related to fermentation times, with differences observed between the two hydrolyzed soybean polysaccharides (microwave ammonium oxalate soy hull polysaccharides (MASP) and soluble soy polysaccharides (SSP)). MASP outperformed SSP in terms of total sugar utilization and added cellular value by intestinal flora. Hydrolyzed soybean polysaccharides decreased interfacial tension with increasing hydrolysis times when modulating the interfacial properties of a Caco-2 cell co-culture system. SCFAs translocation rates increased with fermentation time, from 0 h to 24 h. Also, a negative correlation was observed between SCFAs translocation rates and interfacial tension. Our data provide a foundation for the intestinal absorption of soybean polysaccharides and at the same time bring new insights into the interactions between polysaccharides and food in the future, promoting the application of polysaccharides in food processing and even medicine.

A bioactive bovine whey protein extract improves intestinal barrier function in vitro

The human intestine plays an important role as a barrier against the ingress of pathogens and other harmful antigens. Accordingly, proper regulation of the intestinal barrier is essential for optimal health. Intestinal barrier function is regulated in part by the interactions between dietary compounds and the intestinal immune system. Bioactive whey proteins from bovine milk (such as lactoferrin, lactoperoxidase, and immunoglobulins) are known to exert a range of physiological functions, including modulation of the immune system, and thus have the potential to regulate intestinal barrier function. While the effects of individual whey proteins on intestinal barrier function have been studied to some extent, less is known about the potentially synergistic properties of whey protein mixtures. Here we investigated the effects of a bioactive bovine whey protein (BWP) extract containing all whey proteins with an isoelectric point >6.8 on intestinal barrier function in vitro. Intestinal epithelial cell (Caco-2) monolayers were treated with BWP before measuring the barrier integrity over 48 h by means of trans-epithelial electrical resistance (TEER). Treatment of epithelial monolayers with 1 mg/mL BWP resulted in an increase in TEER compared with untreated epithelial monolayers. To determine whether BWP could mitigate immune-mediated intestinal barrier dysfunction, we challenged differentiated Caco-2 cell monolayers with tumor necrosis factor α (TNFα) to obtain an in vitro model of a “leaky” intestinal epithelium. The TNFα challenge led to a decrease in TEER over time across untreated control monolayers, indicating a loss of barrier function. This loss of barrier function was mitigated in monolayers treated with 1 mg/mL BWP, but not monolayers treated with the equivalent amount of lactoferrin present in 1 mg/mL BWP. These data suggest that naturally co-occurring bioactive proteins together may enhance intestinal barrier integrity and protect against inflammation-induced barrier dysfunction to a greater extent than lactoferrin alone. Further work is required to determine the key proteins and protein combinations within BWP, and the mechanisms through which BWP modulates intestinal barrier function.

Protective effects of Acetobacter ghanensis against gliadin toxicity in intestinal epithelial cells with immunoregulatory and gluten-digestive properties.

The aim of this study was to establish whether Acetobacter ghanensis, the probiotic characteristics of which were evaluated previously, attenuates gliadin-induced toxicity in intestinal epithelial cells with gluten-digestive and immunoregulatory properties. A co-culture model of human intestinal epithelial cell (Caco-2) monolayers on top of peripheral blood mononuclear cells (PBMCs) obtained from patients with celiac disease (CD) was established. The gluten-digestive properties of A. ghanensis were determined by checking bacterial growth in a medium containing gluten as the main nitrogen source. The mRNA levels of genes encoding TJ-associated proteins were measured by quantitative real-time PCR (qRT-PCR). The concentrations of IL-6 and TNFα were determined by enzyme-linked immunosorbent assay (ELISA). We found that PT-gliadin disrupted intestinal barrier integrity by modulating the expression of TJ-associated genes encoding zonulin (increased by ~ 60%), zonula occludens-1 (ZO-1) (decreased by ~ 22%), and occludin (decreased by ~ 28%) in Caco-2 cells. Furthermore, PT-gliadin treatment in Caco-2 cells was associated with increased concentrations of IL-6 (~ 1.6-fold) and TNFα (~ twofold) from PBMCs. These modulatory effects of PT-gliadin, however, were suppressed when Caco-2 cells were subjected to A. ghanensis in the presence of PT-gliadin. As a factor underlying these protective effects, we showed that A. ghanensis could digest gluten peptides. To our knowledge, the current study is the first to demonstrate that A. ghanensis improves intestinal barrier functions by attenuating the modulatory effects of PT-gliadin with immunoregulatory and gluten-digestive properties.

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.

SummaryTwo-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3–4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.

Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers

Recently developed human intestinal epithelial 3D organoid cultures are a useful cell culture model to study intestinal transport physiology. From these, 2D monolayer cultures can be generated in which apical transporters are exposed to the medium, thereby better facilitating in vitro investigation of intestinal absorption processes. However, whether nutrient and electrolyte absorption can be physiologically regulated in human organoid-derived monolayers has not been determined. Constitutive nitric oxide (cNO) is known to regulate multiple gastrointestinal physiological functions. Previous studies using in vivo and in vitro mammalian animal models indicate that enhanced intracellular cNO differentially regulates the two primary apical Na transporters in small intestinal epithelial cells. Here, we generated human jejunal organoid-derived monolayers to determine whether apical nutrient and electrolyte transporter function is regulated by cNO in human enterocytes. Western blot analysis and immunocytochemical staining showed that organoid-derived 2D cultures express markers of enterocyte differentiation and form intact monolayers of apical-basal polarized epithelial cells. Uptake studies demonstrated that jejunal monolayers exhibit functional activity of Na-glucose cotransporter 1 (SGLT1; SLC5A1) and Na-H exchanger 3 (NHE3; SLC9A3). In response to physiological increases in cNO, the two primary apical Na transporters were differentially regulated in human intestinal organoid-derived monolayers, across multiple human specimens. An increase in cNO stimulated SGLT1, while NHE3 was inhibited. These results are similar to what is seen in vivo and in vitro in different animal intestinal models. Thus, human jejunal organoid-derived monolayers are an ideal in vitro model to better understand how intestinal nutrient absorption is regulated.

Mechanistic bottom-up estimation of passive drug absorption from the gastrointestinal tract: Comparison among primary cultured human intestinal cells, Caco-2 cells, artificial membrane, and animal scale-up.

The fraction of drug absorbed (Fa) from the intestine is an important parameter to characterize the pharmacokinetics of a drug. We aimed to search for an experimental system that provides the best parameters for estimating the effective permeability (Peff) used for the bottom-up prediction of Fa. The absorption kinetics of 12 passively absorbed drugs were simulated by a compartment absorption transit (CAT) model using absorption parameters from four different experimental systems: human intestinal epithelial cell (HIEC) monolayer, Caco-2 monolayer, parallel artificial membrane permeability assay (PAMPA), and in situ rat intestinal perfusion. All absorption parameters were obtained from the literature. The in vitro apparent permeability coefficient (Papp) and rat in situ Peff were converted to human Peff using a bottom-up approach for each region, based on the morphological features of the human intestine. The simulated Fa values were compared to the respective observed values. Furthermore, plasma concentration profiles of the drugs were simulated by convolution using the time-course of the absorption rate simulated using the Peff values calculated from the HIEC Papp. The Fa values were best predicted by using the Peff values calculated from HEIC, within a 1.3-fold range of observed Fa in 11 out of 12 drugs. The simulated Cmax values of pharmacokinetic simulation using HIEC Papp fell within a 1.5-fold range of observed values for all the drugs examined. The HIEC monolayer was identified as the most suitable permeation parameter for estimating Fa and Cmax using a morphological feature-based bottom-up approach.

Proteases secreted by Trichinella spiralis intestinal infective larvae damage the junctions of the intestinal epithelial cell monolayer and mediate larval invasion

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

Allicin Improves Intestinal Epithelial Barrier Function and Prevents LPS-Induced Barrier Damages of Intestinal Epithelial Cell Monolayers.

Gut barrier disruption is the initial pathogenesis of various diseases. We previously reported that dietary allicin improves tight junction proteins in the endoplasmic reticulum stressed jejunum. However, whether the allicin benefits the gut barrier within mycotoxin or endotoxin exposure is unknown. In the present study, IPEC-J2 cell monolayers within or without deoxynivalenol (DON) or lipopolysaccharide (LPS) challenges were employed to investigate the effects of allicin on intestinal barrier function and explore the potential mechanisms. Results clarified that allicin at 2 μg/mL increased the viability, whereas the allicin higher than 10 μg/mL lowered the viability of IPEC-J2 cells via inhibiting cell proliferation. Besides, allicin increased trans-epithelial electric resistance (TEER), decreased paracellular permeability, and enhanced ZO-1 integrity of the IPEC-J2 cell monolayers. Finally, allicin supplementation prevented the LPS-induced barrier damages via activating Nrf2/HO-1 pathway-dependent antioxidant system. In conclusion, the present study strongly confirmed allicin as an effective nutrient to improve intestinal barrier function and prevent bacterial endotoxin-induced barrier damages.

Emerging effects of tryptophan pathway metabolites and intestinal microbiota on metabolism and intestinal function.

The metabolism of dietary tryptophan occurs locally in the gut primarily via host enzymes, with ~ 5% metabolized by gut microbes. Three major tryptophan metabolic pathways are serotonin (beyond the scope of this review), indole, kynurenine and related derivatives. We introduce the gut microbiome, dietary tryptophan and the potential interplay of host and bacterial enzymes in tryptophan metabolism. Examples of bacterial transformation to indole and its derivative indole-3 propionic acid demonstrate associations with human metabolic disease and gut permeability, although causality remains to be determined. This review will focus on less well-known data, suggestive of local generation and functional significance in the gut, where kynurenine is converted to kynurenic acid and xanthurenic acid via enzymatic action present in both host and bacteria. Our functional data demonstrate alimited effect on intestinal epithelial cell monolayer permeability and on healthy mouse ileum. Other data suggest a modulatory effect on the microbiome, potentially in pathophysiology. Supportive of this, we found that the expression of mRNA for three kynurenine pathway enzymes were increased in colon from high-fat-fed mice, suggesting that this host pathway is perturbed in metabolic disease. These data, along with that frombacterial genomic analysis and germ-free mice, confirms expression and functional machinery of enzymes in this pathway. Therefore, the host and microbiota mayplay a significant dual role in either the production or regulation of these kynurenine metabolites which, in turn, can influence both host and microbiome, especially in the context of obesity and intestinal permeability.

4-Phenylbutyric acid accelerates rehabilitation of barrier function in IPEC-J2 cell monolayer model.

As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier, the intestinal epithelial barrier plays a determinant role in pigs' health and growth. 4-Phenylbutyric acid (4-PBA), an aromatic fatty acid, was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis. However, whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown. This study aimed to explore the effects of 4-PBA on the intestinal barrier function, using in vitro models of well-differentiated intestinal porcine epithelial cell (IPEC-J2) monolayers in the transwell plates. Cell monolayers with or without 4-PBA (1.0 mmol/L) treatment were challenged with physical scratch, deoxynivalenol (DON, 2.0 μg/mL, 48 h), and lipopolysaccharide (LPS, 5.0 μg/mL, 48 h), respectively. Transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FD-4) permeability were measured to indicate barrier integrity and permeability. Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins. As expected, physical scratch, DON, and LPS challenges decreased TEER and increased FD-4 permeability. 4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage. However, once 48-h DON and LPS challenges were removed, rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment. Also, the relative gene and protein expressions of zonula occludens-1 (ZO-1), occludin, and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation. Taken together, 4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch, DON-, and LPS-induced damage, via enhancing cell mitigation and expressions of tight junction proteins.

JAM‐A signals through the Hippo pathway to regulate intestinal epithelial proliferation

The intestinal mucosa is lined by a columnar epithelial monolayer that serves as a dynamic interface, regulating barrier function and homeostasis. Perturbation of homeostasis, including changes in intestinal epithelial cell (IEC) proliferation and migration, can give rise to pathological states including ulcerative colitis and cancer. Regulation of proliferation and migration is a complex process coordinated, in part, by signaling events emanating from tight junction proteins at cell-cell contacts. One tight junction protein that has been linked to regulation of cellular proliferation is Junctional Adhesion Molecule A (JAM-A). JAM-A forms homodimers in cis and trans through interactions between its extracellular immunoglobulin-like domains. Loss of JAM-A results in enhanced IEC proliferation that has been linked to loss of homophilic interactions and Akt-dependent, beta-catenin signaling. However, JAM-A contributions to other signaling pathways that regulate cell proliferation are poorly understood. Since the Hippo signaling pathway is a well-known regulator of cellular proliferation, differentiation, and apoptosis, we examined whether loss of JAM-A alters Hippo pathway activity. We assessed activity of the Hippo transcriptional coactivator, Yes-1 Associated Protein (YAP), in JAM-A deficient IEC by gene-reporter activity assay and immunoblotting and observed significant increases, suggesting that JAM-A may interact with Hippo pathway constituents upstream of YAP. Immunolabeling studies with monolayers of IEC revealed association of JAM-A with two Hippo pathway constituents, Large Tumor Suppressor Kinase (LATS1) and Merlin. Co-immunoprecipitation experiments confirmed LATS1 and Merlin association with JAM-A. Moreover, phosphoprotein specific immunoblotting studies on JAM-A deficient IEC revealed decreased Hippo pathway activity characterized by decreased LATS1 activity and subsequent increased YAP activity. Such findings are consistent with JAM-A signaling upstream of Merlin and LATS1 and coordinating downstream Hippo signaling. Since there is evidence suggesting that dimerization of JAM-A is a required signal that suppresses IEC proliferation, we performed experiments using an exogenous overexpression system to study effects of a dimerization deficient JAM-A mutant on Hippo signaling. Phosphoprotein specific immunoblotting and EdU incorporation revealed that overexpression of full-length JAM-A resulted in activation of Hippo signaling, suppressing cell proliferation compared to empty vector control cells. However, overexpression of a dimerization defective JAM-A mutant protein lacking the distal immunoglobulin loop (DL1) was unable to activate Hippo signaling and suppress cell proliferation. Based on these observations, we propose that homodimerization of JAM-A functions as a cell-cell contact sensor, initiating downstream Hippo signaling to suppress IEC proliferation. These findings have important implications for understanding effects of altered proliferation on mucosal wound repair, as well as in morbidity associated with certain cancers which have been reported to express decreased levels of JAM-A protein.

Overview of the Effect of Citrobacter rodentium Infection on Host Metabolism and the Microbiota.

Citrobacter rodentium is a natural enteric mouse pathogen that models human intestinal diseases, such as pathogenic E. coli infections, ulcerative colitis, and colon cancer. Upon reaching the monolayer of intestinal epithelial cells (IECs) lining the gut, a complex web of interactions between the host, the pathogen, and the microbiota ensues. A number of studies revealed surprisingly rapid changes in IEC bioenergetics upon infection, involving a switch from oxidative phosphorylation to aerobic glycolysis, leading to mucosal oxygenation and subsequent changes in microbiota composition. Microbiome studies have revealed a bloom in Enterobacteriaceae during C. rodentium infection in both resistant (i.e., C57BL/6) and susceptible (i.e., C3H/HeN) strains of mice concomitant with a depletion of butyrate-producing Clostridia. The emerging understanding that dysbiosis of cholesterol metabolism is induced by enteric infection further confirms the pivotal role immunometabolism plays in disease outcome. Inversely, the host and microbiota also impact upon the progression of infection, from the susceptibility of the distal colon to C. rodentium colonization to clearance of the pathogen, both via opsonization from the host adaptive immune system and out competition by the resident microbiota. Further complicating this compendium of interactions, C. rodentium exploits microbiota metabolites to fine-tune virulence gene expression and promote colonization. This chapter summarizes the current knowledge of the myriad of pathogen-host-microbiota interactions that occur during the progression of C. rodentium infection in mice and the broader implications of these findings on our understanding of enteric disease.

Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tight Junction Assembly.

Intestinal barrier function relies primarily on the assembly and integrity of tight junctions, which forms a size-selective barrier. This barrier restricts paracellular movement of solutes in various types of epithelia. Of note, extracellular Ca2+ concentration affects tight junction assembly. Therefore, the removal and re-addition of Ca2+ into cell culture medium of cultured intestinal epithelial cells causes destabilization and reassembly of tight junction to membrane periphery near apical surface, respectively. Based on this principle, the Ca2+-switch assay was established to investigate tight junction assembly in fully differentiated intestinal epithelial cells. This chapter provides a stepwise protocol for culture of intestinal epithelial cell monolayers using T84 cell line as an in vitro model and the Ca2+-switch assay for evaluating tight junction assembly.

Trichinella spiralis: RNAi-mediated silencing of serine protease results in reduction of intrusion, development and fecundity

In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.

Vitamin E alpha- and gamma-tocopherol mitigate colitis, protect intestinal barrier function and modulate the gut microbiota in mice

Inflammatory bowel diseases (IBDs) including colitis are intestinal disorders characterized by chronic inflammation, barrier dysfunction and dysbiosis. Specific forms of vitamin E have been shown to attenuate colitis, but the mechanisms are not fully understood. The objective of this study is to examine the impact of α-tocopherol (αT) and γ-tocopherol-rich tocopherols (γTmT) on gut inflammation, barrier integrity and microbiota in dextran sulfate sodium (DSS)-induced colitis in mice. We observe that αT and γTmT mitigated DSS-caused fecal bleeding, diarrhea and elevation of IL-6. These vitamin E forms inhibited colitis-induced loss of the tight junction protein occludin, and attenuated colitis-caused elevation of LPS-binding protein in the plasma, a surrogate marker of intestinal barrier dysfunction, suggesting protection of gut barrier integrity. Consistently, αT and γT mitigated TNF-α/IFN-γ-induced impairment of trans-epithelial electrical resistance in human intestinal epithelial Caco-2 cell monolayer. Using 16S rRNA gene sequencing of fecal DNA, we observe that DSS reduced gut microbial evenness and separated microbial composition from healthy controls. In colitis-induced mice, γTmT but not αT separated gut microbial composition from controls, and attenuated DSS-caused depletion of Roseburia, which contains butyrate producing bacteria and is decreased in IBD patients. Canonical correspondence analysis also supports that γTmT favorably altered gut microbial community. In contrast, neither αT nor γTmT affected gut microbes in healthy animals. These results provide evidence supporting protective effects of αT and γT on intestinal barrier function and that γTmT caused favorable changes of the gut microbiota in colitis-induced mice.

Lythrum salicaria Ellagitannins Stimulate IPEC-J2 Cells Monolayer Formation and Inhibit Enteropathogenic Escherichia coli Growth and Adhesion.

Lythrum salicaria herb (LSH) was applied in diarrhea therapy since ancient times. Despite empirically referenced therapeutic effects, the bioactivity mechanisms and chemical constituents responsible for pharmacological activity remain not fully resolved. Taking into consideration the historical use of LSH in treatment of diarrhea in humans and farm animals, the aim of the study was to examine in vitro the influence of LSH and its C-glycosylic ellagitannins on processes associated with maintaining intestinal epithelium integrity and enteropathogenic Escherichia coli (EPEC) growth and adhesion. LSH was not only inhibiting EPEC growth in a concentration dependent manner but also its adhesion to IPEC-J2 intestinal epithelial cell monolayers. Inhibitory activity toward EPEC growth was additionally confirmed ex vivo in distal colon samples of postweaning piglets. LSH and its dominating C-glycosylic ellagitannins, castalagin (1), vescalagin (2), and salicarinins A (3) and B (4) were stimulating IPEC-J2 monolayer formation by enhancing claudin 4 production. Parallelly tested gut microbiota metabolites of LSH ellagitannins, urolithin C (5), urolithin A (6), and its glucuronides (7) were inactive. The activities of LSH and the isolated ellagitannins support its purported antidiarrheal properties and indicate potential mechanisms responsible for its beneficial influence on the intestinal epithelium.

Glutamate metabolism in a human intestinal epithelial cell layer model.

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-13C] or [15N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the 13C was incorporated into alanine, proline, ornithine, and glutamine, and the 15N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8-85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with 13C, 15N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity.

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay.

Evidence That Zika Virus Is Transmitted by Breastfeeding to Newborn A129 (Ifnar1 Knock-Out) Mice and Is Able to Infect and Cross a Tight Monolayer of Human Intestinal Epithelial Cells.

Zika virus (ZIKV) belongs to the Flavivirus genus in the Flaviviridae family. Mainly transmitted via mosquito bites (Aedes aegypti, Aedes albopictus), ZIKV has been classified in the large category of arthropod-borne viruses, or arboviruses. However, during the past two outbreaks in French Polynesia (2013–2014) and Latin America (2015–2016), several cases of ZIKV human-to-human transmission were reported, either vertically via transplacental route but also horizontally after sexual intercourse. Interestingly, high viral burdens were detected in the colostrum and breast milk of infected women and mother-to-child transmission of ZIKV during breastfeeding was recently highlighted. In a previous study, we highlighted the implication of the mammary epithelium (blood–milk barrier) in ZIKV infectious particles excretion in breast milk. However, mechanisms of their further transmissibility to the newborn via oral route through contaminated breast milk remain unknown. In this study, we provide the first experimental proof-of-concept of the existence of the breastfeeding as a route for mother-to-child transmission of ZIKV and characterized the neonatal oral transmission in a well-established mouse model of ZIKV infection. From a mechanistical point-of-view, we demonstrated for the first time that ZIKV was able to infect and cross an in vitro model of tight human intestinal epithelium without altering its barrier integrity, permitting us to consider the gut as an entry site for ZIKV after oral exposure. By combining in vitro and in vivo experiments, this study strengthens the plausibility of mother-to-child transmission of ZIKV during breastfeeding and helps to better characterize underlying mechanisms, such as the crossing of the newborn intestinal epithelium by ZIKV. As a consequence, these data could serve as a basis for a reflection about the implementation of measures to prevent ZIKV transmission, while keeping in mind breastfeeding-associated benefits.