Barrier Function Of Intestinal Epithelial Cells Research Articles

Resveratrol attenuates intestinal epithelial barrier dysfunction via Nrf2/HO-1 pathway in dextran sulfatesodium-induced Caco-2 cells.

The intestinal tract serves as an innate barrier, safeguarding the internal milieu from microorganisms and toxins. Various intestinal inflammatory diseases have a strong association with intestinal barrier dysfunction. The primary functional cells within the intestinal tract, intestinal epithelial cells (IECs) and their tight junctions (TJs), are crucial in preserving the integrity of this mechanical barrier. Resveratrol (Res), a plant-derived phenolic compound, exhibits a range of health-promoting benefits attributed to its anti-inflammatory properties. This study aims to examine Res's efficacy in bolstering IECs barrier function. Dextran sulfate sodium (DSS) was employed to induce barrier dysfunction in IECs. Inflammatory cytokines in supernatants (interleukin [IL]-6, IL-1β, tumor necrotic factor [TNF]-α, and IL-10) were quantified viaenzyme-linked immunosorbent assay (ELISA). Then we assessed monolayer integrity using transepithelial electrical resistance (TEER). TJ protein expression (zonula occludens[ZO]-1 and Occludin) in IECs was evaluated through immunofluorescence and Western blot analysis. Network pharmacology helped identify the biological processes, signaling pathways, and key targets involved in Res's mitigation of DSS-induced IECs barrier dysfunction. The efficacy of the primary target was further corroborated using Western blot. Res was shown to increase cell viability and IL-10 expression while reducing TNF-α, IL-6, and IL-1β levels, thus mitigating the inflammatory response. It enhanced TEER values and upregulated TJ protein expression (ZO-1 and Occludin). Network pharmacology revealed that Res potentially targets the NFE2L2 (nuclear factor erythroid-2-related factor 2, Nrf2), a vital antioxidant factor. Significantly, Res augmented Nrf2 and heme oxygenase 1(HO-1) protein levels, counteracting oxidative stress in the IECs barrier dysfunction model. Overall, our findings suggested that Res ameliorated DSS-induced IECs barrier dysfunction by activating Nrf2/HO-1 pathway, showcasing significant therapeutic potential in the early stages of colitis.

P095 Accelerated degradation of gut barrier-protecting 3-aminobenzoic acid exacerbate colitis in Inflammatory Bowel Disease

Abstract Background Disruption of intestinal epithelial cell (IEC) barrier function is crucial for the development of inflammatory bowel disease (IBD). Decreased diversity and compositional changes in intestinal bacteria, namely dysbiosis, impair IEC barrier function in IBD, but the underlying mechanism is not fully understood. This study aims to identify the impact of dysbiosis-driven changes in intestinal bacterial metabolites on IEC permeability in patients with IBD. Methods Caco2 cells were cultured on the trans-well inserts and stimulated with a library of intestinal bacterial metabolites. Proportional changes of trans-epithelial electrical resistance (TEER) were calculated at 48 hours after stimulation (ΔTEER%) and the permeation of 70-kDa FITC-dextran was evaluated using a Qubit fluorometer. Fecal samples were collected from 10 IBD patients with active colonic inflammation (5 ulcerative colitis and 5 Crohn’s disease) and 5 healthy controls (non-IBD, NIBD). Fecal bacteria-derived metabolites were quantified by LC-MS/MS and compared between IBD patients and NIBD controls. 16SrRNA-based metagenomic analysis was performed on the same fecal samples using QIIME2 followed by a PICRUSt2-based functional analysis of microbial communities. Anti-inflammatory effect of 3-aminobenzoic acid (3-ABA) was examined in C57BL/6 mice using a dextran sodium sulfate (DSS)-induced colitis model. Results Among 119 intestinal bacterial metabolites, 4-ABA decreased epithelial permeability the most with the highest ΔTEER% value (control vs. 4-ABA, 1.8 vs. 30.9 %, p<0.05). 3-ABA, one of three structural isomers of ABA, also decreased epithelial permeability in a dose dependent manner with higher ΔTEER% value than 4-ABA and reduced permeation of FITC-dextran (control vs. 3-ABA, 8.3 vs 3.4 nM, p<0.05). Fecal level of 3-ABA was significantly lower in IBD patients than in NIBD controls (7.0 vs. 505.0 μM). Functional prediction of intestinal microbiota demonstrated that IBD patients were significantly more enriched with the bacteria involved in the degradation of ABA than NIBD controls. Rectal administration of 3-ABA significantly improved body-weight loss, shortening of colon length, histologic scores, and the expression of inflammatory cytokines in mice with DSS-induced colitis (control vs. 3-ABA; body-weight loss,15.33 vs. 18.91 g; colon length, 6.11 vs. 7.35 cm; histologic score, 2.14 vs 0.60). Conclusion Accelerated degradation of luminal 3-ABA by intestinal bacteria may disrupt intestinal epithelial barrier function and exacerbate colitis in patients with IBD.

Differential impact of yeast cell wall products in recovery of porcine intestinal epithelial cell barrier function following Lipopolysaccharide challenge

BackgroundIn swine intestinal barrier deterioration can be caused by exposure to harmful bacteria, toxins or contaminants that can lead to a leaky gut and post weaning diarrhoea. A leaky gut leads to increased infection, inflammation and poor nutrient absorption that can impair piglet growth and ultimately survival. Application of yeast cell wall (YCW) products may offer an opportunity to reduce the intestinal barrier damage caused by microbial challenge. A Mannan rich fraction (MRF) and three YCW products were compared by examining their impact on intestinal barrier function using a Jejunal model of intestine in response to a bacterial challenge using Salmonella LPS.ResultsTrans epithelial electrical resistance (TEER) readings showed MRF had a significantly higher barrier function (P ≤ 0.05) over the positive control while YCW products A, B and C demonstrated no significant improvement to the positive control. Transcriptome analysis of the IPEC-J2 cells showed that differentially expressed genes associated with the gene ontology (GO) term for Structural molecule activity was significantly upregulated in the MRF treated cells over the positive control cells with 56 genes upregulated compared to product B (50 genes), Product C, (25 genes) and the negative control’s 60 genes. Product A had no functional grouping under the structural molecule activity term.Both qPCR and western blotting analysis of tight junction associated genes showed that MRF treated cells demonstrated significantly higher Claudin 3 junctional gene expression (P ≤ 0.05) over the positive control and treatments A, B and C. Occludin expression was significantly higher in MRF treated cells (P ≤ 0.05) over the positive control and product B. A nonsignificant rise in TJP-1 gene expression was observed in the MRF treated cells when compared to the positive control. Protein abundances of Claudin 3, Occludin and TJP-1 were significantly (P ≤ 0.05) higher following MRF application to LPS challenged IPEC-J2 cells over the positive control.ConclusionsThe difference in each YCW products production and composition appeared to influence intestinal barrier integrity. The action of MRF demonstrates its potential ability to raise intestinal barrier integrity of IPEC-J2 intestinal cells on an in vitro level through significantly elevated intracellular connections.

AKR1B8 deficiency drives severe DSS-induced acute colitis through invasion of luminal bacteria and activation of innate immunity.

Dysfunction of intestinal epithelial cells (IECs) promotes inflammatory bowel disease (IBD) and associated colorectal cancer (CRC). AKR1B8 deficiency impairs the IEC barrier function, leading to susceptibility to chronic colitis induced by dextran sulfate sodium (DSS), yet it remains unclear how acute colitic response is in AKR1B8 deficient mice. AKR1B8 knockout (KO) and littermate wild type mice were exposed to oral 1.5% DSS in drinking water for 6 days. Disease activity index and histopathological inflammation scores by H&E staining were calculated for colitic severity; permeability was assessed by fluorescein isothiocyanate dextran (FITC-Dextran) probes and bacterial invasion and transmission were detected by in situ hybridization in mucosa or by culture in blood agar plates. Immunofluorescent staining and flow cytometry were applied for immune cell quantification. Toll-like receptor 4 (TLR4) and target gene expression was analyzed by Western blotting and qRT-PCR. AKR1B8 KO mice developed severe acute colitis at a low dose (1.5%) of DSS in drinking water compared to wild type controls. In AKR1B8 KO mice, FITC-dextran was penetrated easily and luminal bacteria invaded to the surface of IEC layer on day 3, and excessive bacteria translocated into the colonic mucosa, mesenteric lymph nodes (MLNs) and liver on day 6, which was much mild in wild type mice. Hyper-infiltration of neutrophils and basophils occurred in AKR1B8 KO mice, and monocytes in spleen and macrophages in colonic mucosa increased markedly compared to wild type mice. TLR4 signaling in colonic epithelial cells of AKR1B8 KO mice was activated to promote great IL-1β and IL-6 expression compared to wild type mice. AKR1B8 deficiency in IECs drives severe acute colitis induced by DSS at a low dose through activation of the innate immunity, being a novel pathogenic factor of colitis.

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.

EPA and DHA inhibit endocytosis of claudin-4 and protect against deoxynivalenol-induced intestinal barrier dysfunction through PPARγ dependent and independent pathways in jejunal IPEC-J2 cells

Peroxisome proliferator activated receptor gamma (PPARγ) activation has been shown to protect against intestinal injury induced by different stimuli. PPARγ is known to regulate tight junction proteins (TJP) in epithelial cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are natural PPARγ agonists, but the implication of PPARγ in their physiological effects on the gut is poorly understood. Therefore, this study was conducted to investigate the mechanism of EPA and DHA effects on intestinal epithelial cell barrier function in IPEC-J2 cells exposed to deoxynivalenol (DON), a known food mycotoxin that is toxic to both humans and animals. Exposure of cells to EPA and DHA significantly increased mRNA expression of a PPARγ target gene, AP2, and concomitantly increased the protein expression of claudin-4. Treatment with EPA and DHA also reversed the endocytosis and degradation of claudin-4 caused by DON exposure. EPA and DHA also restored the membrane presence of claudin-4 and ZO-1 that was disrupted by DON. However, the protective effects of EPA and DHA against DON exposure was abolished by a specific PPARγ antagonist (T0070907), confirming the importance of PPARγ in regulating TJP expression by the fatty acids. Effect of PPARγ activation by EPA and DHA also included the restoration of transepithelial electrical resistance (TEER) and reduction of fluorescein isothiocyanate-labeled dextran (FITC-dextran) permeability that have been perturbed by DON. However, the effectiveness of EPA and DHA in opposing DON-induced decrease in TEER and the increase in FITC-dextran permeability was not affected by PPARγ inhibition, potentially suggesting the involvement of other PPARγ-independent mechanisms in the observed benefits from EPA and DHA. Collectively, this study shows that the protective effects of EPA and DHA against DON-induced intestinal barrier disruption are through both PPARγ-dependent and-independent pathways. Therefore, EPA and DHA containing ingredients could be used to prevent the DON-induced gut barrier dysfunction in humans and animals.

Claudin‐23 Strengthens the Colonic Epithelial Barrier by Regulating Claudin‐3 and ‐4 proteins in the Tight Junction Plasma Membrane

Colonic epithelial barrier function is controlled by differential expression of Claudin (CLDN) proteins in the crypt‐luminal axis. We recently identified the expression of a CLDN family member, CLDN‐23, in intestinal epithelial cells (IECs). However, the role of CLDN‐23 in regulating epithelial barrier function has not been identified. Analysis of the human and murine intestinal crypt‐luminal axis revealed increased CLDN‐23 expression in differentiated IECs facing the lumen compared to the proliferative crypt‐base IECs. Similarly, CLDN‐23 expression was higher in differentiated human and murine IECs cultured as a monolayer on permeable supports compared to proliferative IECs cultured in a 3D matrigel matrix. To investigate the contribution of CLDN‐23 in regulating IEC barrier function in vivo, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Cldn23 knockout mice (Cldn23ERΔIEC). Functional analysis displayed increased intestinal mucosal permeability to 4kDa FITC dextran, indicating compromised epithelial barrier function. To confirm this observation, we created human IECs in which CLDN‐23 expression was either silenced or enhanced in vitro. CLDN‐23 loss increased paracellular permeability to 4kDa FITC dextran and reduced transepithelial electrical resistance (TEER) consistent with a leaky barrier. Conversely, CLDN‐23 overexpression resulted in improved barrier function. Altogether, these data suggest that CLDN‐23 controls the IEC barrier function.Evaluation of CLDN protein interactions employing co‐culture of HeLa cells expressing CLDN‐23 with those expressing either CLDN‐2, CLDN‐3, or CLDN‐4 showed that CLDN‐23 interacts in trans with barrier‐forming CLDN‐3 and CLDN‐4, but not with pore‐forming CLDN‐2. Using in situ proximity ligation assay, we observed close association in cis between CLDN‐23 and barrier‐forming CLDN‐3 and CLDN‐4 in the TJ plasma membrane, but not with CLDN‐2. These findings suggest that CLDN‐23 interacts in both cis and trans with barrier‐forming CLDN‐3 and CLDN‐4 in differentiated IECs. Interestingly, the overexpression of CLDN‐23 in human IECs revealed up‐regulation of barrier‐forming CLDN‐3 protein expression in the TJ while decreasing the pore‐forming CLDN‐2 protein at this site. In conclusion, we identified that CLDN‐23 strengthens the colonic barrier properties by orchestrating the composition of CLDN protein interactions in epithelial TJs.

Myosin chaperone, UNC‐45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The intestinal epithelium creates a protective barrier separating the luminal content and the immune system in the gut. Leakiness of the intestinal epithelial barrier and inefficient healing of mucosal wounds represent common manifestations of inflammatory bowel diseases (IBD). Integrity of the gut barrier is mediated by different adhesive structures, including adherens junctions (AJ), tight junctions (TJ) and focal adhesion (FA). Functions of these adhesive structures depend on their association with the cortical actin cytoskeleton and particularly, to a major actin motor protein, non‐muscle myosin II (NM‐II). While biochemical mechanisms that regulate activity of NM‐II in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. In this study, we focused on UNC‐45A, a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC‐45A were decreased in colonic epithelium of patients with ulcerative colitis in parallel to diminished expression of NM‐IIA and NM‐IIC isoforms. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired AJ and TJ assembly, and cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC barrier function, junctional assembly and migration. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

Tea Tree Oil Terpinen-4-ol Protects Gut Barrier Integrity by Upregulation of Tight Junction Proteins via the ERK1/2-Signaling Pathway.

Tea tree oil (TTO) exhibits a potent antioxidant, antibacterial, and anti-inflammatory activity and is commonly used in skincare products. However, it is not clear whether TTO can protect gut barrier damage in inflammatory bowel disease (IBD) patients. Herein, we report the impact of terpinen-4-ol (TER, the primary constituent of TTO), on lipopolysaccharide (LPS)-induced intestinal epithelial cell barrier function impairment in intestinal porcine epithelial cell lines (IPEC-J2) and dextran sulfate sodium (DSS)-induced IBD in mice. TER protected against LPS-induced damage in IPEC-J2 cells in vitro and attenuated DSS-induced colitis in vivo. Added TER promoted the tight junction (TJ) proteins expressing in vitro and in vivo and attenuated the LPS-induced upregulation of ERK phosphorylation in IPEC-J2 cells. However, when an inhibitor of ERK phosphorylation was added, TER did not promote the expression of TJ protein, denoting that the ERK signaling pathway mediates the upregulation of TJ proteins. Our data may propose the potential application of TER in treating IBD.

N-Acyl-Homoserine Lactones May Affect the Gut Health of Low-Birth-Weight Piglets by Altering Intestinal Epithelial Cell Barrier Function and Amino Acid Metabolism.

In piglets, low birth weight (LBW) is associated with intestinal dysfunction, which affects their growth performance and causes economic losses. This study was designed to test whether microbial quorum sensing (QS) affects LBW-induced intestinal developmental defects in piglets. Seven normal-birth-weight (NBW; 1.36±0.01 kg) and 7 LBW (0.89±0.01 kg) piglets were selected. Feces were collected from piglets on 2, 21, and 50 days of age for detection of the QS signaling molecules, N-acyl-homoserine lactones (AHLs), and microbiota analysis. The associations between 2 long-chain AHLs [N-3-oxo-dodecanoyl-l-homoserine lactone (3OC12-HSL) and N-3-oxo-tetradecanoyl-l-homoserine lactone (3OC14-HSL)] and the microbes were tested using Spearman correlation coefficients. The effect of 3OC12-HSL and 3OC14-HSL on intestinal porcine epithelial cell-jejunum 2 (IPEC-J2) cell viability was investigated by cholecystokinin octapeptide assay. Transcriptomic analysis was performed by RNA sequencing on cells treated with 3OC12-HSL. The concentrations of 3OC12-HSL and 3OC14-HSL in the feces of LBW piglets were higher than those in NBW piglets at age 50 d by 2.5- and 2.24-fold, respectively (P<0.05). The microbial α diversity (observed species, abundance-based coverage estimator, and Shannon index) of LBW piglets was 81-91% lower than that of NBW piglets (P<0.05). The relative abundance of Ruminococcaceae UCG-002/UCG-013 was 43.0% and 30.0% lower, respectively, in feces from LBW compared with NBW piglets (P<0.05). 3OC12-HSL and Ruminococcaceae UCG-002/UCG-005/UCG-010 abundance were negatively correlated (ρ ≤ -0.58). Treatment with 400 μM 3OC12-HSL markedly reduced IPEC-J2 cell viability by 47.5%. Transcriptomic data showed that 3OC12-HSL mainly changed the "import across plasma membrane" and "arginine and proline metabolism" of IPEC-J2 cells. 3OC12-HSL is a QS signaling molecule with an ability to impair gut health of LBW piglets. This finding adds to our understanding of the mechanisms responsible for gut injury in LBW piglets.

Lactobacillus casei LC01 Regulates Intestinal Epithelial Permeability through miR-144 Targeting of OCLN and ZO1.

Our previous report determined that miR-144 is a key regulator of intestinal epithelial permeability in irritable bowel syndrome with diarrhea (IBS-D) rats. Recent evidence has shown that lactobacilli play an important role in the relief of IBS-D symptoms. However, few studies have addressed the mechanisms by which microRNAs and lactobacilli exert their beneficial effects on intestinal epithelial permeability. Hence, to elucidate whether miRNAs and lactobacilli play roles in intestinal epithelial barrier regulation, we compared miRNA expression levels in intestinal epithelial cells (IECs) under Lactobacillus casei (L. casei LC01) treatment. IECs and L. casei LC01 were co-cultured and then subjected to microRNA microarray assay. qRT-PCR, western blot and ELISA were used to detect the expression of occludin (OCLN) and zonula occludens 1 (ZO1/TJP1). The interaction between miRNAs and L. casei LC01 acting in IECs was investigated through transfection of RNA oligoribonucleotides and pcDNA 3.1 plasmid. The results are as follows: 1) L. casei LC01 decreased the expression of miR-144 and FD4 and promoted OCLN and ZO1 expression in IECs; 2) L. casei LC01 enhanced the barrier function of IECs via downregulation of miR-144 and upregulation of OCLN and ZO1; 3) Under L. casei LC01 treatment, OCLN and ZO1 overexpression could partially eliminate the promoting effect of miR-144 on intestinal permeability in IECs. Our results demonstrate that L. casei LC01 regulates intestinal permeability of IECs through miR-144 targeting of OCLN and ZO1. L. casei LC01 can be a possible therapeutic target for managing dysfunction of the intestinal epithelial barrier.

Intestinal epithelial cells: at the interface of the microbiota and mucosal immunity.

The intestinal epithelium forms a barrier between the microbiota and the rest of the body. In addition, beyond acting as a physical barrier, the function of intestinal epithelial cells (IECs) in sensing and responding to microbial signals is increasingly appreciated and likelyhas numerous implications for the vast network of immune cells within and below the intestinal epithelium. IECs also respond to factors produced by immune cells, and these can regulate IEC barrier function, proliferation and differentiation, as well as influence the composition of the microbiota. The mechanisms involved in IEC-microbe-immune interactions, however, are not fully characterized. In this review, we explore the ability of IECs to direct intestinal homeostasis by orchestrating communication between intestinal microbes and mucosal innate and adaptive immune cells during physiological and inflammatory conditions. We focus primarily on the most recent findings and call attention to the numerous remaining unknowns regarding the complex crosstalk between IECs, the microbiota and intestinal immune cells.

The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition.

Carbon nanomaterials (CNMs) can positively regulate seed germination and enhance plant growth. However, clarification of the impact of plant organs containing absorbed CNMs on animal and human health is a critical step of risk assessment for new nano-agro-technology. In this study, we have taken a comprehensive approach to studying the effect tomato fruits derived from plants exposed to multi-walled carbon nanotubes (CNTs) have on gastrointestinal epithelial barrier integrity and their impact on the human commensal intestinal microbiota using an in vitro cell culture and batch human fecal suspension models. The effects of CNTs on selected pure cultures of Salmonella enterica Typhimurium and Lactobacillus acidophilus were also evaluated. This study demonstrated that CNT-containing fruits or the corresponding residual level of pure CNTs (0.001 μg ml-1) was not sufficient to initiate a significant change in transepithelial resistance and on gene expression of the model T-84 human intestinal epithelial cells. However, at 10 μg ml-1 concentration CNTs were able to penetrate the cell membrane and change the gene expression profile of exposed cells. Moreover, extracts from CNT-containing fruits had minimal to no effect on human intestinal microbiota as revealed by culture-based analysis and 16S rRNA sequencing.

Neutrophils Promote Amphiregulin Production in Intestinal Epithelial Cells through TGF-β and Contribute to Intestinal Homeostasis.

Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-β promoted AREG production by IECs. Mechanistically, TGF-β activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-β-induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.

Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway.

AIMTo explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction (TPSJ) on the epithelial barriers in vitro.METHODSCaco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance (TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respectively used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. Western blotting was also used to evaluate the cellular expression of myosin light chain (MLC), phosphorylated MLC (pMLC), MLC kinase (MLCK), and nuclear factor (NF)-κB p65.RESULTSTPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cytokines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor (TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65.CONCLUSIONOur results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

Cecropin A Modulates Tight Junction-Related Protein Expression and Enhances the Barrier Function of Porcine Intestinal Epithelial Cells by Suppressing the MEK/ERK Pathway.

Inflammatory bowel disease (IBD) in humans and animals is associated with bacterial infection and intestinal barrier dysfunction. Cecropin A, an antimicrobial peptide, has antibacterial activity against pathogenic bacteria. However, the effect of cecropin A on intestinal barrier function and its related mechanisms is still unclear. Here, we used porcine jejunum epithelial cells (IPEC-J2) as a model to investigate the effect and mechanism of cecropin A on intestinal barrier function. We found that cecropin A reduced Escherichia coli (E. coli) adherence to IPEC-J2 cells and downregulated mRNA expression of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). Furthermore, cecropin A elevated the transepithelial electrical resistance (TER) value while reducing the paracellular permeability of the IPEC-J2 cell monolayer barrier. Finally, by using Western blotting, immunofluorescence and pathway-specific antagonists, we demonstrated that cecropin A increased ZO-1, claudin-1 and occludin protein expression and regulated membrane distribution and F-actin polymerization by increasing CDX2 expression. We conclude that cecropin A enhances porcine intestinal epithelial cell barrier function by downregulating the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We suggest that cecropin A has the potential to replace antibiotics in the treatment of IBD due to its antibacterial activity on gram-negative bacteria and its enhancement effect on intestinal barrier function.

P115 THE IBD CANDIDATE GENE, PTPN2, RESTRICTS INTESTINAL BARRIER DEFECTS AND CLAUDIN-2 EXPRESSION VIA STAT INHIBITION

Loss-of-function mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene locus increase IBD susceptibility. We reported that PTPN2 knockdown (PTPN2-KD) in intestinal epithelial cells (IECs): (i) increased IFN-g activation of STAT signaling; (ii) reduced IEC barrier function; (iii) increased expression of the tight junction (TJ) protein, claudin-2, by IFN-g; and that the claudin-2 promoter contains a STAT binding sequence. Increased claudin-2 expression occurs in IBD and increases permeability to cations thus reducing transepithelial electrical resistance (TER). To confirm PTPN2 regulation of barrier function in vivo and identify the mechanisms of STAT regulation of claudin-2 expression. In Ussing chamber studies, TER was significantly reduced in cecum from Ptpn2 knockout (KO) and heterozygous (Het) mice vs. wild-type (WT) littermates indicating increased electrolyte permeability. Immunostaining and western blotting showed progressive increases in claudin-2 expression and STAT-1 phosphorylation (Y701) in Het and KO mice vs. wild-type intestine. Claudin-2 was also elevated in intestinal enteroids from Ptpn2-deficient mice. STAT-1 siRNA significantly decreased the elevated claudin-2 expression in PTPN2-KD IECs. Chromatin immunoprecipitation (ChIP) analysis showed increased STAT-1 binding to the claudin-2 promoter in PTPN2-KD vs. control cells. Claudin-2 promoter-luciferase reporter construct activity was significantly elevated in PTPN2-KD vs. Con-shRNA IEC and was further increased by IFN-g (1000 U/ml). This effect was lost by two different mutations of the STAT binding site thus confirming the requirement of STAT binding for increased claudin-2 transcription in PTPN2-KD IECs. Loss of functional PTPN2 increases susceptibility to inflammation-induced tight junction reorganization, providing an explanation for the association of PTPN2 mutants with UC and CD.

The Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis in Intestinal Epithelial Cells Regulates Intestinal Barrier Function During Intestinal Epithelial Cells–CD4+T-Cell Interactions

Background/Aims: Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function. Methods: In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2–/– mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system. Results: The intestinal mucosal barrier damage in S1PR2–/– mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2–/– mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression. Conclusions: The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.

Enteric glial–mediated enhancement of intestinal barrier integrity is compromised by morphine

BackgroundThe opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability. Materials and methodsRat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the μ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing. ResultsEGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control. ConclusionsMorphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.

The impact of lemon pectin characteristics on TLR activation and T84 intestinal epithelial cell barrier function

Sufficient dietary fibre intake reduces the risk of several diseases, but the mechanisms linking fibre structure and health effects remain unclear. To analyse the influence of the amount of methyl groups esterified to the backbone, lemon pectins of different degrees of methyl esterification (30, 56, and 74DM) were studied for immune receptor activating potential and epithelial barrier protection. In reporter assays, the pectins demonstrated TLR/MyD88 dependent activation of NF-κB/AP-1, which increased with increasing pectin DM. To analyse the importance of backbone structure, the pectins were enzymatically digested into oligomers, which abrogated TLR activating potential. The 30 and 74DM pectins induced strong protection of the epithelial barrier measured by T84 transepithelial electrical resistance (TEER). These results indicate that activation of immune cells by lemon pectins is TLR dependent, and the intact polymer backbone is indispensable for activation. In addition, DM is a determining factor in activating potential and epithelial barrier protective effects.