Barrier Function Of Intestinal Epithelial Cells Research Articles

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.

Toll-Like Receptor 2 Activation by β2→1-Fructans Protects Barrier Function of T84 Human Intestinal Epithelial Cells in a Chain Length–Dependent Manner

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction–mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length–dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan–mediated health effects.

Enteropathogenic Escherichia coli inhibits type I interferon- and RNase L-mediated host defense to disrupt intestinal epithelial cell barrier function.

Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-β is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-β induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-β and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function.

Nod2 improves barrier function of intestinal epithelial cells via enhancement of TLR responses

Intestinal epithelial cells (IECs) form a physical barrier between the internal milieu and the intestinal microflora via the expression of tight junctions. TLR-mediated recognition of intestinal microflora by IECs is important for tight junction preservation, production of chemokines, and cell survival. Disturbance of the IEC barrier function results in bacterial invasion and contributes to the development of inflammatory bowel disease. We observed that muramyl dipeptide (MDP), a breakdown product of bacterial peptidoglycan, strongly enhances subsequent Toll-like receptor (TLR) responses in murine colonic epithelium cell lines. Prior exposure to MDP significantly increased the production of chemokines and cytokines and improved the barrier function induced by different TLR2 and TLR4 ligands. shRNA knock-down studies showed that MDP recognition by Nod2 mediated the enhancement of TLR responses. Our studies indicate that Nod2 stimulation by MDP significantly enhances TLR-mediated IEC barrier function and chemokine production. Failure of this protective mechanism may contribute to the increased risk of Crohn's disease in individuals with a loss-of-function mutation in NOD2.

Active site and frameshift mutants of Protein Tyrosine Phosphatase non‐receptor type 2 inhibit STAT1 dephosphorylation and compromise epithelial barrier function

Mutations in Protein Tyrosine Phosphatase non-receptor type 2 (PTPN2) have been linked to Inflammatory Bowel Disease (IBD) in a subset of patients. Moreover, loss of PTPN2 promotes intestinal epithelial cell (IEC) barrier dysfunction, itself a feature of IBD. We investigated whether loss of function mutations in PTPN2 influence IEC barrier function. HCA7 clone 29 IECs were transiently transfected with constructs encoding for wild type (wt), active site (ast), or deletion frameshift mutations (fs) of PTPN2, all fused to EGFP and under the control of the CMV promoter. Western blotting and densitometry indicated that EGFP-ast or EGFP-fs transfected cells treated with the IBD cytokine, IFNγ (100U/ml, 24hrs), show increased phosphorylation of the signaling molecule, STAT1 when compared to cells transfected with EGFP-wt + IFNγ. TER measurements revealed further decreases of 20% with EGFP-ast and 19% with EGFP-fs, respectively, following IFNγ treatment (days 1–9 post-IFNγ, n=2), compared to cells transfected with EGFP alone + IFNγ. Interestingly, EGFP-wt protected cells from TER loss. These observations were further supported by FITC-dextran permeability measurements. Our data indicate that both PTPN2 mutants act as dominant-negatives, and support our hypothesis that PTPN2 dysfunction plays a role in the intestinal epithelial barrier defects associated with IBD. Supported by the CCFA and NIH T32 DK07202

Deoxynivalenol Impairs Porcine Intestinal Barrier Function and Decreases the Protein Expression of Claudin-4 through a Mitogen-Activated Protein Kinase-Dependent Mechanism ,

Deoxynivalenol (DON) is a common mycotoxin that contaminates cereals and their by-products. The gastrointestinal tract is the first physical barrier against ingested food contaminants. DON contributes to the loss of barrier function of the intestine through the decreased expression of claudin-4 protein, a tight junction protein. The mechanism by which DON alters the intestinal barrier function remains poorly characterized. Therefore, we investigated the involvement of mitogen-activated protein kinases (MAPK) in the DON-induced loss of barrier function. We first verified that 30 µmol/L of DON activated MAPK in a highly sensitive porcine intestinal epithelial cell line (IPEC-1). Inhibition of p44/42 extracellular signal-regulated kinase (ERK) phosphorylation, with 0.5 µmol/L of the specific MAPK pharmacological inhibitor U0126 for 2 h, restored the barrier function of the differentiated intestinal epithelial cell monolayers. The restoration of barrier function was evaluated by trans-epithelial electrical resistance measurements and tracer flux paracellular permeability experiments. The U0126 also restored the intestinal expression of claudin-4 protein, thereby demonstrating that MAPK activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function. Further experiments indicated that p44/42 ERK is not involved in the transcriptional regulation of claudin-4. In conclusion, we demonstrated that DON-induced activation of the p44/42 ERK signaling pathway inhibits the expression of claudin-4 protein, which leads to impaired intestinal barrier function. Given the high levels of DON in cereal grains, these observations of impaired barrier function have implications for human and animal health.

Sphingosine-1-Phosphate Regulates the Expression of Adherens Junction Protein E-Cadherin and Enhances Intestinal Epithelial Cell Barrier Function

The regulation of intestinal barrier permeability is important in the maintenance of normal intestinal physiology. Sphingosine-1-phosphate (S1P) has been shown to play a pivotal role in enhancing barrier function in several non-intestinal tissues. The current study determined whether S1P regulated function of the intestinal epithelial barrier by altering expression of E-cadherin, an important protein in adherens junctions. Studies were performed upon cultured differentiated IECs (IEC-Cdx2L1 line) using standard techniques. S1P treatment significantly increased levels of E-cadherin protein and mRNA in intestinal epithelial cells (IECs) and also led to E-cadherin localizing strongly to the cell-cell border. S1P also improved the barrier function as indicated by a decrease in 14C-mannitol paracellular permeability and an increase in transepithelial electrical resistance (TEER) in vitro. These results indicate that S1P increases levels of E-cadherin, both in cellular amounts and at the cell-cell junctions, and leads to improved barrier integrity in cultured intestinal epithelial cells.

Interferon‐gamma (IFN γ) induced epithelial barrier dysfunction in T84 human intestinal epithelial cells (IECs) occurs via phosphatidylinositol 3‐kinase (PI3‐K) mediated activation of adenosine monophosphate‐activated protein kinase (AMPK)

IFNγ plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD) and some of the effects of IFNγ on intestinal epithelial barrier function are dependent on PI3‐K. Here we investigated the role of the cellular energy sensor, AMPK in the effects of IFNγ on epithelial barrier function.Protein analysis was performed by Western blotting and AMPK knock‐down by siRNA. Transepithelial resistance (TER) was assessed by voltohmeter and epithelial permeability as the flux rate of 10 kDa FITC‐dextran.IFNγ (1000 U/ml) induced activation of AMPK in a manner independent of changes in intracellular ATP (the classic trigger for AMPK activation). Inhibition of PI3‐K with LY294002 (20 µM) blocked IFNγ‐induced AMPK phosphorylation (n=9; p&lt;0.05; 6h), and mitigated the IFNγ‐induced decrease in TER across T84 monolayers (n=9; p&lt;0.05). Knock‐down of AMPK diminished the IFNγ‐induced increase in epithelial permeability (72 h; n=3; p&lt;0.05) and prevented IFNγ‐induced downregulation of the tight junction proteins occludin and ZO‐1 (n=3; p&lt;0.05).We conclude that IFNγ sequentially activates PI3‐K and AMPK to impair tight junction protein expression and thus barrier function in IECs. Our data indicate a novel role for AMPK as a mediator of cytokine signalling, independent of changes in cellular energy status, and thus may have implications for mucosal defense in the setting of inflammation.Supported by the CCFA and NIH.

Mammalian Scribble regulates the assembly of epithelial tight junctions

Scribble, Disc‐large (DLG), and Lethal giant larvae (LGL) tumor suppressor proteins have been implicated in the regulation of epithelial cell polarity. However, the mechanisms by which these proteins contribute to epithelial polarity are not well understood. Since tight junctions (TJs) play an important role in regulating intestinal epithelial cell (IEC) polarity and barrier function, we examined if these proteins participate in TJ dynamics and in the establishment of the IEC barrier function. Model IECs, SK‐CO15 and T84 cells were used for these studies, and TJ assembly/disassembly was analyzed using the calcium switch assay. Fluorescence labeling/confocal microscopy demonstrated that Scribble and LGL1 but not DLG1 co‐localized with TJ proteins, occludin and zonula occludens‐1. Additionally, Scribble and LGL1 co‐internalized with the TJ proteins during TJ disassembly induced by calcium depletion. Furthermore, siRNA‐mediated downregulation of Scribble, but not LGL1 or DLG1 significantly attenuated the functional and morphologic assembly of TJs during calcium repletion. Downregulation of Scribble did not influence expression of DLG1 or LGL1 and vise versa, and these proteins did not co‐associate as determined by immunoprecipitation. In conclusion, we demonstrate a unique role of mammalian Scribble in regulation of IEC TJ assembly and in the development of epithelial barrier function.Supported by CCFA Career Development Award (AII), NIH DK‐59888 (AN), and NIH DK‐55567 (AN)

Ste20-related proline/alanine-rich kinase: A novel regulator of intestinal inflammation

Recently, inflammatory bowel disease (IBD) has been the subject of considerable research, with increasing attention being paid to the loss of intestinal epithelial cell barrier function as a mechanism of pathogenesis. Ste20-related proline/alanine-rich kinase (SPAK) is involved in regulating barrier function. SPAK is known to interact with inflammation-related kinases (such as p38, JNK, NKCC1, PKCtheta, WNK and MLCK), and with transcription factor AP-1, resulting in diverse biological phenomena, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and regulation of chloride transport. This review examines the involvement of Ste20-like kinases and downstream mitogen-activated protein kinases (MAPKs) pathways in the pathogenesis and control of intestinal inflammation. The primary focus will be on the molecular features of intestinal inflammation, with an emphasis on the interaction between SPAK and other molecules, and the effect of these interactions on homeostatic maintenance, cell volume regulation and increased cell permeability in intestinal inflammation.

Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C

Background & Aims: Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria. Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites. The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs. Methods: TLR2 agonist (synthetic bacterial lipopeptide Pam 3CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays—combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2. Transepithelial electrical resistance characterized intestinal epithelial barrier function. Results: Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCα and PKCδ). Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant. Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists. This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident. Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation. Conclusions: The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation.

Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration.

Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lacking eae and espB (eae- and espB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion than eae- and espB-positive STEC or enteropathogenic E. coli. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.

PI3'-kinase- and fas-dependent pathways regulate apoplosis, barrier function and chloride secretion in intestinal epithelial cells (IEC): Implications for inflammatory diarrheal states

BACKGROUND/OBJECTIVES: The epithelial lining of the intestine serves as a critical barrier to lumenal bacteria and may be compromised by pathotogic Fas-mediated IEC apoptosis. Phosphatidylinositol (PI)3'-kinase has been described to limit apoptosis in other systems. We hypothesized that PI3'-kinase dependent pathways regulate Fas-mediated apoptosis and barrier function in IEC. METHODS: lEO lines (HT-29, T84) were exposed to agonist anti-Fas Ab in the presence or absence of chemical inhibitors of PI3'-kinase (LY294002, wortmannin). Apoptosis, barrier function, short-circuit current (Alsc) and expression of adhesion molecules were assessed. RESULTS: Inhibition of PI3'-kinase strongly sensitized lEG to Fas-mediated apoptosis. Inhibition of PI3'-kinase combined with cross-linking Fas was associated with barrier dysfunction ()transepithelial electrical resistance (TER), permeability). Confocal analyses revealed disruption of actin and adhesion/junctional molecules (EpCAM, E-cadherin, ZO-1, desmoplakin) in monolayers exposed to anti-Fas Ab in the context of PI3'-kinase inhibition. Caspase inhibition prevented apoptosis in response to cross-linking Fas in the presence or absence of PI3'-kinase inhibitors. In spite of preventing apoptosis, caspase inhibition did not effect PI3'-kinase-dependent changes in TER. To explore additional mechanisms for diminished TER, we measured changes in Isc in T84 monolayers. Whereas cross-linking Fas alone had no effect on L~Isc, cross-linking Fas in the presence of LY294002 led to synergistic increases in L~Isc consistent with increased CIsecretion. To determine downstream effectors mediating PI3'-kinase-dependent protection from Fas-mediated apoptosis, we expressed constitutivelyactive Akt, a principal downstream effector of the PI3'-kinase pathway. Akt protected against Fas-mediated apoptosis to an extent that was comparable to expression of a genetic caspase inhibitor p35. CONCLUSIONS: PI3'-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediatad apoptosis. Inhibition of PI3'-kinase in the context of Fas signaling results in increased chloride secretion. These findings suggest that agonists of PI3'-kinase such as growth factors may have a dual effect on intestinal inflammation by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.

Extracellular regulated mitogen activated kinase activity is required for the regulation of the intestinal epithelial cell barrier function by IL-17

Extracellular regulated mitogen activated kinase activity is required for the regulation of the intestinal epithelial cell barrier function by IL-17

Regulation of tight junction formation in intestinal epithelial cells by the IL-15 ligand-receptor system

Background: Human intestinal epithelial cells express IL-15 as well as IL-15 receptors consisting of the IL-15 receptor ct, IL-2 receptor 13 and 7c subunits. However the functional role of the IL-15 ligand receptor system in the regulation of intestinal epithelial cell function is unknown but may modulate the intestinal epithelial barrier. We assessed the regulation of protein levels and cellular distribution of the tight junction associated proteins ZO-1, ZO-2, and occludin in intestinal epithelial ceils by an IL-15 and IL-2 receptor 13 chain dependent pathway. Methods: The human intestinal epithelial cell line T-84, a model of intestinal crypt epithelium, which lacks the expression of the IL-2 receptor 13 chain constitutively but expresses IL-15, was stably transfected with the IL-2 receptor 13 chain to substitute a complete IL-15 receptor complex. Formation of tight junctions was determined by the measurment of transepithelial electrical resistance (TER). Expression of ZO-1, ZO-2, occludin, and E-cadherin as well as phosphotyrosine kinase activity was assessed by Western blotting of membrane and cytosol associated proteins. Cell proliferation was determined by 3H Thymidine uptake and MTS assays. Results: Expression of the IL-2 receptor 13 chain in T-84 cells resulted in a 3 fold increase of steady state TER within 4 four days after replating above the levels reached by the parental T-84 cells. The development TER in IL-2 receptor 13 chain transfected T-84 cells could be further enhanced by exogenous IL-15 and blocked by addition of anti IL-2 receptor antibodies. In contrast cell proliferation of IL-2 receptor 13 chain transfected and parental T-84 ceils was not altered. IL-2 receptor 13 chain transfected T-84 ceils demonstrated strong upregulation of tyrosine phosphorylation of a number of proteins with molecular masses of 120-100 and 60-40 kDa. The protein levels of ZO-1 and phosphorylated and unphosphorylated Occludin were increased 10 fold in the membrane associated protein fractions of IL-2 receptor 13 chain transfected T-84 cells compared to parental cells. In contrast, the levels of ZO-2 in membrane associated fractions of the stable transfectans was decreased 3 fold. The protein levels of E-cadherin were not regulated by the expression of the IL-2 receptor 13 chain in T-84 cells. Conclusion: Reconstition of a complete IL-15 receptor complex in T-84 cells results in autocrine regulation of the tight junction associated proteins ZO-1, ZO-2 and Occludin leading to the marked increase of TER. IL-15 may be an important regulator of intestinal epithelial cell barrier function.