Intestinal Epithelial Tight Junction Permeability Research Articles

Lactobacillus acidophilus inhibits the TNF-α-induced increase in intestinal epithelial tight junction permeability via a TLR-2 and PI3K-dependent inhibition of NF-κB activation.

Defective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in thepathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-α (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapies with benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown. The primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNF-α-induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems. Pre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-κB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-κB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-κB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-κB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-α phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-κB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability.

Bifidobacterium bifidum Strain BB1 Inhibits Tumor Necrosis Factor-α–Induced Increase in Intestinal Epithelial Tight Junction Permeability via Toll-Like Receptor-2/Toll-Like Receptor-6 Receptor Complex–Dependent Stimulation of Peroxisome Proliferator-Activated Receptor γ and Suppression of NF-κB p65

Bifidobacterium bifidum (BB) strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. TNF-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. Previously reported, TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. The addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α induced increase in intestinal TJ permeability by interfering the with TNF-α induced enterocyte NF-κB p50/p65 and MLCK gene activation. The BB1 protective effect against the TNF-α induced increase in intestinal permeability was mediated by TLR-2/TLR-6 heterodimer complex activation of PPAR-γ and PPAR-γ pathway inhibition of TNF-α induced IKK-α activation, which in turn resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, MLCK-induced opening of the TJ barrier. In conclusion, these studies unravel novel intracellular mechanisms of BB1 protection against the TNF-α induced increase in intestinal TJ permeability. Our data show that BB1 protects against the TNF-α induced increase in intestinal epithelial TJ permeability via a PPAR-γ dependent inhibition of NF-κB p50/p65 and MLCK gene activation.

Bifidobacterium bifidum inhibits the TNF-α induced increase in intestinal epithelial tight junction permeability via a TLR-2/TLR-6 and PPAR-γ dependent inhibition of NF-κB p50/p65

Background: Bifidobacterium bifidum (BB) is one of the most widely used probiotic bacterial species and BB has been shown to have a protective effect against intestinal inflammation by an enhancement of the intestinal epithelial tight junction (TJ) barrier. Previous studies from our laboratory showed that a specific BB strain, referred to as BB1, caused a marked enhancement of intestinal epithelial TJ barrier, while strain BB4 had no effect. TNF-α contributes to the development of intestinal inflammation in inflammatory conditions of the gut, in part by increasing intestinal permeability and antigenic penetration. TNF-α induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB p50/p65 activation of myosin light chain kinase (MLCK) gene and MLCK-induced opening of the intestinal TJ barrier. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor known to have anti-inflammatory activity by interfering with NF-κB activation. However, the protective effect of BB1 against TNF-α induced increase in intestinal TJ permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to elucidate the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were the model systems used to assess intestinal TJ permeability. Results: 1) TNF-α caused an increase Caco-2 TJ permeability, as assed by Caco-2 trans-epithelial resistance (TER) and trans-epithelial flux of paracellular marker, FITC-Dextran 10kD. 2) Addition of BB1 (1x108 CFU/ml) to the apical membrane compartment inhibited the TNF-α increase in Caco-2 epithelial TJ permeability, while strain BB4 had no effect. 3) BB1 but not BB4, caused an increase in Caco-2 TLR-2 expression on the apical membrane surface, and siRNA induced knockdown of TLR-2 prevent the BB1 inhibition of TNF-α induced increase in Caco-2 TJ permeability. 4) TNF-α caused a rapid phosphorylation and degradation of Caco-2 IKK-α, as well as and phosphorylation and nuclear translocation of NF-κB p50/p65, indicating NF-κB activation. BB1, but not BB4, inhibited the NF-κB activation. 5) TNF-α activation of NF-κB was associated with an increase in MLCK gene (increase in MLCK promoter activity and mRNA transcription) and kinase activity. BB1, but not BB4, inhibited the TNF-α induced increase in MLCK gene and kinase activity. 6) BB1 but not BB4, caused a rapid PPAR-γ activation (PPAR-γ phosphorylation and nuclear translocation), and siRNA induced knockdown of PPAR-γ inhibited the TNF-α induced activation of NF-κB and increase in Caco-2 TJ permeability. 7) Intraperitoneal injection of TNF-α (5 mg) also caused time-dependent increase in mouse small intestinal permeability (IP) to FITC-Dextran 10kD. TNF-α caused a degradation of IkB-α in intestinal tissue and nuclear translocation of NF-kB in mouse enterocyte. 8) BB1, but not BB4, selectively activated PPAR-γ in mouse enterocytes and inhibited the TNF-α-induced activation of NF-κB, resulting in a decrease in mouse IP. 9) BB1 inhibition of TNF-α activation of mouse enterocyte NF-κB and increase in IP was prevented in mice with intestinal epithelial cell-specific knockout of PPAR-γ Villin-cre and TLR-2 Villin-cre mice. Conclusion: BB1 protects against the TNF-α induced increase in intestinal TJ permeability in a strain-specific manner. BB1 protects against the TNF-α induced increase in intestinal TJ permeability via a novel intracellular mechanism involving BB1/TLR-2 pathway activation of PPAR-γ, and PPAR-γ mediated inhibition of NF-κB p50/p65 and MLCK gene in enterocyte. This research project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MATRIX METALLOPROTEINASE (MMP-9) INDUCED INCREASE IN INTESTINAL EPITHELIAL TIGHT JUNCTION BARRIER IS MEDIATED BY EGFR ACTIVATION

Abstract BACKGROUND The defective intestinal tight junction (TJ) barrier has a key pathogenic factor in Crohn’s disease (CD) and other inflammatory conditions of the gut. Clinical studies in CD patients have shown that a persistent increase in intestinal permeability is predictive of poor clinical outcome. Matrix metalloproteinase-9 (MMP-9) has been shown to be markedly increased in intestinal tissues of patients with CD and it has been postulated that MMP-9 plays an important role on the pathogenesis of intestinal inflammation in CD. Our recent studies suggested that MMP-9 at clinically relevant concentrations causes an increase in intestinal epithelial TJ permeability in-vitro and in-vivo by inducing intracellular signaling pathways via activating distinct basolateral membrane receptors. However, the specific protein receptors and the intracellular signaling cascades involved have not been fully identified. One of the potential protein receptors that might be a target of MMP-9 on the basolateral membrane of the intestinal epithelial cells is the epidermal growth factor receptor (EGFR), which has been shown to have opposing roles in the pathophysiology of CD. AIMS Therefore, our aims in this study were to examine the effect of MMP-9 on EGFR activation and delineate the role of EGRF in MMP-9 induced increase in intestinal epithelial TJ permeability by using Caco-2 monolayers as an in-vitro model system. RESULTS MMP-9 (400 ng/ml) caused a time-dependent increase in EGFR activation as assessed by EGFR phosphorylation, and by in-vitro kinase activity measurement of EGFR in filter-grown Caco-2 monolayers. Inhibition of EGFR by pharmacologic inhibitor, PD 153035, prevented the MMP-9 induced increase in Caco-2 TJ permeability measured by trans-epithelial resistance (TER) and mucosal-to-serosal flux of a paracellular marker dextran 10kd. MMP-9 caused a decrease in the TJ protein occludin expression. Inhibition of EGFR by PD 153035 prevented the MMP-9 induced degradation of occludin expression. In addition, the effect of EGFR activation by a known activator, NSC 228155 25, in the absence of MMP-9, caused an increase in Caco-2 TJ permeability and a decrease in occludin expression. CONCLUSION Our data showed that MMP-9 induced increase in intestinal epithelial TJ permeability is mediated in part by the activation of EGFR and the subsequent degradation of occludin expression. Identifying the specific membrane receptors that mediate the MMP-9 induced increase in intestinal TJ permeability could be important in developing future therapeutic strategies in IBD.

AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability

ABSTRACT The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXФ) (67–70 and 148–151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation. Abbreviations: Amil: amiloride; AP2: adaptor protein complex 2; AP2A1: adaptor related protein complex 2 subunit alpha 1; AP2M1: adaptor related protein complex 2 subunit mu 1; ATG7: autophagy related 7; CAL: calcitriol; Cas9: CRISPR-associated protein 9; Con: control; CPZ: chlorpromazine; DSS: dextran sodium sulfate; EBSS: Earle’s balanced salt solution; IBD: inflammatory bowel disease; TER: trans-epithelial resistance; KD: knockdown; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MβCD: Methyl-β-cyclodextrin; MET: metformin; MG132: carbobenzoxy-Leu-Leu-leucinal; MTOR: mechanistic target of rapamycin kinase; NT: non target; RAPA: rapamycin; RES: resveratrol; SMER: small-molecule enhancer 28; SQSTM1: sequestosome 1; ST: starvation; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Matrix Metalloproteinase-9 (MMP-9) induced disruption of intestinal epithelial tight junction barrier is mediated by NF-κB activation.

BackgroundMatrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear.AimsThe main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system.ResultsMMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis.ConclusionOur data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s).

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 > 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene

Lipopolysaccharides (LPSs) are a major component of Gram-negative bacterial cell wall and play an important role in promoting intestinal inflammatory responses. Recent studies have shown that physiologically relevant concentrations of LPS (0 to 2000 pg/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability without causing cell death. However, the intracellular pathways and the mechanisms that mediate LPS-induced increase in intestinal TJ permeability remain unclear. The aim was to delineate the intracellular pathways that mediate the LPS-induced increase in intestinal permeability using invitro and invivo intestinal epithelial models. LPS-induced increase in intestinal epithelial TJ permeability was preceded by an activation of transforming growth factor-β-activating kinase-1 (TAK-1) and canonical NF-κB (p50/p65) pathways. The siRNA silencing of TAK-1 inhibited the activation of NF-κB p50/p65. The siRNA silencing of TAK-1 and p65/p50 subunit inhibited the LPS-induced increase in intestinal TJ permeability and the increase in myosin light chain kinase (MLCK) expression, confirming the regulatory role of TAK-1 and NF-κB p65/p50 in up-regulating MLCK expression and the subsequent increase in TJ permeability. The data also showed that toll-like receptor (TLR)-4/myeloid differentiation primary response (MyD)88 pathway was crucial upstream regulator of TAK-1 and NF-κB p50/p65 activation. In conclusion, activation of TAK-1 by the TLR-4/MyD88 signal transduction pathway and MLCK by NF-κB p65/p50 regulates the LPS-induced increase in intestinal epithelial TJ permeability.

Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability.

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.

264 - Tocopherylquinone-Mediated Activation of Aryl Hydrocarbon Receptor Reduces Intestinal Epithelial Tight Junction Permeability and Severity of Experimental Colitis

264 - Tocopherylquinone-Mediated Activation of Aryl Hydrocarbon Receptor Reduces Intestinal Epithelial Tight Junction Permeability and Severity of Experimental Colitis

Lipopolysaccharide-Induced Increase in Intestinal Epithelial Tight Permeability Is Mediated by Toll-Like Receptor 4/Myeloid Differentiation Primary Response 88 (MyD88) Activation of Myosin Light Chain Kinase Expression

Lipopolysaccharides (LPSs) are a major component of the Gram-negative bacterial cell wall and play an important role in mediating intestinal inflammatory responses in inflammatory bowel disease. Although recent studies suggested that physiologically relevant concentrations of LPS (0 to 1 ng/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability, the mechanisms that mediate an LPS-induced increase in intestinal TJ permeability remain unclear. Herein, we show that myosin light chain kinase (MLCK) plays a central role in the LPS-induced increase in TJ permeability. Filter-grown Caco-2 intestinal epithelial monolayers and C57BL/6 mice were used as an invitro and invivo intestinal epithelial model system, respectively. LPS caused a dose- and time-dependent increase in MLCK expression and kinase activity in Caco-2 monolayers. The pharmacologic MLCK inhibition and siRNA-induced knock-down of MLCK inhibited the LPS-induced increase in Caco-2 TJ permeability. The LPS increase in TJ permeability was mediated by toll-like receptor 4 (TLR-4)/MyD88 signal-transduction pathway up-regulation of MLCK expression. The LPS-induced increase in mouse intestinal permeability also required an increase in MLCK expression. The LPS-induced increase in intestinal permeability was inhibited in MLCK-/- and TLR-4-/- mice. These data show, for the first time, that the LPS-induced increase in intestinal permeability was mediated by TLR-4/MyD88 signal-transduction pathway up-regulation of MLCK. Therapeutic targeting of these pathways can prevent an LPS-induced increase in intestinal permeability.

Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway.

The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins and intestinal secretions, prevents passage of luminal substances and antigens through the paracellular space. Dysfunction of the intestinal barrier integrity induced by toxins and pathogens is associated with a variety of gastrointestinal disorders and diseases. Although butyrate is known to enhance intestinal health, its role in the protection of intestinal barrier function is poorly characterized. Therefore, we investigated the effect of butyrate on intestinal epithelial integrity and tight junction permeability in a model of LPS-induced inflammation in IPEC-J2 cells. Butyrate dose-dependently reduced LPS impairment of intestinal barrier integrity and tight junction permeability, measured by trans-epithelial electrical resistance (TEER) and paracellular uptake of fluorescein isothiocyanate-dextran (FITC-dextran). Additionally, butyrate increased both mRNA expression and protein abundance of claudins-3 and 4, and influenced intracellular ATP concentration in a dose-dependent manner. Furthermore, butyrate prevented the downregulation of Akt and 4E-BP1 phosphorylation by LPS, indicating that butyrate might enhance tight junction protein abundance through mechanisms that included activation of Akt/mTOR mediated protein synthesis. The regulation of AMPK activity and intracellular ATP level by butyrate indicates that butyrate might regulate energy status of the cell, perhaps by serving as a nutrient substrate for ATP synthesis, to support intestinal epithelial barrier tight junction protein abundance. Our findings suggest that butyrate might protect epithelial cells from LPS-induced impairment of barrier integrity through an increase in the synthesis of tight junction proteins, and perhaps regulation of energy homeostasis.

Caco-2 세포에서 커큐민 처리에 의한 IL-1α로 유도된 소장 상피세포의 tight junction 투과성 저해

The intestinal tight junction (TJ) plays an important role as a paracellular barrier. Impaired TJ permeability and enhanced proinflammatory cytokine production are crucial pathophysiological mechanisms in inflammatory bowel diseases (IBDs). Although proinflammatory cytokines, tumor necrosis factor-alpha and interluekin-1 beta, which are markedly increased in IBD patients, have been reported to increase intestinal TJ permeability, the role of interleukin-1 alpha (IL-1α) in the TJ has not been studied. Phytochemicals could prevent proinflammatory cytokine-caused TJ alteration. Curcumin (CCM), a biologically active component of turmeric, has a strong anti-inflammatory activity. The purpose of this study was to elucidate the effect of IL-1α on intestinal epithelial TJ permeability and the role of CCM in IL-1α’s action on TJ in an in vitro intestinal epithelial system, Caco-2 monolayers. The TJ integrity of Caco-2 monolayers was estimated by measuring the flux of FITC-labeled dextran and transepithelial electrical resistance (TEER). Apical IL-1α (100 ng/ml) treatment elevated TJ permeability and suppressed TEER of Caco-2 monolayers. Pretreatment with CCM (20 μM) for 30 min significantly inhibited IL-1α-induced TJ alterations, such as increased TJ permeability and decreased in TEER values. These results demonstrated that IL-1α-induced increases in Caco-2 TJ permeability and CCM blocked the action of IL-1α in the TJ.

TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway

Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of invitro and invivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-β. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.

Matrix metalloproteinase 9-induced increase in intestinal epithelial tight junction permeability contributes to the severity of experimental DSS colitis.

Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9(-/-) mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9(-/-) mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9(-/-) mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK(-/-) mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9(-/-) mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.

MicroRNA-21 regulates intestinal epithelial tight junction permeability.

Increased tight junction (TJ) barrier permeability, induced by tumour necrosis factor (TNF)-α, may lead to the defects in TJ barrier and subsequent development of inflammation. Recent evidence suggests that miR-21 is implicated in inflammatory diseases. However, the physiological role of miR-21 in intestinal permeability remains elusive. This study aimed to explore the role of miR-21 in intestinal epithelial tight junction permeability. The filter-grown Caco-2 monolayers model system was established to mimic intestinal barrier defect. The tight junction proteins were detected by immunofluorescence and western blot analysis. The expression of miR-21 was assessed by real-time polymerase chain reaction (PCR). We found that the expression of miR-21 was increased significantly in TNF-α induced intestinal TJ barrier defect model. miR-21 overexpression significantly enhanced while miR-21 knockdown significantly decreased intestinal permeability. In addition, miR-21 overexpression significantly increased while miR-21 knockdown significantly decreased the levels of interleukin-6, interleukin-8 and prostaglandin E2 in cell culture medium. Furthermore, miR-21 positively regulated Akt phosphorylation and negatively regulated Phosphatase and tensin homolog (PTEN) expression in Caco-2 cells. Our results suggest that miR-21 may regulate intestinal epithelial tight junction permeability through PTEN/PI3K/Akt signalling pathway. This promotes the feasibility of targeting miR-21 in the clinical to preserve the intestinal barrier.

513 Matrix Metalloproteinase MMP-12 Increases Intestinal Epithelial Tight Junction Permeability and Severity of Experimental Colitis

Background: Matrix Metalloproteinase (MMP)-induced extracellular matrix remodeling modulates intestinal inflammation. MMP-12 is a human macrophage elastase capable of degrading basement membrane (BM). Defective intestinal barrier leading to increased intestinal permeability is an important pathogenic factor for intestinal inflammation. The role of MMP-12 in intestinal barrier function and intestinal inflammation remains unclear. We hypothesize that MMP-12 induces degradation of BM and helps macrophage transmigration thereby compromising intestinal barrier and augmenting intestinal inflammation. Aim: The aim of this study was to investigate the role of MMP-12 in intestinal epithelial tight junction (TJ) permeability and macrophage transmigration in experimental dextran sodium sulfate (DSS) colitis and in vitro Caco-2 cell model. Methods: Nine weeks-old wild type (WT) and MMP-12-/mice were administered 3% DSS in drinking water for 7 days. An in vivo colonic recycling perfusion and in vitro epithelial cell model was used to study epithelial permeability. Results: DSS treatment resulted in a marked increase in MMP-12 protein expression in WT colon. DSS administration significantly increased the colonic permeability in WT but not MMP-12-/mice (p<0.01). The loss of body weight, disease activity index, and histological lesion score of colitis was significantly attenuated in MMP-12-/DSS group compared to WT DSS group (p<0.01). In immunohistochemical study, the BM laminin was significantly lost in WT DSS colon but not in MMP-12-/DSS colon. The epithelial infiltration of macrophages in DSS colitis, as assessed by macrophage marker CD68 staining, was found to be significantly lower in MMP-12-/mice than WT mice. To further investigate the role of MMP-12 in intestinal TJ permeability, human intestinal epithelial Caco-2 cells were co-cultured with phorbol myristate acetate activated, MMP-12 secreting human macrophage U937 cells. The co-culture resulted into progressive and significant decrease in Caco-2 transepithelial resistance (TER) and concomitant increase in paracellular inulin flux, indicating loss of Caco-2 TJ barrier. Furthermore, siRNA-induced knock down of MMP-12 expression in U937 macrophage cells attenuated loss of Caco-2 TER and increase in inulin flux after U937 co-culture. Also, siRNA-induced knock down of MMP-12 significantly prevented U937 macrophage transmigration across the Caco-2 cells. Conclusion:The clinical severity, colonic permeability, and macrophage infiltration in colitis was attenuated in MMP-12-/mice. Macrophage derived MMP-12 increased intestinal epithelial permeability and enables macrophage transmigration in a cell culture model. These data suggest that MMP-12-induced macrophage transmigration and loss of intestinal epithelial TJ barrier contributes to the development of colitis.

515 microRNA-375-KLF5 Regulation of Intestinal Epithelial CFTR Function

Background: Matrix Metalloproteinase (MMP)-induced extracellular matrix remodeling modulates intestinal inflammation. MMP-12 is a human macrophage elastase capable of degrading basement membrane (BM). Defective intestinal barrier leading to increased intestinal permeability is an important pathogenic factor for intestinal inflammation. The role of MMP-12 in intestinal barrier function and intestinal inflammation remains unclear. We hypothesize that MMP-12 induces degradation of BM and helps macrophage transmigration thereby compromising intestinal barrier and augmenting intestinal inflammation. Aim: The aim of this study was to investigate the role of MMP-12 in intestinal epithelial tight junction (TJ) permeability and macrophage transmigration in experimental dextran sodium sulfate (DSS) colitis and in vitro Caco-2 cell model. Methods: Nine weeks-old wild type (WT) and MMP-12-/mice were administered 3% DSS in drinking water for 7 days. An in vivo colonic recycling perfusion and in vitro epithelial cell model was used to study epithelial permeability. Results: DSS treatment resulted in a marked increase in MMP-12 protein expression in WT colon. DSS administration significantly increased the colonic permeability in WT but not MMP-12-/mice (p<0.01). The loss of body weight, disease activity index, and histological lesion score of colitis was significantly attenuated in MMP-12-/DSS group compared to WT DSS group (p<0.01). In immunohistochemical study, the BM laminin was significantly lost in WT DSS colon but not in MMP-12-/DSS colon. The epithelial infiltration of macrophages in DSS colitis, as assessed by macrophage marker CD68 staining, was found to be significantly lower in MMP-12-/mice than WT mice. To further investigate the role of MMP-12 in intestinal TJ permeability, human intestinal epithelial Caco-2 cells were co-cultured with phorbol myristate acetate activated, MMP-12 secreting human macrophage U937 cells. The co-culture resulted into progressive and significant decrease in Caco-2 transepithelial resistance (TER) and concomitant increase in paracellular inulin flux, indicating loss of Caco-2 TJ barrier. Furthermore, siRNA-induced knock down of MMP-12 expression in U937 macrophage cells attenuated loss of Caco-2 TER and increase in inulin flux after U937 co-culture. Also, siRNA-induced knock down of MMP-12 significantly prevented U937 macrophage transmigration across the Caco-2 cells. Conclusion:The clinical severity, colonic permeability, and macrophage infiltration in colitis was attenuated in MMP-12-/mice. Macrophage derived MMP-12 increased intestinal epithelial permeability and enables macrophage transmigration in a cell culture model. These data suggest that MMP-12-induced macrophage transmigration and loss of intestinal epithelial TJ barrier contributes to the development of colitis.