Intestinal Tight Junction Permeability Research Articles

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.

Melatonin ameliorates imidacloprid-induced intestinal injury by negatively regulating the PGN/P38MAPK pathway in the common carp (Cyprinuscarpio)

Imidacloprid (IMI), one of the most frequently used neonicotinoid insecticides in agriculture, is resided in surface water worldwide and poses a threat to aquatic organisms. Melatonin (MT) provides effective protection against insecticide-induced toxicity, nevertheless, the toxic effects and whether MT attenuates intestinal injury caused by IMI exposure in the common carps remains poorly explored. Previous studies have reported adverse effects of IMI exposure on intestinal health status. Therefore, we first demonstrated that IMI altered the composition and function of the intestinal microbiota, destroying the integrity of intestinal ultrastructure, increasing intestinal permeability. Meanwhile, metagenomic sequencing and ELISA kits results hypothesized that peptidoglycan (PGN) is an IMI-triggered intestinal microbial metabolite. Subsequently, we thus further elucidated that IMI induced an increase in intestinal tight junction permeability by inducing PGN secretion in vitro model. MT addition dramatically attenuated IMI-induced intestinal toxicity by remitting PGN synthesis and thus resecuring tight junction permeability, thereby reducing intestinal injury. SB203580 was supplied as a P38MAPK inhibitor to alleviate the increased permeability of tight junctions induced by IMI/PGN. Therefore, these findings confirmed that MT protects against IMI-induced intestinal injury by negatively regulating PGN/P38MAPK pathway to antagonize the increased tight junction permeability.

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.

IL-1β and the Intestinal Epithelial Tight Junction Barrier.

The intestinal epithelial tight junction (TJ) barrier controls the paracellular permeation of contents from the intestinal lumen into the intestinal tissue and systemic circulation. A defective intestinal TJ barrier has been implicated as an important pathogenic factor in inflammatory diseases of the gut including Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and celiac disease. Previous studies have shown that pro-inflammatory cytokines, which are produced during intestinal inflammation, including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ, have important intestinal TJ barrier-modulating actions. Recent studies have shown that the IL-1β-induced increase in intestinal TJ permeability is an important contributing factor of intestinal inflammation. The IL-1β-induced increase in intestinal TJ permeability is mediated by regulatory signaling pathways and activation of nuclear transcription factor nuclear factor-κB, myosin light chain kinase gene activation, and post-transcriptional occludin gene modulation by microRNA and contributes to the intestinal inflammatory process. In this review, the regulatory role of IL-1β on intestinal TJ barrier, the intracellular mechanisms that mediate the IL-1β modulation of intestinal TJ permeability, and the potential therapeutic targeting of the TJ barrier are discussed.

PROTON PUMP INHIBITORS (PPI) INDUCES COLONIC TIGHT JUNCTION BARRIER (TJ) DYSFUNCTION VIA AN UPREGULATION OF TJ PORE FORMING CALUDIN-2 PROTEIN

Abstract Background Proton pump inhibitors (PPIs) are highly effective antagonists of gastric acid secretion and are widely used to treat a number of gastroesophageal disorders, including peptic ulcer disease, GERD, and Barrett’s esophagus. PPI-induced elevation in intra-gastric pH and subsequent alterations of gastrointestinal physiology are thought to cause undesired effects on the entire GI tract. Defective intestinal Tight Junction (TJ) barrier is an important pathogenic factor for intestinal inflammation. Claudin-2 is a pore forming TJ protein whose overexpression causes selective increase in TJ permeability to small molecules. Claudin-2 expression is known to be up-regulated in intestinal inflammation and inflammation-associated colon carcinogenesis. The effect of PPI on Intestinal TJ barrier is not known. Aim: The aim of the present study was to study the effect of PPI on Intestinal TJ permeability. Methods A cell culture model of filter grown human intestinal epithelial Caco-2 monolayers, was used to study intestinal epithelial TJ barrier function. The mouse colonic permeability was measured by Ussing chambers studies. Western blot (WB) and Immunofluorescence (IF) was used to study the protein expression of claudin-2 in Caco-2 cells and in mouse colon. Cell surface biotinylation was used to study intracellular trafficking of TJ proteins. Results PPIs, caused a concentration- and time-dependent decrease in transepithelial resistance (TER), and increase in urea flux in filter-grown Caco-2 cells. Further studies on intestinal TJ revealed that PPI caused a significant increase in pore forming TJ protein claudin-2 protein level (western blot), but not mRNA (RT-PCR) in Caco-2 monolayers. Claudin-2 was co-localized to its known intercellular trafficking vesicles, clathrin pits. PPI treatment caused an increase in expression and junctional localization of claudin-2 and a decrease in its cytoplasmic co-localization with clathrin. Similarly, pulse-chase assay showed that the claudin-2 half-life was increased by PPI. In cell surface biotinylation assay, the rate of claudin-2 endocytosis was significantly reduced by PPI. In ex-vivo, Ussing chamber studies, PPI administration for 45 days resulted in a significant decrease in murine colonic TER and increase in urea flux. Immunofluorescence and Western blot analysis showed that PPI also caused an increase in TJ protein claudin-2 expression in mice colonic mucosa. Conclusion Our results suggest that the PPIs induces colonic TJ permeability via increasing pore forming claudin-2 levels. PPIs affect intracellular trafficking to reduce claudin-2 degradation and increased its junctional localization.

Selective brain cooling achieves peripheral organs protection in hemorrhagic shock resuscitation via preserving the integrity of the brain-gut axis.

Background: Although whole-body cooling has been reported to improve the ischemic/reperfusion injury in hemorrhagic shock (HS) resuscitation, it is limited by its adverse reactions following therapeutic hypothermia. HS affects the experimental and clinical bowel disorders via activation of the brain-gut axis. It is unknown whether selective brain cooling achieves beneficial effects in HS resuscitation via preserving the integrity of the brain-gut axis.Methods: Male Sprague-Dawley rats were bled to hypovolemic HS and resuscitated with blood transfusion followed by retrograde jugular vein flush (RJVF) with 4 °C or 36 °C normal saline. The mean arterial blood pressure, cerebral blood flow, and brain and core temperature were measured. The integrity of intestinal tight junction proteins and permeability, blood pro-inflammatory cytokines, and multiple organs damage score were determined.Results: Following blood transfusion resuscitation, HS rats displayed gut barrier disruption, increased blood levels of pro-inflammatory cytokines, and peripheral vital organ injuries. Intrajugular-based infusion cooled the brain robustly with a minimal effect on body temperature. This brain cooling significantly reduced the HS resuscitation-induced gut disruption, systemic inflammation, and peripheral vital organ injuries in rats.Conclusion: Resuscitation with selective brain cooling achieves peripheral vital organs protection in hemorrhagic shock resuscitation via preserving the integrity of the brain-gut axis.

IL1B Increases Intestinal Tight Junction Permeability by Up-regulation of MIR200C-3p, Which Degrades Occludin mRNA

Defects in the epithelial tight junction (TJ) barrier contribute to development of intestinal inflammation associated with diseases. Interleukin 1 beta (IL1B) increases intestinal permeability in mice. We investigated microRNAs that are regulated by IL1B and their effects on expression of TJ proteins and intestinal permeability. We used Targetscan to identify microRNAs that would bind the 3' untranslated region (3'UTR) of occludin mRNA; regions that interacted with microRNAs were predicted using the V-fold server and Assemble2, and 3-dimensional models were created using UCSF Chimera linked with Assemble2. Caco-2 cells were transfected with vectors that express microRNAs, analyzed by immunoblots and real-time polymerase chain reaction (PCR), and grown as monolayers; permeability in response to IL1B was assessed with the marker inulin. Male C57BL/6 mice were given intraperitoneal injections of IL1B and intestinal recycling perfusion was measured; some mice were given dextran sodium sulfate to induce colitis and/or gavage with an antagonist to MIR200C-3p (antagomiR-200C) or the nonspecific antagomiR (control). Intestinal tissues were collected from mice and analyzed by histology and real-time PCR; enterocytes were isolated by laser capture microdissection. We also analyzed colon tissues and organoids from patients with and without ulcerative colitis. Incubation of Caco-2 monolayers with IL1B increased TJ permeability and reduced levels of occludin protein and mRNA without affecting the expression of other transmembrane TJ proteins. Targetscan identified MIR122, MIR200B-3p, and MIR200C-3p, as miRNAs that might bind to the occludin 3'UTR. MIR200C-3p was rapidly increased in Caco-2 cells incubated with IL1B; the antagomiR-200c prevented the IL1B-induced decrease in occludin mRNA and protein and reduced TJ permeability. Administration of IL1B to mice increased small intestinal TJ permeability, compared with mice given vehicle; enterocytes isolated from mice given IL1B had increased expression of MIR200C-3p and decreased levels of occludin messenger RNA (mRNA) and protein. Intestinal tissues from mice with colitis had increased levels of IL1B mRNA and MIR200C-3p and decreased levels of occludin mRNA; gavage of mice with antagomiR-200C reduced levels of MIR200C-3p and prevented the decrease in occludin mRNA and the increase in colonic permeability. Colon tissues and organoids from patients with ulcerative colitis had increased levels of IL1B mRNA and MIR200C-3p compared with healthy controls. Using 3-dimensional molecular modeling and mutational analyses, we identified the nucleotide bases in the occluding mRNA 3'UTR that interact with MIR200C-3p. Intestine tissues from patients with ulcerative colitis and mice with colitis have increased levels of IL1B mRNA and MIR200C-3p, which reduces expression of occludin by enterocytes and thereby increases TJ permeability. Three-dimensional modeling of the interaction between MIR200C-3p and the occludin mRNA 3'UTR identified sites of interaction. The antagomiR-200C prevents the decrease in occludin in enterocytes and intestine tissues of mice with colitis, maintaining the TJ barrier.

Probiotics Prevents Sensitization to Oral Antigen and Subsequent Increases in Intestinal Tight Junction Permeability in Juvenile-Young Adult Rats.

Increased intestinal permeability is thought to underlie the pathogenesis of food allergy. We explore the mechanism responsible for changes in the morphology and function of the intestinal barrier using a rat model of food allergy, focusing on the contribution of intestinal microbiota. Juvenile–young adult rats were sensitized with ovalbumin and treated with antibiotics or probiotics (Clostridium butyricum and Lactobacillus reuteri), respectively. The serum ovalbumin-IgE levels, intestinal permeability, histopathological features, tight junction (TJ)-associated proteins, Th2 cytokines, and gut microbiota in feces were analyzed in each group. Sensitized rats showed an increase in ovalbumin-IgE levels and intestinal permeability with gut mucosal inflammation, whereas rats that received probiotics were only mildly affected. Rats given ovalbumin, but not those given probiotics, showed a reduction in both TJ-related protein expression and localization. Th2 cytokine levels were increased in the sensitized rats, but not in those given probiotics. TJs in rats treated with ovalbumin and antibiotics were disrupted, but those in rats administered probiotics were undamaged. Clostridiaceae were increased in the probiotics groups, especially Alkaliphilus, relative to the ovalbumin-sensitized group. Gut microbiota appears to play a role in regulating epithelial barrier function, and probiotics may help to prevent food sensitization through the up-regulation of TJ proteins.

The role of the gut microbiome in chronic liver disease: the clinical evidence revised.

SummaryRecent research has suggested a role for the intestinal microbiota in the pathogenesis and potential treatment of a wide range of liver diseases. The intestinal microbiota and bacterial products may contribute to the development of liver diseases through multiple mechanisms including increased intestinal permeability, chronic systemic inflammation, production of short-chain fatty acids and changes in metabolism. This suggests a potential role for pre-, pro- and synbiotic products in the prevention or treatment of some liver diseases. In addition, there is emerging evidence on the effects of faecal microbial transplant. Herein, we discuss the relationship between the intestinal microbiota and liver diseases, as well as reviewing intestinal microbiota-based treatment options that are currently being investigated.

Lipopolysaccharide at low concentration suppressed tight junction assembly in intestinal epithelial cells via AMPK suppression and MAPK pathway

Intestinal tight junction acts as a physical barrier and selective filter in intestine. Effect of low concentration of lipopolysaccharide (LPS) on AMPK activity, which is central to tight junction regulation, and its mechanism in intestinal epithelial cells (IEC) are not well characterized. This study aimed to investigate the effect of low‐concentration of LPS on AMPK suppression and intestinal tight junction permeability. To test whether low‐concentration of LPS inhibits AMPK phosphorylation, western blot analysis was performed. The result revealed that low dose LPS (1 ng/ml) suppresses AMPK activation in T84 cells. In order to investigate whether LPS suppressed intestinal tight junction assembly, calcium switch assays were performed. The result showed that LPS significantly suppressed tight junction assembly at 6 to 24 hours post‐treatment, indicating that low‐concentration of LPS inhibits tight junction assembly in IEC. Moreover, this effect was disappeared in the presence of U0126, a MAPK inhibitor. This data indicate that low‐concentration of LPS inhibits tight junction assembly via MAPK pathways. In conclusion, LPS induced AMPK suppression and tight junction permeability via MAPK pathway in IEC. This study shed light on the mechanism of intestinal diseases involving gram negative bacterial infection.Support or Funding InformationThis work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University. (BRG5980008)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

MMP-9-induced increase in intestinal epithelial tight permeability is mediated by p38 kinase signaling pathway activation of MLCK gene.

Matrix metalloproteinase-9 (MMP-9) has been implicated as being an important pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is markedly elevated in intestinal tissue of patients with IBD, and IBD patients have a defective intestinal tight-junction (TJ) barrier manifested by an increase in intestinal permeability. The loss of intestinal epithelial barrier function is an important contributing factor in the development and prolongation of intestinal inflammation; however, the role of MMP-9 in intestinal barrier function remains unclear. The purpose of this study was to investigate the effect of MMP-9 on the intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved by using in vitro (filter-grown Caco-2 monolayers) and in vivo (mouse small intestine recycling perfusion) systems. MMP-9 caused a time- and dose-dependent increase in Caco-2 TJ permeability. MMP-9 also caused an increase in myosin light-chain kinase (MLCK) gene activity, protein expression, and enzymatic activity. The pharmacological MLCK inhibition and siRNA-induced knockdown of MLCK inhibited the MMP-9-induced increase in Caco-2 TJ permeability. MMP-9 caused a rapid activation of the p38 kinase signaling pathway and inhibition of p38 kinase activity prevented the MMP-9-induced increase in MLCK gene activity and the increase in Caco-2 TJ permeability. MMP-9 also caused an increase in mouse intestinal permeability in vivo, which was accompanied by an increase in MLCK expression. The MMP-9-induced increase in mouse intestinal permeability was inhibited in MLCK-deficient mice. These data show for the first time that the MMP-9-induced increase in intestinal TJ permeability in vitro and in vivo was mediated by the p38 kinase signal transduction pathway upregulation of MLCK gene activity and that therapeutic targeting of these pathways can prevent the MMP-9-induced increase in intestinal TJ permeability. NEW & NOTEWORTHY MMP-9 is highly elevated in patients with IBD. IBD patients have compromised intestinal TJ barrier function manifested by an increase in intestinal permeability and intestinal inflammation. This study shows that MMP-9, at clinically achievable concentrations, causes an increase in intestinal TJ permeability in vitro and in vivo. In addition, a MMP-9-induced increase in intestinal TJ permeability was mediated by an increase in MLCK gene and protein expression via the p38 kinase pathway.

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.

Effects of Flaxseed Supplementation on Intestinal Tight Junction Protein and TNF‐alpha Gene Expression in Obese Mice

Increased gut permeability may lead to lipopolysaccharides translocation to the blood and causes endotoxemia and inflammation. The objective of this study was to determine the effect of the health promoting compounds of flaxseed on intestinal tight junction permeability by measuring intestinal tight junction protein (ZO‐1) and tumor necrosis factor (TNF)‐α expression. Sixty C57BL/6J male mice were randomly assigned into one of the following diet groups and fed for eight weeks: 1) 45% Kcal fat; 2) 45% Kcal fat, 10% whole flaxseed; 3) 45% Kcal fat, 6% defatted flaxseed; 4) 45% Kcal fat, 4% flaxseed oil; and 5) 16% Kcal fat. ZO‐1 and TNF‐α gene expression in mouse intestine samples were determined by real‐time quantitative PCR. The fold change in ZO‐1 and TNF‐α expression was calculated using 2−ΔΔCT method. TNF‐α expression was significantly decreased in whole flaxseed, defatted flaxseed, or flaxseed oil diet group compared to the high fat diet group (p<0.05). ZO‐1 expression was significantly decreased in defatted flaxseed, flaxseed oil, or low fat diet group compared to the high fat diet group (p=0.0034). The results indicated that health promoting compounds of flaxseed down‐regulate ZO‐1 and TNF‐α expression. The specific mechanisms of down regulation of ZO‐1 and TNF‐α expression or gut permeability regulation by flaxseed and its health promoting compounds are unknown and need further study.Support or Funding InformationNDSU HDE College and HNES Department

183 - Lipopolysaccharide Induced Increase in CACO-2 Intestinal Tight Junction Permeability is Mediated by MIR-429 Induced Degradation of Occludin MRNA

183 - Lipopolysaccharide Induced Increase in CACO-2 Intestinal Tight Junction Permeability is Mediated by MIR-429 Induced Degradation of Occludin MRNA

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.

617 Molecular Mechanism of microRNA Regulation of IL-1β Induced Increase in Intestinal Tight Junction Permeability

617 Molecular Mechanism of microRNA Regulation of IL-1β Induced Increase in Intestinal Tight Junction Permeability

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.