Maren Amasheh

Epithelial Tight Junctions in Intestinal Inflammation

The epithelium in inflamed intestinal segments of patients with Crohns disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro‐erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin‐2. Th1‐cytokine effects by interferon‐γ in combination with TNFα are important for epithelial damage in Crohns disease, while interleukin‐13 (IL‐13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin‐2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from α‐hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon‐γ and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients

Background and aims: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. Methods: Epithelial barrier function was characterised by impedance spectroscopy and [3H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. Results: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) Ω cm2, p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor α (TNFα) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFα and IL13 reduced the transepithelial resistance of rat jejunal mucosa. Conclusions: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

TNFα-Induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFκB signaling.

TNFα-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFα effect on barrier- or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNFα-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNFα to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNFα-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFα removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFα-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NFκB pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.

Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (Repi). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest Repi, followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (Rtrans or TER) overestimated true Repi by ~60%. In colon, strongest expression of “tightening” claudins 1, 3, 4, 5, and 8 was detected. In accordance with Repi the most proximal of the small intestinal segments, duodenum exhibited highest expression of “tightening” claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.

Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines

Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in Ussing chambers. Transepithelial resistance (Rt) and 3H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. Rt was decreased (101±10 versus 189±10 Ω·cm2) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45±10%, 16±7%, 42±8%, and 42±13% of controls, respectively), while claudin-2 increased to 208±32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73±0.09 to 8.55±2.92 pmol·h−1·cm−2. Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFα and IFNγ alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.

Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation.

In distal colon, the limiting factor for Na(+) absorption is represented by the epithelial sodium channel (ENaC). During absorption, high transepithelial Na(+) gradients are observed. In human colon and in HT-29/B6-GR cells, we investigated whether Na(+) back-leakage is prevented by paracellular sealing. Tissues and cells were incubated with corticosteroids. Barrier properties were analyzed in electrophysiological experiments. Subsequently, analysis of ENaC and tight junction protein expression, localization, and regulation was performed. In colon, nanomolar aldosterone induced sodium absorption via ENaC. Concomitantly, paracellular (22)Na(+) permeability was reduced by half and claudin-8 within the tight junction complex was nearly doubled. Real-time PCR validated an increase of claudin-8 transcripts. Two-path impedance spectroscopy following ENaC induction in HT-29/B6-GR revealed a specific increase of paracellular resistance. These results represent an important physiological implication: Na(+) absorption is paralleled by claudin-8-mediated sealing of the paracellular barrier to prevent Na(+) back-leakage, supporting steep Na(+) gradients in distal colon.

Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells

Sodium caprate is a promising candidate for inducing drug absorption enhancement. The mechanism of that uptake-enhancing effect is not fully understood so far. We investigated how caprate acts in an established human intestinal cell line, HT-29/B6, on the transient opening of transcellular (across the cell membranes) and paracellular (across the tight junction) pathways. Sodium caprate (10 mm) caused a rapid and reversible decrease of transepithelial resistance which is based, as measured by two-path impedance spectroscopy, exclusively on resistance changes of the paracellular pathway. Measurements of paracellular marker fluxes revealed an increased permeability for fluorescein (330 Da) and FITC-dextran (4 and 10 kDa), indicating an opening of the paracellular barrier. Confocal microscopy revealed a marked reduction of tricellulin in tricellular tight junctions and of claudin-5 in bicellular tight junctions. This was not due to altered protein expression, as occludin, claudins or tricellulin were not significantly changed in Western blots. Visualization of the translocation site of the cell membrane-impermeable marker molecule sulpho-NHS-SS-biotin (607 Da) indicated the tricellular tight junction to be the predominant pathway. We suggest that caprates known enhancing effect on intestinal drug uptake is based on increased permeability in tricellular cell contacts, mediated by reversible removal of tricellulin from the tricellular tight junction.

Transforming Growth Factor-β, a Whey Protein Component, Strengthens the Intestinal Barrier by Upregulating Claudin-4 in HT-29/B6 Cells

TGFβ (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFβ-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFβ in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFβ1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFβ(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFβ. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFβ1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.

Barrier Effects of Nutritional Factors

High dietary intake of fruits and vegetables is associated with a reduced disease risk. Therefore, clinical interest is growing in therapies based on dietary supplements and effects of food components. Immune‐modulatory and barrier‐protective effects have been described for the amino acid glutamine and the trace element zinc. In Caco‐2‐cells, zinc is necessary to maintain the expression of proteins like ZO‐1 and occludin, and experimental evidence exists that glutamine has enterocyte‐protective effects and modulates intestinal barrier function in stressed animals and humans. Polyunsaturated fatty acids (PUFA) improve paracellular permeability after IL‐4 incubation. Enhancement of barrier properties by long‐chain PUFA is discussed controversially, but a beneficial role preventing the redistribution of occludin and ZO‐1 and reduction of epithelial resistance by IFN‐γ and TNF‐α exists. In addition, a group of secondary plant compounds, the polyphenols, are supposed to be important in this respect. The flavonoid quercetin and its metabolite DHBA increased epithelial resistance of Caco‐2‐cells to 157 ± 4% of control values, and DHBA up to 119 ± 4% of control values, respectively. This is due to a 2.3 ± 0.1‐fold expression rate of the tight junction protein claudin‐4.

Tight junction proteins as channel formers and barrier builders.

Tight junctions form the paracellular barrier for ions and uncharged solutes not only in “tight” but also in “leaky” epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin‐1 and many others, strengthen the barrier, while a few claudins, such as claudin‐2 or ‐10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin‐2 represents the prototype of a paracellular, channel‐forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin‐5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin‐8, another barrier builder, was demonstrated to be regulated by Na+ uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na+ absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na+ by upregulating claudin‐8.