Erzsebet Paszti-Gere

Acute Oxidative Stress Affects IL-8 and TNF-α Expression in IPEC-J2 Porcine Epithelial Cells

Reactive oxygen species are implicated in cell and tissue damage in a number of diseases including acute and chronic inflammation of the gut. Effects of H2O2 exposure on non-carcinogenic porcine epithelial cell line, IPEC-J2 cells cultured on collagen-coated membrane inserts were monitored based on transepithelial electrical resistance (TER) change, extent of necrotic cell damage, gene expression of inflammatory cytokines IL-8 and TNF-α. Furthermore, the junction proteins claudin-1 and E-cadherin were also investigated by immunohistochemistry. Peroxide (1mM) increased IL-8 and TNF-α gene expression levels significantly allowing 1 h recovery time without affecting the cellular distribution of junction proteins, TER and cell survival rate. In conclusion, the IPEC-J2 cell line on membrane insert was introduced as a fast and reliable investigation tool for oxidative stimuli-triggered intestinal inflammation and in the future as a screening method for antioxidant and probiotic candidates.

Chlorogenic Acid Combined with Lactobacillus plantarum 2142 Reduced LPS-Induced Intestinal Inflammation and Oxidative Stress in IPEC-J2 Cells.

This study was carried out to investigate protective effect of chlorogenic acid against lipopolysaccharide-induced inflammation and oxidative stress in intestinal epithelial cells. As a marker of inflammatory response, IL-6, IL-8, TNF-α mRNA and protein levels, furthermore, COX-2 mRNA level were followed up. Intracellular redox status and extracellular H2O2 level were also monitored by two fluorescent assays (DCFH-DA, Amplex Red). Moreover, the effect of gut microbiota metabolites in the above mentioned processes was taken into account in our model using Lactobacillus plantarum 2142 bacterial strain. Our data revealed that chlorogenic acid had significant lowering effect on the inflammatory response. Treatment with chlorogenic acid (25–50 μM) significantly decreased gene expression and concentration of proinflammatory cytokines IL-6 and IL-8 compared to LPS-treated cells. COX-2 and TNF-α mRNA levels were also reduced. Furthermore, chlorogenic acid reduced the level of reactive oxygen species in IPEC-J2 cells. Simultaneous application of chlorogenic acid and Lactobacillus plantarum 2142 supernatant resulted protective effect against LPS-induced inflammation and oxidative stress as well.

Polymethoxyflavone Apigenin-Trimethylether Suppresses LPS-Induced Inflammatory Response in Nontransformed Porcine Intestinal Cell Line IPEC-J2

The in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 μM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

Changes in the Distribution of Type II Transmembrane Serine Protease, TMPRSS2 and in Paracellular Permeability in IPEC-J2 Cells Exposed to Oxidative Stress

The effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2 (TMPRSS2) were studied using porcine epithelial IPEC-J2 cells on membrane inserts. Increased paracellular permeability of FITC-dextran 4 kDa (fluorescence intensity 43,508 ± 2,391 versus 3,550 ± 759) and that of gentamicin (3.41 ± 0.06 % increase to controls) were measured parallel with the reduced transepithelial electrical resistance (23.3 ± 4.06 % decrease) of cell layers 6 h after 1 h 1 mM H2O2 treatment. The immunohistochemical localization of adherens junctional β-catenin was not affected by reactive oxygen species (ROS) up to 4 mM H2O2. Peroxide-triggered enhanced paracellular permeability of IPEC-J2 cell layer was accompanied by predominantly cytoplasmic occurrence of TMPRSS2 embedded in cell membrane under physiological conditions. These results support that ROS can influence paracellular gate opening via multifaceted mode of action without involvement of β-catenin redistribution in adherens junction. Altered distribution pattern of TMPRSS2 and relocalized transmembrane serine protease activity may contribute to weakening of epithelial barrier integrity under acute oxidative stress.

Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro

Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER) causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h) treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.

Interaction exists between matriptase inhibitors and intestinal epithelial cells

Abstract The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.

Reinforced Epithelial Barrier Integrity via Matriptase Induction with Sphingosine-1-Phosphate Did Not Result in Disturbances in Physiological Redox Status

Objectives. The relationship among matriptase function, cellular redox status, and maintenance of intestinal barrier integrity has not been established yet. The aim of this study is to reveal if the crosstalk between matriptase activators and intestinal epithelial monolayers can lead to perturbations in physiological redox regulation in vitro. Methods. The effects of suramin and sphingosine-1-phosphate (S1P) were tested on viability of intestinal porcine epithelial IPEC-J2 cells using MTS assay. Measurements of transepithelial electrical resistance (TER) were performed to determine changes in barrier integrity of cell monolayers. Amplex Red assay was used to monitor extracellular hydrogen peroxide production. Occludin distribution pattern was detected prior to and after matriptase activation using immunofluorescent staining technique. Results. TER reduction was observed in suramin-treated IPEC-J2 cell monolayers, which could be attributed to cell cytotoxic properties of 48 hr 50 μM suramin administration. In contrast, S1P treatment increased TER significantly and elevated occludin accumulation in tight junctions. It was also found that extracellular hydrogen peroxide levels were maintained in IPEC-J2 cells exposed to matriptase activators. Discussion. S1P administration not accompanied by redox imbalance might be one of the key strategies in the improvement of barrier function and consequently in the therapy of intestinal inflammations.

In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells

Abstract The transmembrane serine protease, TMPRSS2 is an important target in the treatment of seasonal influenza infections and contributes to prostate carcinogenesis and metastasis. In this study, the effect of the synthetic TMPRSS2 inhibitor I-432 on jejunal IPEC-J2 cell monolayers cultured on membrane inserts was characterized. Using a fluorogenic substrate, it was found that the apical addition of I-432 could suppress trypsin-like activity in the supernatants of IPEC-J2 cells. The inhibition of TMPRSS2 did not affect physiologically produced hydrogen peroxide levels in the apical and in basolateral compartments. Loss of expression of the TMPRSS2 serine protease domain (28 kDa) was also observed when cells were pre-exposed to I-432. Partial decrease in immunofluorescent signal intensities derived from the altered distribution pattern of TMPRSS2 was detected after a 48 h long incubation of IPEC-J2 cells with the inhibitor indicating the efficacy of TMPRSS2 inhibition via I-432 administration in vitro.

The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21 ± 26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6 ± 14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice.

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer.

Gentamicin is an aminoglycoside antibiotic widely used in combination with dimethyl sulphoxide (DMSO) in topical drug formulations. It is not known, however, whether DMSO can enhance the permeation of gentamicin through biological membranes, leading to oto- and nephrotoxic side effects. A simple and reliable high-performance liquid chromatographic (HPLC) method was applied for the quantitative determination of gentamicin collected from the apical and basolateral compartments of the porcine intestinal epithelial cell line IPEC-J2 cell monolayer using fluorometric derivatisation of the analyte with fluorenylmethyloxycarbonyl chloride (FMOC) prior to chromatographic run in the presence and absence of 1% DMSO. The lack of change in transepithelial electrical resistance (TER) demonstrated that gentamicin and 1% DMSO did not affect IPEC-J2 cell monolayer integrity via the disruption of cell membranes. Chromatographic data also ascertained that gentamicin penetration across the cell monolayer even in the presence of 1% DMSO was negligible at 6 h after the beginning of apical gentamicin administration. This study further indicates that the addition of this organic solvent does not increase the incidence of toxic effects related to gentamicin permeation.