The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro

Scope: Ellagitannins are polyphenols found in numerous fruits, nuts and seeds. The elagitannin punicalagin and its bioactive metabolites ellagic acid and urolithins are discussed to comprise a high potential for therapeutically or preventive medical application such as in intestinal diseases. The present study characterizes effects of punicalagin, ellagic acid and urolithin A on intestinal barrier function in the absence or presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha). Methods and Results: Transepithelial resistance (TER), fluorescein and ion permeability, tight junction protein expression and signalling pathways were examined in Caco-2 and HT-29/B6 intestinal epithelial cell models. Punicalagin had less or no effects on barrier function in both cell models. Ellagic acid was most effective in ileum-like Caco-2 cells, where it increased TER and reduced fluorescein and sodium permeabilities. This was paralleled by myosin light chain kinase two mediated expression down-regulation of claudin-4, -7 and -15. Urolithin A impeded the TNF alpha-induced barrier loss by inhibition of claudin-1 and -2 protein expression upregulation and claudin-1 delocalization in HT-29/B6. Conclusion: Ellagic acid and urolithin A affect intestinal barrier function in distinct ways. Ellagic acid acts preventive by strengthening the barrier per se, while urolithin A protects against inflammation-induced barrier dysfunction

Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function

PS reduces transepithelial, transcellular, and paracellular resistance of IPEC-J2.

<p>(<b>A</b>) Time course of transepithelial resistance (TER) of IPEC-J2, which were cultured using different culture media (FBS, fetal bovine serum; ABS, adult bovine serum; GS, adult goat serum; PS, adult porcine serum; ‘+’ indicates supplementation with ITS and EGF) as indicated in the key. PS prevented cells from developing extremely high TER values over a time period of at least three weeks. Note the TER time course of PS (5% +), which developed a maximum around day 7 and reached a lower plateau level at day 14 post seeding. (n = 4–5 each) (<b>B</b>) TER values of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) were corrected for surface enlargement by villi and crypts of porcine jejunum (factor 10) (TER^corr) for comparison with pig jejunal values (dark grey bar). IPEC-J2/FBS exhibited higher TER^corr values (n = 20) than pig jejunum (n = 15; ***, p<0.001), whereas TER^corr of IPEC-J2/PS (n = 20) was not significantly (n.s.) different from porcine values. (<b>C</b>) TER time courses of IPEC-J2/FBS and IPEC-J2/PS were monitored two weeks before and after culture conditions were exchanged as indicated by ‘change’. IPEC-J2 immediately started to develop respective serum-typical TER values. (n = 4–5 each) (<b>D</b>) Two-path impedance spectroscopy was employed to determine R^para (light grey bars) and R^trans (dark grey bars) of IPEC-J2/FBS and IPEC-J2/PS. Significant reduction of both, R^para (*, p<0.05, n = 6) as well as R^trans (**, p<0.01, n = 6) occurred in PS when compared to FBS culture. R^epi (white bars) is calculated as R^para·R^trans/R^para+R^trans.</p

PS optimizes cell dimension of IPEC-J2.

<p>(<b>A</b>) Horizontal and (<b>B</b>) vertical aspects of IPEC-J2 were visualized by live cell imaging using FITC-dextran 4000 (FD4), those of pig jejunocytes by immunofluorescence staining of ZO-1 (<b>A</b>) and of E-cadherin (E-cad, <b>B</b>). Scale bar: 20 µm.</p

PS elevates active ion transport and membrane capacitance of IPEC-J2.

<p>(<b>A</b>) Forskolin-induced short-circuit current (▵I_SC) of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) were corrected for surface enlargement by villi and crypts of porcine jejunum (factor 10) (▵I_SC^corr) for comparison with pig jejunal values (dark grey bar). Stimulation by forskolin resulted in an increased chloride secretory response in IPEC-J2/PS (n = 11; **, p<0.01) compared to IPEC-J2/FBS (n = 10), which almost reached porcine values (n = 9; n.s.). (<b>B</b>) Epithelial capacitance (C^epi) of IPEC-J2/FBS (white bar) and IPEC-J2/PS (light grey bar) was determined via impedance spectroscopy. In IPEC-J2/PS, C^epi was increased (n = 15; ***, p<0.001) compared to IPEC-J2/FBS (n = 22).</p

PS affects permeability to fluorescein but not charge selectivity of IPEC-J2.

<p>(<b>A</b>) Paracellular permeability to fluorescein (P_FLU) of IPEC-J2/FBS (n = 7) and IPEC-J2/PS (n = 8). (<b>B</b>) Permeability ratio for sodium and chloride (P_Na/P_Cl), indicating paracellular charge selectivity, as determined by dilution potentials measurements. The broken line indicates no charge selectivity. Pig jejunal tissue (n = 12), IPEC-J2/PS (n = 13), IPEC-J2/FBS (n = 11); n.s., not significant; ***, p<0.001.</p

PS approximates porcine jejunal marker patterns of IPEC-J2 to that of jejunum.

<p>(<b>A</b>) Confocal immunofluorescence images of IPEC-J2/FBS, IPEC-J2/PS, and cryosectioned pig jejunal mucosae. Porcine jejunocyte marker proteins are presented in green, counterstain in red, as indicated. Nuclei are presented in blue (DAPI). In IPEC-J2, GLUT2 and Na/K-ATPase could be detected within the basolateral membrane, whereas ezrin and SGLT1 could be verified within the apical membrane of IPEC-J2/PS only. In addition, IPEC-J2 were positive for vimentin. The broken line indicates that counterstain choice differed between IPEC-J2 and pig jejunum. Scale bar: 20 µm. (<b>B</b>) Marker proteins of IPEC-J2/FBS and IPEC-J2/PS (n = 3 to 4 different cell passages) were analyzed by Western blotting and were subsequently densitometrically quantified. To allow for different cell architecture, values were normalized to E-cadherin, with the exception of vimentin, which was normalized to β-actin. All signals of IPEC-J2/PS are given in relation to IPEC-J2/FBS values (100%). *, p<0.05; **, p<0.01.</p

PS does not affect junctional protein localization within cell-cell contacts but controls tight junction protein quantity of IPEC-J2.

<p>(<b>A</b>) Confocal immunofluorescence images of IPEC-J2/FBS, IPEC-J2/PS, and cryosectioned pig jejunal mucosae. Cldn5, tric, and occl are presented in green, counterstain in red, as indicated. Nuclei are presented in blue (DAPI). The broken line indicates different counterstain between IPEC-J2 and pig jejunum. Scale bar: 20 µm. (<b>B</b>) Tight junction proteins of IPEC-J2/FBS and IPEC-J2/PS (n = 3 to 4 different cell passages) were analyzed by Western blotting and were subsequently densitometrically quantified. To allow for different cell architecture, values were normalized to E-cadherin. All signals of IPEC-J2/PS are given in relation to IPEC-J2/FBS values (100%). *, p<0.05.</p

PS approximates tight junction ultrastructure of IPEC-J2 to pig jejunum.

<p>(<b>A</b>) Freeze-fracture images of IPEC-J2/FBS, IPEC-J2/PS, and pig jejunal tissue (scale bar: 200 nm). (<b>B</b>) Morphometric analysis of the TJ meshwork depth, (<b>C</b>) the number of horizontal strands, and (<b>D</b>) the TJ strand type (continuous vs. particle type). IPEC-J2/PS, n = 22; IPEC-J2/FBS, n = 23; pig jejunum, n = 20; n.s., not significant; *, p<0.05; **, p<0.01.</p