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 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 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

IL-7 promotes IEC proliferation and survival.

<p>(<b>A, B</b>) Rag⁻IL-7⁻ (IL-7⁻; n = 3) and Rag⁻IL-7R⁻ (IL-7R⁻; n = 3) mice were treated with PBS (white bars) or IL-7/anti-IL-7 (black bars) twice a week for 2 weeks. (<b>A, C</b>) Colon wall thickness (µm) and (<b>B, D</b>) the percentage of Ki67⁺ cells in crypts were determined in colon sections from (<b>A, B</b>) IL-7-treated Rag⁻IL-7⁻ and Rag⁻IL-7R⁻ mice and (<b>C, D</b>) untreated WT (n = 6) and IL-7tg (n = 5) mice. (<b>A</b>) 30–54, (<b>B</b>) 15–23, (<b>C</b>) 47–68 and (<b>D</b>) 29–32 individual measurements were performed per experimental group. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (<b>E</b>) Colon sections from PBS-treated (upper row) and IL-7/anti-IL-7-treated Rag⁻IL-7⁻ (lower row; n = 3) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3) or β-catenin (βcat). (<b>F</b>) Colon sections from WT (n = 5) and IL-7tg mice (n = 6) were stained with DAPI and antibodies for β-catenin. (<b>E, F</b>) White arrows indicate nuclei. Bar diagrams show the percentage of luminal IEC with nuclear β-catenin. 130–280 nuclei per experimental group were counted. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (<b>A–F</b>) Data represent one experiment with a total of 23 individual mice and 2–3 independent staining reactions per mouse.</p

IL-7R signaling protects Rag⁻ mice from DSS-induced colitis.

<p>(<b>A, B</b>) WT (n = 4), Rag⁻ (n = 8), Rag⁻IL-7R⁻ (n = 7) and Rag⁻OT-I⁺ mice (n = 6) received dextran sulfate sodium (DSS) via the drinking water. From day 5 on, DSS-free drinking water was provided. (<b>A</b>) Body weight was determined every day and calculated in relation to the initial body weight. Shown are the mean relative body weight ± SEM and the time after onset of DSS treatment. (<b>B, C</b>) Colon samples were taken at day 8 and analyzed histologically. Shown are histological scores for groups of untreated (open symbols; n = 4) and DSS-treated mice (closed symbols; n = 6–8). (<b>C</b>) Shown are representative colon sections from the indicated mice. (<b>A–C</b>) Data represent one experiment.</p

Elevated levels of IL-7 expression and IEC hyperplasia in the colon of Rag⁻ mice.

<p>(<b>A</b>) Representative bioluminescence (BL) images for Rag-competent (Rag⁺; n = 31) and Rag-deficient IL-7GCDL mice (Rag⁻; n = 21) are shown. BL was determined (<b>B</b>) in the intestine and (<b>C</b>) thymus, heart, lung, liver, skin and kidney of Rag⁺ (n = 12) and Rag⁻ IL-7GCDL mice (n = 12). (<b>A–C</b>) BL is shown in photons per s per cm² per steradian. (<b>D, E</b>) Colon sections from Rag⁺ (n = 5–8) and Rag⁻ IL-7GCDL mice (n = 6–8) were stained with (<b>D</b>) periodic acid-Schiff (PAS)/Alcian blue (AB) or (<b>E</b>) DAPI and antibodies for IL-7 and EpCam. (<b>D</b>) Differentiated goblet cells stain positive for PAS (red) and appear purple/magenta. Acidic mucopolysaccharides/glycosaminoglycans are visualized by AB. Arrows indicate the distance between the basis of the crypts and the colon lumen. (<b>D, E</b>) Data are representative for 3 independent experiments and 2–3 staining reactions per mouse.</p