Deoxynivalenol, but not E. coli lipopolysaccharide, changes the response pattern of intestinal porcine epithelial cells (IPEC-J2) according to its route of application.

The porcine intestinal epithelium is a primary target for mycotoxin deoxynivalenol (DON) and lipopolysaccharides (LPS). Although epithelial cells are exposed to these toxins mainly from the luminal-chyme compartment an exposure from the blood side resulting from systemic absorption cannot be excluded. Thus, we investigated the effect of DON and LPS, alone or combined, on porcine intestinal epithelial cells IPEC-J2 on a transcriptional, translational and functional level when administered either from apical or basolateral. IPEC-J2 cells were cultured on 12-well inserts in complete medium at 5% CO2 and 39°C and subjected to following treatments: control (CON), 2000 ng/mL DON, 1 µg/mL LPS or DON+LPS for 72 h, either from apical or basolateral. Transepithelial electrical resistance (TEER), protein and IL-8 content were measured and microarray analysis, qRT-PCR (IL-8, zonula occludens-1 ZO-1, ß-actin), Western Blot (ZO-1, ß-actin) and immunofluorescence (ZO-1) were performed. Data of at least three independent experiments were analysed with ANOVA and Dunnett's post hoc test. Basolateral DON resulted in significantly lower cell counts (p<0.05) with larger cells (p<0.01), whereas apical DON reduced total (p<0.001) and specific protein content (IL-8 content CON vs. DON: 2378 pg/3 mL vs. 991 pg/3 mL; p<0.001). Transcripts of ß-actin and ZO-1 were significantly upregulated in response to DON, irrespective of direction, whereas IL-8 mRNA remained unaffected. However, ZO-1 spatial distribution in the tight junction and its function (TEER) were detrimentally affected by basolateral DON only. In conclusion, direction of DON exposure affected IPEC-J2 differently on a translational and functional level, but was mainly inconsequential on a transcriptional level.

Deoxynivalenol affects the composition of the basement membrane proteins and influences en route the migration of CD16(+) cells into the intestinal epithelium.

The numerous pores in the basement membrane (BM) of the intestinal villi are essential for the communication of enterocytes with cells in the lamina propria, an important mechanism for the induction of intestinal immune responses. The intestinal epithelial barrier is affected by the mycotoxin deoxynivalenol (DON) from both the apical (luminal) and basolateral (serosal) side. The pig is the most susceptible species to the anorectic and immune-modulating effects of DON, which is most prevalent in crops. We analysed in pigs the effect of DON-contaminated feed on the composition and perforation of the BM and the presence of CD16(+) cells or their dendrites in the epithelium. In addition to in vivo experiments, in vitro studies were carried out. Using microarray analyses, the effects of DON on IPEC-J2 cells were studied with the focus on the BM. Our in vivo results showed in the control pigs: (1) a significant increased pore number (p ≤ 0.001) in the jejunum in comparison to ileum, (2) no difference in the pore size, and (3) comparable frequency of intraepithelial CD16(+) cells/dendrites in the jejunum and ileum. There was a marked trend that DON feeding increases: (1) the pore number in jejunum, and (2) the number of CD16(+) cells/dendrites in the epithelium (Tukey-Kramer; p = 0.055 and p = 0.067, respectively). The in vivo results were extended with microarray analyses of epithelial cell (IPEC-J2 cells). The down-regulation of genes like syndecan, fibulin 6 and BM-40 was observed. These proteins are important factors in the BM composition and in formation of pores. Our results provide evidence that already low basolateral concentrations of DON (50 ng/mL) influence the production of the BM protein laminin by epithelial cells. Thus, DON affects the composition of the BM.

Interactions between the Fusarium toxin deoxynivalenol and lipopolysaccharides on the in vivo protein synthesis of acute phase proteins, cytokines and metabolic activity of peripheral blood mononuclear cells in pigs.

The in vivo effects of the Fusarium toxin deoxynivalenol (DON) on albumin and fibrinogen synthesis in pigs and metabolic activity of porcine peripheral blood mononuclear cells (PBMCs) were studied alone or in combination with lipopolysaccharides (LPSs) in order to examine proposed synergistic effects of both substances. A total of 36 male castrated pigs (initial weight of 26 kg) were used. Uncontaminated (Control) and naturally DON-contaminated (chronic oral DON, 3.1mg/kg diet) wheat was fed for 37 days. On the day of protein synthesis measurement, pigs recruited from the Control group were treated once intravenously with (iv) DON (100 µg/kg live weight (LW)/h), iv LPS (7.5 µg/kgLW/h) or a combination of both substances, and six pigs from the chronic oral group were treated once with iv LPS. A treatment with DON alone exhibited no alterations of acute phase protein synthesis and metabolic activity of PBMC. There was no evidence that the chosen dosing regimen of DON had influences on the induced sub-acute stage of sepsis, as the LPS challenge, irrespective of DON co-exposure, mediated an acute phase reaction with a typical decrease of albumin synthesis, as well as changes in cytokine concentration and a loss of metabolic activity in PBMC.

Deoxynivalenol and E.coli lipopolysaccharide alter epithelial proliferation and spatial distribution of apical junction proteins along the small intestinal axis.

We investigated a proposed synergistic effect of deoxynivalenol (DON) and lipopolysaccharides (LPS) on small intestinal architecture and epithelial barrier integrity in pigs. Crypt depth and intestinal cell proliferation were analyzed, as well as expression of zonula occludens protein-1 (ZO-1) and ß-catenin of the apical junction complex along the small intestine. Barrows (26.2±4.1 kg) were fed restrictedly either a control diet (CON) or a diet naturally contaminated with 3.1 mg DON/kg feed (DON) for 37 d. At d 37, the control group was infused for 1 h either with 100 µg/kg BW of DON (CON-DON, n=6), 7.5 µg/kg BW of LPS (CON-LPS, n=6), a combination of both (CON-DON+LPS, n=7), or 0.9% NaCl (CON-CON, n=6) and the DON group with 7.5 µg/kg BW of LPS (DON-LPS, n=8) or 0.9% NaCl (DON-CON, n=6). Pigs were euthanized 3.25 h after start of infusion. Immunohistochemistry (5'-bromo-2'-deoxyuridine for proliferation) and immunofluorescence (ZO-1 and ß-catenin) from duodenum, proximal jejunum, mid-jejunum, proximal ileum, and terminal ileum were analyzed for crypt depth, cell proliferation, and apical junction proteins. Duodenal crypts were deeper compared with the other 4 intestinal regions, and proximal jejunal crypts were deeper than those of mid-jejunum and proximal ileum (P<0.001). Epithelial proliferation showed a bell-shaped distribution along the small intestinal axis. Duodenal proliferating cells had the least number compared with jejunal sections and proximal ileum (P<0.001). Neither DON nor LPS affected these variables. Zonula occludens-1 displayed a distinct spatial distribution in the epithelium with an apical and a cytosolic component. Apical expression of ZO-1 was severely damaged in the mid-jejunum (P<0.001) of CON-DON compared with animals treated with LPS. Also, in all animals receiving LPS systemically, the cytosolic ZO-1 fraction in the 3 upper gut sections disappeared completely. This effect was independent of DON presence. Control pigs had a greater basolateral ß-catenin accumulation (P<0.05) in the cells, whereas the protein distribution did not differ in CON-DON pigs. In conclusion, results of this experiment demonstrated that epithelial proliferation has a distinct pattern along the small intestine and is not necessarily positively linked to crypt depth in pigs. Furthermore, results indicate that LPS changed the spatial distribution of ZO-1. A synergistic effect of DON and LPS on intestinal architecture could not be verified in the present study.

Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC).

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism's internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air-liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.