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 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.