Porcine Intestinal Epithelium Research Articles

Hyperosmotic Stress Induces the Expression of Organic Osmolyte Transporters in Porcine Intestinal Cells and Betaine Exerts a Protective Effect on the Barrier Function.

Background/objectives: The porcine intestinal epithelium plays a fundamental role as a defence interface against pathogens. Its alteration can cause severe inflammatory conditions and diseases. Hyperosmotic stress under physiological conditions and upon pathogen challenge can cause malabsorption. Different cell types counteract the osmolarity increase by accumulating organic osmolytes such as betaine, taurine, and myo-inositol through specific transporters. Betaine is known for protecting cells from hyperosmotic stress and has positive effects when fed to pigs. The aim of this study is to demonstrate the modulation of osmolyte transporters gene expression in IPEC-J2 during osmolarity changes and assess the effects of betaine. Methods: IPEC-J2 were seeded in transwells, where differentiate as a polarized monolayer. Epithelial cell integrity (TEER), oxidative stress (NO) and gene expression of osmolyte transporters, tight junction proteins (TJp) and pro-inflammatory cytokines were evaluated. Results: Cells treated with NaCl hyperosmolar medium (500 mOsm/L) showed a TEER decrease at 3 h and detachment within 24 h, associated with an osmolyte transporters reduction. IPEC-J2 treated with mannitol hyperosmolar medium (500 mOsm/L) upregulated taurine (TauT), myo-inositol (SMIT) and betaine (BGT1) transporters expression. A decrease in TJp expression was associated with a TEER decrease and an increase in TNFα, IL6, and IL8. Betaine could attenuate the hyperosmolarity-induced reduction in TEER and TJp expression, the NO increase and cytokines upregulation. Conclusions: This study demonstrates the expression of osmolyte transporters in IPEC-J2, which was upregulated upon hyperosmotic treatment. Betaine counteracts changes in intracellular osmolarity by contributing to maintaining the epithelial barrier function and reducing the inflammatory condition. Compatible osmolytes may provide beneficial effects in therapies for diseases characterized by inflammation and TJp-related dysfunctions.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Effect of atractylenolide III on zearalenone-induced Snail1-mediated epithelial–mesenchymal transition in porcine intestinal epithelium

BackgroundThe intestinal epithelium performs essential physiological functions, such as nutrient absorption, and acts as a barrier to prevent the entry of harmful substances. Mycotoxins are prevalent contaminants found in animal feed that exert harmful effects on the health of livestock. Zearalenone (ZEA) is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals. Here, we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium.ResultsTreatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin, which induced Snail1-mediated epithelial-to-mesenchymal transition (EMT). In addition, ZEA induces Snail-mediated EMT through the activation of TGF-β signaling. The treatment of IPEC-J2 cells with atractylenolide III, which were exposed to ZEA, alleviated EMT.ConclusionsOur findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epithelial cells and ways to mitigate it.

Glutamine Promotes Porcine Intestinal Epithelial Cell Proliferation through the Wnt/β-Catenin Pathway.

Glutamine (Gln) is a critical nutrient required by neonatal mammals for intestinal growth, especially for newborn piglets. However, the mechanisms underlying the role of Gln in porcine intestinal epithelium development are not fully understood. The objective of the current study was to explore the possible signaling pathway involved in the promotion of porcine intestinal epithelial cell (IPEC-J2) proliferation by Gln. The results showed that 1 mM Gln promoted IPEC-J2 cell proliferation, and tandem mass tag proteomics revealed 973 differentially expressed proteins in Gln-treated IPEC-J2 cells, 824 of which were upregulated and 149 of which were downregulated. Moreover, gene set enrichment analysis indicated that the Wnt signaling pathway is activated by Gln treatment. Western blotting analysis further confirmed that Gln activated the Wnt/β-catenin signaling pathway. In addition, Gln increased not only cytosolic β-catenin but also nuclear β-catenin protein expression. LF3 (a β-catenin/TCF4 interaction inhibitor) assay and β-catenin knockdown demonstrated that Gln-mediated promotion of Wnt/β-catenin signaling and cell proliferation were blocked. Furthermore, the inhibition of TCF4 expression suppressed Gln-induced cell proliferation. These findings further confirmed that Wnt/β-catenin signaling is involved in the promotion of IPEC-J2 cell proliferation by Gln. Collectively, these findings demonstrated that Gln positively regulated IPEC-J2 cell proliferation through the Wnt/β-catenin pathway. These data greatly enhance the current understanding of the mechanism by which Gln regulates intestinal development.

Protective effects of methylsulfonylmethane (MSM) on barrier function injury of porcine intestinal epithelial cells (IPEC-J2) induced by lipopolysaccharide (LPS)

Methylsulfonylmethane (MSM) is a natural organic sulfur component that has anti-inflammatory and antioxidant properties. In this study, injury of porcine intestinal epithelial cell (IPEC-J2) models were used to investigate the effect of MSM on lipopolysaccharide (LPS)-induced porcine intestinal epithelium barrier damage. The results of the cell cycle showed that the cells in the G2/M phase decreased significantly with the supplementation of 300 mmol/L MSM ( P < 0.05). The ELISA assay revealed that MSM could significantly inhibit the expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 ( P < 0.01). Meanwhile, MSM could significantly increase the value of cell monolayer transepithelial electrical resistance while reducing the FITC-dextran flux permeability and lactate dehydrogenase activity in IPEC-J2 cells ( P < 0.01). Additionally, 300 mmol/L MSM significantly increased both mRNA and protein expression of occludin, claudin-1, and ZO-1 ( P < 0.05). Furthermore, MSM prevented the downregulation of epidermal growth factor receptor (EGFR) by LPS, indicating that MSM might enhance tight junction function through mechanisms of activation of EGFR-mediated protein synthesis in IPEC-J2 cells. Therefore, our findings suggested that MSM has protective effects on inflammation and epithelial barrier injury in LPS-induced IPEC-J2 cells, indicating that MSM might be used as a potential therapeutic agent in the pig industry.

Glutamine alleviated heat stress-induced damage of porcine intestinal epithelium associated with the mitochondrial apoptosis pathway mediated by heat shock protein 70.

The present study aimed to investigate the effect of glutamine (Gln) addition on the damage of porcine intestinal epithelial cells (IPEC-J2) induced by heat stress (HS). IPEC-J2 cultured in logarithmic growth period in vitro were firstly exposed to 42 °C for 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for cell viability and cultured with 1, 2, 4, 6, 8, or 10 mmol Gln per L of culture media for heat shock protein 70 (HSP70) expression to determine the optimal disposal strategy (HS, 42 °C for 12 h and HSP70 expression, 6 mmol/L Gln treatment for 24 h). Then IPEC-J2 cells were divided into three groups: control group (Con, cultured at 37 °C), HS group (HS, cultured at 42 °C for 12 h), and glutamine group (Gln+HS, cultured at 42 °C for 12 h combined with 6 mmol/L Gln treatment for 24 h). The results showed that HS treatment for 12 h significantly decreased the cell viability of IPEC-J2 (P < 0.05) and 6 mmol/L Gln treatment for 12 h increased HSP70 expression (P < 0.05). HS treatment increased the permeability of IPEC-J2, evidenced by the increased fluorescent yellow flux rates (P < 0.05) and the decreased transepithelial electrical resistance (P < 0.05). Moreover, the downregulated protein expression of occludin, claudin-1, and zonula occludens-1 was observed in HS group (P < 0.05), but Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS (P < 0.05). In addition, HS resulted in the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) (P < 0.05); however, the reductions in mitochondrial membrane potential expression and B-cell lymphoma-2 expression were induced by HS (P < 0.05). But Gln treatment attenuated HS-induced adverse effects mentioned above (P < 0.05). Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which may be associated with the mitochondrial apoptosis pathway mediated by HSP70.

Gene expression profiling after ochratoxin A treatment in small intestinal epithelial cells from pigs.

Ochratoxin A (OTA) is a well-known mycotoxin that causes disease through the ingestion of contaminated food or feed, for example, in the porcine industry. The intestinal epithelium acts as the first barrier against food contamination. We conducted a study on the exposure of the porcine intestinal epithelium to OTA. We used the intestinal porcine epithelial cell line IPEC-J2 as an in vitro model to evaluate the altered molecular mechanisms following OTA exposure. Gene expression profiling revealed that OTA upregulated 782 genes and downregulated 896, totalling 1678 differentially expressed genes. Furthermore, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting confirmed that OTA damages the tight junction protein ZO-1. Moreover, OTA activated the expression of inflammatory genes (IL-6, IL-8, IL-10, NF-kB, TLR4, and TNF-α). In summary, this study confirmed that OTA alters various molecular mechanisms and has several adverse effects on IPEC-J2 cells.

Protective effect and mechanism of betaine against hyperosmotic stress in porcine intestinal epithelium

• Betaine exerts osmoprotective role in small intestine. • Betaine ameliorated hyperosmotic stress induced cell shrinkage and elevation ROS of intestinal epithelium. • Betaine attenuated mitochondria-related apoptosis caused by hyperosmolarity via maintaining mitochondrial homeostasis. • Betaine alleviated hyperosmolarity-induced autophagy with the decrease of autophagosomes and autolysosomes. Hyperosmolarity under physiological conditions resulting from lumen contents is a big challenge to the normal function of intestinal epithelium. Betaine is a potent osmolyte which is mostly studied in kidney. This study was aimed to gain insight into the osmoprotectant role of betaine in intestinal epithelium under hyperosmotic condition with intestinal porcine epithelial cells (IPEC-J2) and piglets. The result showed that betaine could attenuate the hyperosmolarity-induced reduction of cell viability after treating 12 h. Betaine ameliorates hyperosmotic stress in intestinal epithelium via decreasing reactive oxygen species (ROS) with the alleviation of cell shrinkage. Moreover, betaine prevented mitochondria from membrane collapse and alleviated mitochondrial-related apoptosis induced by hyperosmolarity in vivo and vitro . In addition, betaine ameliorated autophagy caused by hyperosmolarity by up-regulation LC3 II and down-regulation p62. In summary, betaine protects porcine small intestinal epithelium from hyperosmotic stress by alleviating cell shrinkage, ROS accumulation as well as apoptosis and autophagy.

Effects of spermine on the proliferation and migration of porcine intestinal epithelial cells

Whether spermine promotes the repair of porcine intestinal epithelium damage through Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase C-γ1 (PLC-γ1) signaling remains unclear. The current study investigated the effects of spermine addition on the proliferation and migration of IPEC-J2 cells and the effects of Rac1/PLC-γ1 signaling on cell migration. We showed that the inhibitors of Rac1 (NSC-23766) and PLC-γ1 (U73122) reduced cell migration and decreased the protein levels of Rac1 and PLC-γ1 in the cells. Moreover, spermine promoted the proliferation and migration of the IPEC-J2 cells, that is, 1 µM spermine exhibited the best effect, and spermine treatment increased the protein levels of Rac1 and PLC-γ1. Further experiments showed that spermine treatment increased cell migration and enhanced Rac1 and PLC-γ1 protein levels, compared with NSC-23766 and U73122 treatments with spermine. In conclusion, spermine treatment promoted the repair of damaged porcine intestinal epithelium by accelerating cell proliferation and migration mediated by Rac1/PLC-γ1 signaling.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Transferrin receptor 1 is a supplementary receptor that assists transmissible gastroenteritis virus entry into porcine intestinal epithelium

BackgroundTransmissible gastroenteritis virus (TGEV), the etiologic agent of transmissible gastroenteritis, infects swine of all ages causing vomiting and diarrhea, in newborn piglets the mortality rate is near 100%. Intestinal epithelial cells are the primary target cells of TGEV. Transferrin receptor 1 (TfR1), which is highly expressed in piglets with anemia, may play a role in TGEV infection. However, the underlying mechanism of TGEV invasion remains largely unknown.ResultsOur study investigated the possibility that TfR1 can serve as a receptor for TGEV infection and enables the invasion and replication of TGEV. We observed that TGEV infection promoted TfR1 internalization, clustering, and co-localization with TfR1 early in infection, while TfR1 expression was significantly down-regulated as TGEV infection proceeded. TGEV infection and replication were inhibited by occluding TfR1 with antibodies or by decreasing TfR1 expression. TGEV infection increased in TGEV-susceptible ST or IPEC-J2 cell lines and TGEV-resistant Caco-2 cells when porcine TfR1 was over-expressed. Finally, we found that the TGEV S1 protein interacts with the extracellular region of TfR1, and that pre-incubating TGEV with a protein fragment containing the extracellular region of TfR1 blocked viral infection.ConclusionsOur results support the hypothesis that TfR1 is an additional receptor for TGEV and assists TGEV invasion and replication.

Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

Transmissible gastroenteritis virus (TGEV) causes severe diarrhea and high mortality in newborn piglets. It is well established that porcine intestinal epithelium is the target of the TGEV infection, however the mechanism that TGEV invades the host epithelium remains largely unknown. Aminopeptidase N (APN) is a known receptor of TGEV. This study discovered that the extracellular receptor binding domain 1 pertaining to epidermal growth receptor (EGFR) interact with TGEV spike protein. APN and EGFR synergistically promote TGEV invasion. TGEV promotes APN and EGFR clustering early in infection. Furthermore APN and EGFR synergistically stimulate PI3K/AKT as well as MEK/ERK1/2 endocytosis signaling pathways. TGEV entry is via clathrin and caveolin mediated endocytosis in IPEC-J2 cells. TGEV binds with EGFR, and subsequently promotes EGFR internalization by a clathrin-mediated endocytosis pathway. These results show that EGFR is a co-factor of TGEV, and that it plays a synergistic role with APN early in TGEV infection.

Infection of porcine precision cut intestinal slices by transmissible gastroenteritis coronavirus demonstrates the importance of the spike protein for enterotropism of different virus strains

TGEV is a coronavirus that is still widely spread in pig farming. On molecular level this virus has been studied in detail. However, studying TGEV infection within the complexity of the porcine intestinal epithelium reveals difficulties due to limiting infection models. Here we established a new ex vivo model to analyze the enterotropism of TGEV in porcine intestinal tissue. Precision cut intestinal slices (PCIS) were produced and ATP level was measured to proof vitality of the slices. ATP measurements and HE staining revealed living tissue in culture for up to 24h. PCIS were infected with three different TGEV strains. TGEV PUR 46-MAD is a commonly used TGEV strain that is known to be attenuated. TGEV Miller was passaged in piglets several times to reveal high infection. Finally, TGEV GFP is a recombinant strain that obtained its main body from TGEV PUR 46-MAD, but its spike protein from TGEV PUR-C11 that showed high mortality in piglets in vivo. Our results were in complete consensus of these statements. TGEV Miller mildly and TGEV GFP extensively infected the cells in the jejunum based on the amount of positive stained epithelial cells. However, for TGEV PUR 46-MAD no nucleocapsid protein was detected in the epithelial cells of the tissue. This shows that differences in TGEV strains and their infectious potential are highly dependent on their S protein.

Doxycycline Induces Mitophagy and Suppresses Production of Interferon-β in IPEC-J2 Cells

Previous reports have demonstrated that the second-generation tetracycline derivative doxycycline (DOX) interrupts mitochondrial proteostasis and physiology, inhibits proliferation of many cell types, and induces apoptosis. However, the effects of DOX, which is widely used in porcine husbandry by feed, on the porcine intestinal epithelium are unclear. In this study, we demonstrated that DOX damaged mitochondrial morphology and induced the co-localization of mitochondria with autophagosomes, suggesting that DOX induces mitophagy in IPEC-J2 cells. We also found evidence that DOX increased intracellular levels of reactive oxygen species (ROS) or mitochondrial-specific ROS in a dose dependent manner. Moreover, 50 μg/ml DOX significantly decreased production of interferon-β and facilitated replication of transmissible gastroenteritis coronavirus in IPEC-J2 cells. These results demonstrated that DOX induced mitophagy and ROS production, which damaged the intestinal epithelium. As DOX is used extensively in pig husbandry, uncontrolled application poses a significant threat of viral infection, so stricter policies on its usage should be required.

Postnatal regulation of MAMDC4 in the porcine intestinal epithelium is influenced by bacterial colonization.

The MAM domain‐containing 4 (MAMDC4) protein is associated with the unique endocytotic mechanism observed in the intestine of mammals during the immediate postnatal period. Transcriptional expression of MAMDC4 was substantially upregulated at birth in both the piglet jejunum and ileum and its expression decreases after birth. The protein was found localized specifically to the apical region of the luminal epithelium, however, MAMDC4 protein expression was lost at day 10 and 15 in the jejunum and ileum, respectively, and was not associated with “fetal” enterocyte replacement. Although spatial variation in the subcellular localization of Claudin 1 (CLDN1) was noted at day 3, the loss of MAMDC4 at later stages of development did not appear to have any effect on the tight junction structure. Germ‐free (GF) piglets and piglets whose gastrointestinal flora consists exclusively of Escherichia coli (EC) or Lactobacillus fermentum (LF) maintained MAMDC4 protein expression to 14 days of age in distal regions of the small intestine whereas those with conventionalized intestinal flora (CV) showed no MAMDC4 protein at this age. MAMDC4 protein expression was most pronounced in the LF and GF colonized piglets which showed staining in the epithelial cells at 75% and 95% of the length of the small intestine, respectively, which matched that of the newborn. In contrast, EC animals showed only a low abundance at these regions as well as a discontinuous staining pattern. Collectively these results suggest that maturation of MAMDC4 expression in the porcine epithelium occurs more rapidly than what is reported in previously studied rodent species. Furthermore, intestinal bacterial colonization is a major regulator of MAMDC4 in a manner specific to bacterial species and independent of enterocyte turnover.

In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells.

Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization and TGEV entry. The TGEV spike protein is capable of binding with EGFR, activating the downstream phosphoinositide-3 kinase (PI3K), then causing the phosphorylation of cofilin and F-actin polymerization via Rac1/Cdc42 GTPases. Inhibition of EGFR and PI3K decreases the entry of TGEV. EGFR is also the upstream activator of mitogen-activated protein kinase (MAPK) signaling pathways that is involved in F-actin reorganization. Additionally, lipid rafts act as signal platforms for the EGFR-associated signaling cascade and correlate with the adhesion of TGEV. In conlusion, these results provide valuable data of the mechanisms which are responsible for the TGEV pathogenesis and may lead to the development of new methods about controlling TGEV.

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, β-actin), Western Blot (ZO-1, β-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.

Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium.

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

Cell type-dependent internalization of theEscherichia coliSTb enterotoxin

Previous studies have suggested that internalization of the Escherichia coli STb enterotoxin in human and rat intestinal epithelial cells is involved in STb pathogenesis, but toxin uptake in porcine jejunum epithelium, the in vivo target tissue, still remains elusive. Using flow cytometry, we studied the internalization of fluorescein isothiocyanate-labelled STb in porcine intestinal epithelial IPEC-J2 and murine fibroblast NIH-3T3 cell lines. In contrast to the selective pronase resistance of STb in NIH-3T3 cells at 37 °C, but not at 4 °C, indicative of toxin internalization, most of the toxin was pronase-sensitive at both temperatures in IPEC-J2 cells, indicating reduced uptake, but significant cell surface binding. Actin reorganization is required for STb internalization by NIH-3T3 cells, confirming STb endocytosis in these cells. The toxin receptor, sulfatide, could not explain these internalization differences because both cell lines possessed surface sulfatide and internalized antisulfatide antibodies over time at 37 °C. Inhibition of lipid rafts endocytosis, known to contain sulfatide, with methyl-β-cyclodextrin or genistein, did not influence toxin uptake by either cell line. STb internalization is therefore differentially regulated depending on the cell type, possibly by factors other than sulfatide. Although a small STb fraction could be internalized by porcine intestinal epithelial cells, our findings suggest the ability of STb to induce, from the cell surface, intracellular signalling leading to fluid secretion in porcine intestinal epithelium.