Ulrike Lodemann

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1α, −6, −7, −8, −18, TNF-α and GM-CSF, but not TGF-β or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen–host cell interactions.

Effects of Enterococcus faecium NCIMB 10415 as probiotic supplement on intestinal transport and barrier function of piglets

Abstract Many studies report positive effects of probiotic supplementation on the performance and health of piglets. The intention of this study was to describe the effects of Enterococcus faecium NCIMB 10415 on the transport and barrier functions of pig small intestine to improve our understanding of the underlying mechanisms of this probiotic. Ussing chamber studies were conducted with isolated jejunal epithelia of piglets at the age of 14, 28, 35 and 56 days. Jejunal tissues of the control group were compared with epithelia of piglets that had received a diet supplemented with the probiotic Enterococcus faecium NCIMB 10415. Transport properties (absorption and secretion) of the epithelia were examined by mucosal addition of glucose or L-glutamine or by serosal addition of PGE2. Electrophysiology of the epithelia was continuously recorded and the change in short circuit current (Isc) was determined. Paracellular permeability was measured by measuring the flux rates of mannitol. The increase of Isc caused by mucosal addition of glucose was, at all glucose concentrations, higher in the probiotic group compared with the control group. However, the difference (up to 100% of the control) was not significant. The increase of Isc after the mucosal addition of L-glutamine (12 mmol/l) was higher in the tissues of the probiotic group but did not reach significance. Serosal PGE2 induced a significantly higher increase of Isc in tissues of the probiotic group at the age of 28 days. No consistent differences were observed in mannitol transport rates between the feeding groups. Significant age-dependent alterations of absorptive and secretory properties of the jejunal epithelium were observed; these were independent of the treatment. A probiotic supplementation seems to influence transport properties of small intestine epithelium. The increased absorption of glucose could be interpreted as a positive effect for the animal.

Effects of Bacillus cereus var. toyoi as probiotic feed supplement on intestinal transport and barrier function in piglets

The objective of the study was to assess the effects of feed supplementation with the probiotic Bacillus cereus var. toyoi on transport and barrier properties of pig jejunum. Sows and their respective piglets were randomly assigned to two feeding groups: a control group and a probiotic group in which the standard diet was supplemented with Bacillus cereus var. toyoi. At the age of 14, 28, 35 and 56 days, 5 piglets per subgroup were killed and tissue samples from the mid jejunum were mounted in conventional Ussing chambers. Absorptive and secretory properties of the jejunum epithelia were assessed by stimulation of Na-coupled glucose and L-glutamine transport and stimulation of ion secretion by PGE2. Kinetic parameters maximal transport velocity (Vmax) and Michaelis Menten constant (Km) were calculated for glucose and PGE2-stimulated ion secretion. Mannitol fluxes and tissue resistance were measured to evaluate barrier function. With respect to absorption, glucose transport was not changed by treatment and only a slightly higher L-glutamine transport was observed in the probiotic group compared with the control group. The PGE2-stimulated the short circuit current (ΔIsc) in the small intestine and Vmax were higher in the probiotic group at days 28 and 35 compared with the control group. The probiotic seems to have a stabilising (decreasing) effect on the variability of the data. Changes of absorptive and secretory transport properties dependent on age were observed.

A High Amount of Dietary Zinc Changes the Expression of Zinc Transporters and Metallothionein in Jejunal Epithelial Cells in Vitro and in Vivo but Does Not Prevent Zinc Accumulation in Jejunal Tissue of Piglets

High dietary zinc concentrations are used to prevent or treat diarrhea in piglets and humans, but long-term adaptation to high zinc supply has yet not been assessed. Intestinal zinc uptake is facilitated through members of zinc transporter families SLC30 (ZnT) and SLC39 (ZIP). Whereas in rodents, regulation of zinc homeostasis at low or adequate zinc supply has been described, such mechanisms are unclear in piglets. A total of 54 piglets were fed diets containing 57 [low dietary zinc (LZn)], 164 [normal dietary zinc (NZn)], or 2425 [high dietary zinc (HZn)] mg/kg dry matter zinc. After 4 wk, 10 piglets/group were killed and jejunal tissues taken for analysis of zinc transporters SLC30A1 (ZnT1), SLC30A2 (ZnT2), SLC30A5 (ZnT5), SLC39A4 (ZIP4), divalent metal transporter 1 (DMT1), and metallothionein-1 (MT). Weight gain was higher (P < 0.05) in pigs fed HZn than in the LZn and NZn groups during the first 2 wk. Food intake did not differ between groups. The digesta and jejunal tissue zinc concentrations were higher (P < 0.05) in the HZn pigs than in NZn and LZn pigs. Expression of ZnT1 was higher (P < 0.05) and ZIP4 lower (P < 0.05) in HZn pigs than in the 2 other groups, whereas expression of ZnT5 and DMT1 did not differ between treatments. Expression of ZnT2 was lower (P < 0.05) in the LZn group than in the HZn and NZn groups. The mRNA expression and protein abundance of MT was higher (P < 0.05) in the HZn group than in the NZn and LZn groups. Studies with intestinal porcine cell line intestinal epithelial cell-J2 confirmed the dose-dependent downregulation of ZIP4 and upregulation of ZnT1 and MT (P < 0.05) with increasing zinc concentration within 24 h. In conclusion, high dietary zinc concentrations increase intracellular zinc, promote increased zinc export from intestinal tissues into extracellular compartments, and decrease zinc uptake from the gut lumen. The adaptive process appears to be established within 24 h; however, it does not prevent tissue zinc accumulation.

Effects of zinc on epithelial barrier properties and viability in a human and a porcine intestinal cell culture model.

Zinc is an essential trace element with a variety of physiological and biochemical functions. Piglets are commonly supplemented, during the weaning period, with doses of zinc above dietary requirements with positive effects on health and performance that might be attributed to anti-secretory and barrier-enhancing effects in the intestine. For a better understanding of these observations increasing zinc sulfate (ZnSO4; 0-200μM) concentrations were used in an in vitro culture model of porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells and effects on barrier function, viability, and the mRNA expression of one selected heat shock protein (Hsp) were assessed. When treated apically with zinc sulfate, the transepithelial electrical resistance (TEER) did not change significantly. In contrast, cell viability measured by lactate dehydrogenase (LDH) leakage, by ATP and by WST-1 conversion in postconfluent IPEC-J2 monolayers was affected after a 24-h treatment with 200μM ZnSO4. Caco-2 cells were more resistant to Zn. ZnSO4 did not induce any effect on viability, except when it was used at the highest concentration (200μM), and only in preconfluent cells. Furthermore, ZnSO4 induced Hsp70 mRNA expression at 200μM and was more pronounced in preconfluent cells. The observed dose-related effects of zinc are cell-line specific and depended on the differentiation status of the cells. The IPEC-J2 cell line appears to be a suitable in vitro model to characterize specific effects on porcine intestinal cells.

Effects of diet and osmotic pressure on Na+ transport and tissue conductance of sheep isolated rumen epithelium

The intention of this study was to determine the effects of mucosal osmotic pressure on transport and barrier functions of the rumen epithelium of sheep, which were fed various diets: hay ad libitum, or 600, 1200 or 1800 g day−1 of a supplemented diet plus hay ad libitum. The experiments were conducted by using the conventional Ussing chamber technique. Mucosal osmolarity was adjusted to 300 (control), 375 or 450 mosmol l−1. Feeding of a supplemented diet led to a significant increase of mucosal to serosal Na+ transport and net Na+ transport, probably because of an increase of apical Na+–H+ exchange activity. An increase in mucosal osmotic pressure: (a) reduced net Na+ transport in all feeding groups, the remaining net Na+ transport being higher in tissues of sheep fed a supplemented diet; (b) increased transepithelial tissue conductance, this rise being smallest with a high intake of the supplemented diet; and (c) enhanced the serosal to mucosal Na+ transport in tissues of hay‐fed sheep and sheep fed with 600 g day−1 of the supplemented diet, while higher intakes of the supplemented diet (1200 and 1800 g) did not produce any effect. All these changes indicate a diet‐dependent adaptation to luminal hypertonicity.

Enterococcus faecium NCIMB 10415 Modulates Epithelial Integrity, Heat Shock Protein, and Proinflammatory Cytokine Response in Intestinal Cells

Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium), a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli) strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2) and human (Caco-2) intestinal cells were incubated without bacteria (control), with E. faecium, with enteropathogenic (EPEC) or enterotoxigenic E. coli (ETEC) each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER) and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.

Characterization of the effects of Enterococcus faecium on intestinal epithelial transport properties in piglets1

Probiotics have been shown to have positive effects on growth performance traits and the health of farm animals. The objective of the study was to examine whether the probiotic strain Enterococcus faecium NCIMB 10415 (E. faecium) changes the absorptive and secretory transport and barrier properties of piglet jejunum in vitro and thereby to verify tendencies observed in a former feeding trial with E. faecium. Further aims were to assess a potential mechanism of probiotics by testing effects of IL-α, which is upregulated in the peripheral blood mononuclear cells of E. faecium-supplemented piglets, and to test the hypothesis that IL-1α induces a change in ion transport. Sows and their piglets were randomly assigned to a control group and a probiotic group supplemented with E. faecium. The sows received the probiotic supplemented feed from d 28 before parturition and the piglets from d 12 after birth. Piglets were killed at the age of 12 ± 1, 26 ± 1, 34 ± 1, and 54 ± 1 d. Ussing chamber studies were conducted with isolated mucosae from the mid jejunum. Samples were taken for mRNA expression analysis of sodium-glucose-linked transporter 1 (SGLT1) and cystic fibrosis transmembrane conductance regulator (CFTR). The Na(+)/glucose cotransport was increased in the probiotic group compared with the control group at 26 (P = 0.04) and 54 d of age (P = 0.01). The PGE2-induced short circuit current (Isc) was greater at 54 d of age in the probiotic group compared with the control group (P = 0.03). In addition, effects of age on the absorptive (P < 0.01) and secretory (P < 0.01) capacities were observed. Neither SGLT1 nor CFTR mRNA expression was changed by probiotic supplementation. Mannitol flux rates as a marker of paracellular permeability decreased in both groups with increasing age and were less in the probiotic group at the 26 d of age (P = 0.04), indicating a tighter intestinal barrier. The ΔIsc induced by IL-1α was inhibited by bumetanide (P < 0.01), indicating an induction of Cl(-) secretion. Thus, in this experimental setup, E. faecium increased the absorptive and secretory capacity of jejunal mucosae and enhanced the intestinal barrier. Furthermore, the results indicated that IL-1α induces bumetanide-sensitive chloride secretion. The effects of cytokines as potential mediators of probiotic effects should, therefore, be the subject of further studies.

Cry1Ab Treatment Has No Effects on Viability of Cultured Porcine Intestinal Cells, but Triggers Hsp70 Expression

In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.

Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenic Escherichia coli (EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods.