Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic Escherichia coli and Probiotic Enterococcus faecium

Abstract

The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC-induced decrease in transepithelial resistance (R_t) (p ≤ 0.05). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation (p ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein (p ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells (p ≤ 0.05). These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC.

Figure 1

Experimental set-up.

Figure 2

R _t in cells incubated with bacterial strains (Ecf, ETEC, and Ecf + ETEC) and in control cells not exposed to bacteria [means ± SEM]. Groups differ significantly (P ≤ 0.05) when marked with different letters, (e.g. a, b) in the figure. N = 5 independent experiments.

Figure 3

Protein expression (Western blot) of claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-8 after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC). (a) Protein expression relative to respective controls [means ± SEM]. Samples were taken after 8 h. No differences were observed between treatment groups. N = 3 independent experiments per bar. (b) Exemplarily, data of one Western blot is shown. 1 = control (8 h), 2 = Ecf (8 h), 3 = ETEC (8 h), 4 = Ecf (8 h) + ETEC (6 h), and 5 = Ecf (10 h) + ETEC (8 h). Sample 4 was included as a control to rule out effects of longer total bacterial incubation (preincubation) and was not included in the statistical analysis shown in (a).

Figure 4

Confocal images of postconfluent IPEC-J2 cells at 6 h after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC) and controls (red: claudin-3, green: claudin-4). Under all conditions, claudin-4 was localized in the tight junction and in the lateral membrane. N = 5 independent experiments.

Figure 5

Confocal images of postconfluent IPEC-J2 cells 6 h after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC) and controls (red: occludin, green: claudin-4). N = 5 independent experiments.

Figure 6

Apoptosis (a) and cytotoxicity (b) of IPEC-J2 cells after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC) or without bacteria (Con = control) as assessed by caspase-3/7 activity in luminescence assay for apoptosis and by a dead cell protease fluorescence assay for cytotoxicity [means ± SEM]. Measurements were taken after 6 h. Groups differ significantly (P ≤ 0.05) when marked with different letters, (e.g. a, b) in the figure. N = 4 (apoptosis assay) and N = 5 (cytotoxicity assay) independent experiments.

Figure 7

mRNA expression of IL-6 (a), IL-1α (c), and TNF-α (e) and cytokine release of IL-6 (b), IL-1α (d), and TNF-α (f) from IPEC-J2 cells after treatment with bacterial strains (Ecf, ETEC, and Ecf + ETEC) or without bacteria (Con = control) [means ± SEM]. Samples were taken after 4 h (mRNA) or 8 h (protein release). Groups differ significantly (P ≤ 0.05) when marked with different letters, (e.g. a, b) in the figure. N = number of independent experiments as indicated in the figure caption.

Figure 8

Intracellular cAMP concentrations in lysates of IPEC-J2 cells measured by ELISA at 4 h after commencement of incubation with bacterial strains (Ecf, ETEC, and Ecf + ETEC) compared with control [means ± SEM]. Groups differ significantly (P ≤ 0.05) when marked with different letters, (e.g. a, b) in the figure. N = 5 independent experiments.