Publikationsdatenbank

Probiotic effects on epithelial barrier properties and inflammatory response in an infection model of the porcine intestine (2022)

Art

Hochschulschrift

Autor

Kern, Martina (WE 2)

Quelle

Berlin: Mensch & Buch Verlag, 2022 — XI, 113 Seiten

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/35655

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The probiotic Enterococcus faecium NCIMB 10415 (hereafter called E. faecium) is a licensed feed additive for pigs in the EU. Piglets supplemented with E. faecium showed a decreased incidence and severity of post-weaning diarrhea in previous studies. The main cause of postweaning diarrhea in piglets is infection with enterotoxigenic Escherichia coli (ETEC). However, the underlying cellular mechanisms of the protective effects of E. faecium against ETEC infection have only rarely been analyzed. Therefore, the present study investigated the effects of the probiotic E. faecium on epithelial barrier properties and cytokine expression in the intestinal porcine epithelial cell line J2 (IPEC-J2) during an infection with ETEC in vitro. A decrease of the transepithelial electrical resistance (Rt) in ETEC-infected cells was detected that was not seen in cells pre-incubated with E. faecium. These observations confirmed the results of a former study conducted at our institute. The expression of tight junction (TJ) proteins analyzed in the present study was not influenced by incubation with the bacterial strains. However, a modulation of the cell structure was seen in ETEC-infected cells, i.e. cells arranged on top of each other, indicating increased cell shedding. This was not seen in ETEC-infected cells pre-incubated with E. faecium. The rate of apoptosis was not modulated by ETEC. However, the cytotoxicity that significantly increased in ETEC-infected cells, was ameliorated by pre-incubation with E. faecium. At the mRNA level, ETEC upregulated the proinflammatory cytokine tumor necrosis factor (TNF)-α and increased the release of the cytokines TNF-α, interleukin (IL)-1α, and IL-6 at the protein level. A protective effect of the probiotic could be detected for the ETEC-induced increased TNF-α mRNA expression and IL-6 protein release. Both were decreased by preincubation of cells with E. faecium. The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) was also elevated by ETEC, but this was counteracted in cells preincubated with E. faecium. In a further part of the present study, the inflammasome was analyzed as a potential mediator of probiotic effects. First, the basal expression of inflammasome components was analyzed at the mRNA level in the intestine of piglets and fattening pigs. The expression of nucleotidebinding oligomerization domain-like receptor protein 3 (NLRP3), NLRP6, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1β, and IL-18 was detected in the jejunum, ileum, and colon of pigs. A characteristic expression pattern was revealed for NLRP6, ASC, and caspase-1 in 29-day-old piglets that showed a decreasing expression of these components in an oral-aboral direction. Such expression pattern could not be detected in 70-day-old fattening pigs. Furthermore, the effects of the probiotic E. faecium on the mRNA expression of inflammasomes and inflammasome components were analyzed in a feeding trial. Piglets supplemented with E. faecium showed increased mRNA expression of ASC in the jejunum. The mRNA expression of NLRP3, NLRP6, caspase-1, IL-1β, and IL-18 was not modulated by the probiotic. In order to analyze probiotic effects during an infection with a pathogen, the jejunum of 80-dayold pigs was incubated with E. faecium, ETEC, E. faecium and ETEC, or without bacteria in an Ussing chamber experiment ex vivo. The mRNA expression of the inflammasome components was not modulated by the bacterial strains. However, at the protein level, the release of IL-1β was significantly increased by ETEC. A protective effect was found for the probiotic E. faecium. ETEC-induced increased release of IL-1β protein was significantly reduced by pre-incubation with E. faecium. In conclusion, several protective effects of the probiotic E. faecium have been detected at the cellular level during ETEC infection in the present work. The probiotic effects were mediated via the reduced decrease in Rt, the downregulated expression and release of inflammatory cytokines, the decreased intracellular concentrations of cAMP, the maintenance of the physiological epithelial cell structure, and the lessening of cytotoxic effects during ETEC infection. A characteristic oral-aboral expression pattern was revealed for the inflammasome NLRP6 and the inflammasome component ASC and caspase-1 in the intestine of piglets. The detected expression pattern was similar to the expression pattern in humans thereby reinforcing the use of pigs as an animal model for preclinical and translational inflammasome research. Pre-incubation with E. faecium reduced the ETEC-induced release of IL-1β protein. IL-1β release is associated with inflammasome activation. Hence, the present study provides evidence for the inflammasome as a mediator of protective probiotic effects at the functional level during infection with ETEC ex vivo. The protective effects of E. faecium demonstrated in this study represent important findings concerning the cellular mode of action of the probiotic during ETEC infection. Based on these findings, the prophylactic use of E. faecium can be considered for the reduction of the incidence and severity of ETEC infections in piglets as a very promising strategy. The latter has the potential to decrease the necessity of the therapeutic use of antibiotics in pig farming.