Publikationsdatenbank

Inflammasomes as potential mediators of probiotic effects in porcine intestinal immune and epithelial cells (2019)

Art

Hochschulschrift

Autor

Loß, Henriette (WE 2)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2019 — IV, 110 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/25774

Kontakt

Institut für Veterinär-Physiologie

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

Abstract / Zusammenfassung

Post-weaning diarrhea is a widespread problem in pig rearing. Common causative agents are enterotoxigenic E. coli bacteria. Since post-weaning diarrhea is associated with a high morbidity and mortality rate, innovative prevention and therapy strategies are required. The probiotic strain E. faecium NCIMB 10415 has been demonstrated to be a promising tool to counter this disease. With regard to immunological events, host-pathogen interactions include recognition via innate immune receptors, such as NLR, some of which form inflammasomes. These multiprotein complexes license caspase-1 to process the pro-inflammatory cytokines IL-1β and IL-18. Little is known about the involvement of inflammasome signaling pathways in post-weaning diarrhea in piglets or about the role of the NLRP3 inflammasome in promoting probiotic effects. The objective of the present work was to analyze inflammasome responses to pathogenic ETEC IMT4818 with relevance for post-weaning diarrhea and probiotic E. faecium NCIMB 10415, an authorized feed additive for sows and piglets, in in vitro and ex vivo experiments in various porcine cells and tissues. Since myeloid cells are well suited for investigations into the functions of inflammasomes, the first study of the current thesis was carried out on porcine DC derived from blood monocytes. To determine whether inflammasome signaling contributes to probiotic effects of E. faecium NCIMB 10415, porcine MoDC were pretreated with the probiotic prior to a pathogenic ETEC challenge. Moreover, inflammasome activation processes were further monitored in the presence and absence of a priming signal displayed by LPS. LPS priming induced the transcription of inflammasome components, a characteristic of the first step of inflammasome activation. Inflammasome stimulation occurred upon incubation with ETEC, but not with E. faecium. As compared with LPS-preincubated cells, the observed ETEC effects appeared at later time points when the MoDC were left unprimed. In the applied experimental setup, preincubation with probiotic E. faecium did not mediate protective effects during a pathogenic ETEC challenge via the NLRP3 inflammasome in porcine DC. In the second study, a porcine intestinal co-culture model consisting of IEC (cell line IPEC-J2) and immune cells (MoDC) was established in order to mimic the bidirectional interplay between these two cell types. The aim was to unravel any alterations in the immune response patterns of IPEC-J2 cells and DC to the added bacteria attributable to mutual IEC/immune cell interactions. In addition, the question was addressed as to which soluble factors mediate this communication. Furthermore, the expression of caspase-13 was analyzed, as it has been suggested as a potential candidate driving non-canonical inflammasome activation in pigs. MoDC revealed a more tolerogenic phenotype in the presence of IPEC-J2 cells showing attenuated inflammasome and IL-8 responses. Porcine caspase-13 was affected by bacterial incubation in each cell type. In the cell line IPEC-J2, non-canonical inflammasome signaling appeared to be initiated by ETEC infection and by co-cultivation with DC. With regard to possible mediators of IPEC-J2/MoDC crosstalk, evidence was found for TSLP secretion by IPEC-J2 cells and MoDC. The detected tolerogenic activity of co-cultured MoDC might be partly explained by an autocrine TSLP regulation in these cells. The third part of the present thesis comprises a systematic investigation in which inflammasome components have been examined in tissues from the small and large intestine of pigs at two different ages. A feeding trial was aimed at testing the impact of probiotic E. faecium on inflammasome expression in piglets. To verify the results obtained in vitro, porcine jejunal epithelia were incubated ex vivo with the aforementioned bacterial strains in mono- and coincubation setups (probiotic preincubation and a subsequent challenge with ETEC) employing the Ussing chamber technique. The systematic analysis showed that, similar to their human counterparts, the expression of certain inflammasome components (particularly NLRP6, ASC, and caspase-1) decreased gradually from the jejunum to the colon. Probiotic supplementation had only a weak impact on inflammasome expression levels. However, E. faecium was capable of reducing ETEC-triggered IL-1β protein liberation in the experiments challenging jejunal tissues. In contrast to the in vitro results, this indicated the involvement of the inflammasome pathway in probiotic effects of E. faecium NCIMB 10415. In conclusion, infection with ETEC IMT4818 caused inflammasome activation in vitro and ex vivo. The results of probiotic treatment with E. faecium NCIMB 10415 varied between in vitro and ex vivo approaches, with inflammasome-related advantageous effects being detected solely in the ex vivo study involving the ETEC challenge of porcine jejunum. In the established IPEC-J2/MoDC co-culture model, the presence of IPEC-J2 cells induced a tolerogenic phenotype in co-cultured MoDC indicating that reciprocal interactions between IEC and underlying immune cells orchestrate immunological responses.