Identifizierung von Wirkungsmechanismen eines Probiotikums auf porcine adaptive Immunzellen
Immunologie
Tierzucht, Tierernährung, Tierhaltung
Zusammenfassung der Projektergebnisse
Weaning of piglets is a stress situation often resulting in a multi-factorial disease, the Post-Weaning Syndrome, a serious threat affecting the life/health status of piglets as well as the swine industry worldwide. To overcome the disease and to increase the performance of pigs and piglets, antibiotics were widely used before there banishment from farming throughout European Union in 2006 as a result of the antibiotic-associated increase of bacterial resistance. Probiotics represent a widely used alternative, showing the modulation of the gastrointestinal tract-associated immune cell compartments which is associated with beneficial effects in pigs. However, the underlying mechanisms have not yet evaluated in detail. In this study, simplified cell culture experiments were conducted with primary cultured porcine immune cells (peripheral blood mononuclear cells, PBMCs) of German Landrace sows and lymphocytes isolated from mesenteric lymph nodes (LNLs) of weaned piglets and slaughter pigs. PBMC and lymphocytes were cocultured with the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) in different concentrations, preparations and for different time points. After treatment, changes of the immune cell phenotype and the transcript amount of immune relevant genes were explored by flow cytometry, qPCR and RNA-Sequencing. We detected a higher relative cell count of CD8β+ cytotoxic T-cells after treatment with vital E. faecium bacteria within PBMCs in pigs and piglets. Additionally, we observed a higher activation of cytotoxic T-cells within LNLs, which were co-cultured with vital E. faecium. That hints towards a stimulation of cytotoxic T-cells through secreted substances of vital E. faecium and therefore an enhancement of immune defense mechanisms is probable. Furthermore, we found a higher relative cell count of CD21+ B-cells and correspondingly a higher relative cell count of CD79+ B-cells in treatments with UV-inactivated E. faecium, while there was no effect with vital E. faecium, but rather a trend towards a lower relative cell count with vital E. faecium bacteria. In MACS-separated B-cells, we found a higher relative cell count of activated CD21+CD2- and a lower relative cell count of naive CD21+CD2+ B-cells. In addition, we measured lower expression of B-cell regulatory and activation marker genes as IGLC, IGKC, CD40, and CD2 in treatments with vital E. faecium. Comparative RNA-Sequencing of PBMCs either untreated or treated with vital or UV-inactivated E. faecium compared with vital vs. inactivated E. faecium revealed 537 significantly differentially expressed genes and five differentially expressed miRNAs in all three comparison groups. Interestingly, the majority of genes differentially expressed between vital and UV-treated E. faecium were also differentially expressed between UV-treated bacteria and the control, but not between vital E. faecium and control. That hints towards different mode of actions of inactivated/dead and vital bacteria on the immune cells. Thus, we suggest, that E. faecium influences the direction of immune response towards an enhanced response of cytotoxic T-cells at the expense of B-cells, which are rather reduced. This is also reflected by data of former animal trails and hints towards a specific immunomodulatory effect of E. faecium on adaptive immune cells. Taken together, this in vitro pilot study demonstrates the modulation of the porcine immune cell compartment triggered by the probiotic strain E. faecium NCIMB 10415. Furthermore, this study shows the importance of performing in vitro experiments with primary cells of several donor pigs, considering the potential diversity within the cohort of genetically identical pigs. We found a high inter pig diversity of secreted proteins, as well as a different immune status, although our sows were from the same genetic background and were kept under the same environmental conditions. Also a hierarchical clustering of treated PBMC according to similarities of gene expression revealed a strong clustering towards the individual pig. Nevertheless were we able to provide evidence of a direct immunomodulatory effect of the Enterococcus faecium NCIMB 10415 strain on adaptive immune cells in vitro.
Projektbezogene Publikationen (Auswahl)
- “Feeding of Enterococcus faecium NCIMB 10415 to piglets affects B-cells on cell, gene and miRNA level” EAAP2016 - 67th Annual Meeting of the Federation of Animal Science, Belfast/UK, 29.08. – 02.09.2016
Kreuzer-Redmer S., Bekurtz J., Arends D., Brockmann G.A.
- “Effects of Enterococcus faecium NCIMB 10415 on porcine immune cells” Animal Genetics and Disease 2017, Wellcome Genome Campus Conference Centre, Hinxton/Cambridgeshire/UK, 20.09. – 22.09.2017
Kreuzer-Redmer S., Wöltje N., Larsberg F., Hildebrandt K., Brockmann G.A.
- “In Vitro Effects of Enterococcus Faecium on Primary Cultured Porcine Immune Cells“ 11th International Scientific Conference on Probiotics and Prebiotics - IPC2017, Budapest/Hungary, 20.06. – 22.06.2017
Kreuzer-Redmer S., Wöltje N., Larsberg F., Hildebrandt K., Brockmann G.A.
- Effects of a probiotic on B-cells from German Landrace sows. Tagung Gesellschaft für Ernährungsphysiology, Göttingen, 13.03 – 15.03.2018; Proc. Soc. Nutr. Physiol. (2018) 27
Larsberg F., Korkuc P., Wöltje N., Hildebrandt K., Brockmann G.A., Kreuzer-Redmer S.
- Effects of the probiotic Enterococcus faecium NCIMB 10415 on primary cultured porcine adaptive immune cells. Tagung Gesellschaft für Ernährungsphysiology, Göttingen, 13.03 – 15.03.2018; Proc. Soc. Nutr. Physiol. (2018) 27
Kreuzer-Redmer S., Larsberg F., Korkuc P., Wöltje N., Hildebrandt K., Brockmann G.A.
- In vitro effects of the probiotic Enterococcus faecium NCIMB 10415 on adaptive immune cells from German Landrace sows 11th World Congress on Genetics Applied to Livestock Production, Auckland/New Zealand 11.-16.02.2018
Kreuzer-Redmer S., Larsberg F., Korkuc P., Hildebrandt K., Wöltje N., Brockmann G.A.
