Please cite this article in press as: Siepert, B., et al., Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets. Vet. Immunol. Immunopathol. (2013), http://dx.doi.org/10.1016/j.vetimm.2013.10.013 ARTICLE IN PRESS G Model VETIMM-9120; No. of Pages 13 Veterinary Immunology and Immunopathology xxx (2013) xxx–xxx Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology j ourna l h omepa ge: www.elsevier.com/locate/vetimm Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets Bianca Siepert a , Nicole Reinhardt a,f , Susanne Kreuzer d , Angelika Bondzio c , Sven Twardziok g , Gudrun Brockmann d , Karsten Nöckler e , Istvan Szabó e , Pawel Janczyk e , Robert Pieper b , Karsten Tedin a,∗ a Institute of Microbiology and Epizootics, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany b Institute of Animal Nutrition, Freie Universität Berlin, Königin-Luise-Str. 49, 14195 Berlin, Germany c Institute of Veterinary Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany d Breeding Biology and Molecular Genetics, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115 Berlin, Germany e Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277 Berlin, Germany f Institute of Anatomy, Medical Faculty, Otto-von-Guericke Universität, Leipziger Str. 44, 39120 Magdeburg, Germany g Molecular Biology and Bioinformatics, Charité University Medicine, Arnimallee 22, 14195 Berlin, Germany a r t i c l e i n f o Article history: Received 3 June 2013 Received in revised form 11 October 2013 Accepted 21 October 2013 Keywords: Probiotics Enterococcus faecium Salmonella infections Porcine Innate immunity a b s t r a c t In a Salmonella challenge study of weaned piglets supplemented with the probiotic Entero- coccus faecium NCIMB 10415 (SF68), we observed a delayed, post-infection proliferative response of purified blood mononuclear cell fractions towards Salmonella antigens. In order to clarify this observation, we examined the patterns of immune-associated gene expres- sion in long-term feeding trials of both pre- and post-weaning piglets. Piglets supplemented with E. faecium NCIMB 10415 showed a post-weaning dysregulation in the expression patterns of both pro- and anti-inflammatory cytokine expression in intestinal tissues and spleen. Piglets of the supplemented group showed significantly reduced levels of IL-8, IL- 10 and the co-stimulatory molecule CD86 mRNA expression in ileal Peyer’s patches. The expression of CTLA4, an inhibitor of T-cell activation/proliferation, showed similar levels of expression in all tissues examined, particularly in ileal Peyer’s patches post-weaning where IL-8, IL-10 and CD86 transcript levels were significantly reduced relative to control animals. Blood serum cytokine protein levels showed elevated TGF in pre-weaning piglets which, together with IL-6, may have suppressed IFN production in the probiotic-fed animals. In a second Salmonella challenge study, post-weaning, E. faecium-fed animals showed signif- icantly elevated levels of IL-8 gene expression in mesenteric lymph nodes, but reduced levels in the spleen. At early times post-infection, the probiotic-fed group showed simi- lar levels of IL-10, CD86 and CTLA4 mRNA expression as the control animals in intestinal Peyer’s Patches, despite high relative levels of IL-8 expression in mesenteric lymph nodes. The sum of the observations suggests that supplementation of pre-weaning piglets with E. faecium affects intestinal immune-associated gene expression, which is aggravated post- weaning when the animals receive increased levels of the probiotic in feed. We suggest the post-weaning reductions in gene expression may delay the host response to infec- tions, and provide pathogenic bacteria such as Salmonella with a “window of opportunity”, leading to the increased bacterial loads and shedding observed in challenge trials. Possible mechanisms explaining these effects of E. faecium NCIMB 10415 are discussed. © 2013 Elsevier B.V. All rights reserved. ∗ Corresponding author at: Institute of Microbiology and Epizootics, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, D-14163, Berlin. Tel.: +49 30 838 59705; fax: +49 30 838 451 851. E-mail address: karsten.tedin@fu-berlin.de (K. Tedin). 0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2013.10.013