title: Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.
abstract: Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.
authors.affiliation: Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Länggassstrasse 128, CH-3012 Bern, Switzerland. michelle.imhof@kkh.unibe.ch
authors.forename: Michelle
authors.initials: M
authors.lastname: Im Hof
authors.forename: Lina
authors.initials: L
authors.lastname: Williamson
authors.forename: Artur
authors.initials: A
authors.lastname: Summerfield
authors.forename: Vreni
authors.initials: V
authors.lastname: Balmer
authors.forename: Virginie
authors.initials: V
authors.lastname: Dutoit
authors.forename: Ekambar R
authors.initials: ER
authors.lastname: Kandimalla
authors.forename: Dong
authors.initials: D
authors.lastname: Yu
authors.forename: Andreas
authors.initials: A
authors.lastname: Zurbriggen
authors.forename: Marcus G
authors.initials: MG
authors.lastname: Doherr
authors.forename: John
authors.initials: J
authors.lastname: Peel
authors.forename: Petra J
authors.initials: PJ
authors.lastname: Roosje
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D003208:Concanavalin A / Q000276:immunology / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004285:Dogs / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D051217:Toll-Like Receptor 9 / Q000819:agonists* / Q000235:genetics / Q000276:immunology
chemical_list: C408982:CPG-oligonucleotide; D016207:Cytokines; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D051217:Toll-Like Receptor 9; D003208:Concanavalin A