Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro

Synthetic agonists of TLR9 containing novel DNA structures and R′pG (wherein R = 1-(2′-deoxy-β-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate TH-1-type-immune responses and potently reverse allergen-induced TH-2 responses to TH-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-γ, IL-10, TGF-β, 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-γ on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-β 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 TH-1 immune response including T- and B-cell proliferation.