ODN 2216 broadly influence the gene expression profile in primary feline immune cells of adult cats
In an effort to assess both the breadth of the effects conferred by treatment with a CpG-A and possible variability in the responses obtained in individual cats, the mRNA expression of ten genes relevant to early immune responses was measured in ODN 2216 stimulated PBMCs of fourteen cats divided in four different age groups (group 1: 10 weeks (n = 4), group 2: 1.5 years (n = 4), group 3: 7 years (n = 4), group 4: 14 years (n = 2)). Only slight individual variability in gene induction was observed after 24 h stimulation of immune cells from adult cats ranging between 1.5 and 14 years of age (Figure2B-D). Overall, the mRNA expression profile measured in stimulated PBMCs of these cats corroborated their induction of strong antiviral immune responses. The expression of type I IFN mRNA, including IFNα, IFNβ and IFNω was substantially increased in the immune cells subjected to ODN 2216 stimulation from every adult cat, with minimal inductions of 490, 60 and 1600-fold respectively observed in the older animals of group 4 (Figure2D). Moreover, all individual IFNα subtypes tested were induced at similar levels in the cells of four adult cats from group 2 (Figure2E). Increased levels of proinflammatory cytokine mRNA were also measured in most individuals of groups 2–4, with IL-6 more systematically increased than TNFα. The cells from these cats indicated a typical Th1 orientation after stimulation, with enhanced transcription of IL-12 in 9/10 and IFNγ in 6/10 animals, together with absent or only low induction of IL-4. Stimulation of PBMCs with ODN 2216 also created an optimal environment for NK cell activity, as indicated by the increases in mRNA expression of the NK cell stimulator IL-15 by up to 20-fold and of the NK cell effector Granzyme B by up to 7-fold. The cells of those cats (c08, c09, c12) that had most effectively proliferated (Figure1A) and/or exhibited the strongest expression of co-stimulatory molecules (Figure1B-E) following ODN 2216 stimulation also consistently expressed the highest mRNA levels of IFNα, IFNω, IL-6, Il-12, IL-15 and Granzyme B. PBMCs of cat c08 (group 2, Figure1B) were also by far most responsive to stimulation with ODN 2216. When cells from kittens of group 1 were stimulated, higher individual variability was observed than in adult animals (Figure2A). The PBMCs of only 2 out of 4 cats from this group could be stimulated with ODN 2216 to increase mRNA expression of the tested genes. In both individuals, not only the mRNA expression of type I IFN genes was enhanced at much lower levels than observed in adult cats but the overall immune response favored a Th2 direction, characterized by upregulated IL-4 and downregulated IL-12 mRNA levels (Figure2A).
In order to determine whether a discrepancy in expression of TLR9 between the PBMCs of adult animals and kittens could play a role in these observations, mRNA levels of this gene were measured in immune cells of each cat. Although basal TLR9 expression was similar in the PBMCs of the cats of all age groups (Figure2F), ODN 2216 stimulation increased TLR9 transcription in the cells of kittens, but decreased transcription of this gene in the cells of adult cats so that differences in the mRNA levels of this receptor were significantly higher in young cats (group 1) than in adult cats of groups 2 and 3 (p = 0.0286) (Figure2G).