Immunostimulatory effects of non-CpG oligodeoxynucleotides on cobia ( Rachycentron canadum )

Oligodeoxynucleotides (ODNs) with unmethylated CpG dinucleotides mimic the immunostimulatory activity of bacterial DNA in vertebrates through the recognition of cellular Toll-like receptor 9 (TLR9). We previously demonstrated that CpG motif, 5′-GACGTT-3′ (1668), and 5′-GTCGTT-3′ (2395) were highly immunostimulatory in cobia. In this study, motifs containing CpA, CpC, and CpT were used to evaluate their effects on cobia TLR9 (RCTLR9) gene expression. RCTLR9 expression increased significantly in response to CpT ODN in comparison with other ODN motifs. The optimal dosage of CpT ODN was 10 µg/fish. Furthermore, when the endosomal acidification was blocked using chloroquine immersion, the CpT ODN-induced expression of RCTLR9 in spleen and liver was inhibited in a dose-dependent manner. Our results demonstrated that ODNs containing motifs other than CpG, such as CpT ODN, can stimulate RCTLR9 expression, which is mediated, at least partially, by the interaction between CpT ODN and RCTLR9.