Immunomodulatory Effects Of A Novel Immunostimulant CpG DNA On Grass Carp

In mice and humans, B cells, antigen-presenting cells (APCs) including monocytes, macrophages and dendritic cells and natural killer (NK) cells can be stimulated directly or indirectly by the bacterial DNA and oligodeoxynucleotides (ODNs) containing the CpG dinucleotides (CpG DNA).In Part I, we used head kidney macrophages of grass carp (Ctenopharyngodon idellus) as an in vitro model and investigated the effects of several CpG-ODNs, Escherichia coli DNA and Bifidobacterium adolescentis DNA on fish immunocytes. The CpG-ODNs included the optimal motifs: the ODN-1826 (GACGTT) and -2006 (GTCGTT) for the mice and humans cells, the ODN-1670 (AACGTT) used in Atlantic salmon, the ODN-D containing two repeats motif of those in 1670 and the ODN-R with an inverted CpG. The results showed that CpG DNA has an immunomodulatory role on a dose-dependent way in grass carp, and all the ODNs and bacteria DNAs except the ODN-R could activate macrophages, increasing the levels of superoxide anion (O~2), hydrogen peroxide (H2O2), acid phosphatase and bactericidal activity. New evidence was provided that CpG DNA could induce the cell-mediated immunity in fish. Interestingly, no significant differences among the ODNs tested could be found and the ODN-D was not more efficient than 1670. There is also no remarkable differences between bacteria DNAs. It suggests that the sequence which contains the unmethylated 'CG' dinucleotides could make contribute to this immunostimulatory effect. These findings indicate that CpG DNA could be useful tools for understanding the important anti-bacterial defense mechanism in fish.In Part II, we studied the effects of E.coli DNA on expression of some important immune-related cytokines including MHCâ…¡, pre-B-cell enhancing factor(PBEF), natural killer cell enhancement factor(NKEF) and MX on grass carp. After 48h of E.coli DNA infection, we isolated total RNA of head kidney and used RT-PCR to amplified cDNA of MHCâ…¡ alpha-2,PBEF, NKEF and MX. Using semi-quartitative RT-PCR, we found the upregulated expression of MHC â…¡ alpha-2 in CpG DNA-induced head kidneys compared with those of controls. It was shown that the remarkable bands of PBEF,NKEF and MX from CpG DNA-stimulated fish, while there is no band tested from controls. It suggests that bacteria DNA may be an effective activator on immune moleculars of grass carp.In a word, we observed that CpG DNA is a novel immunostimulatory in vivo and in vitro on fish as well as those on higher vertebrates, which promote the cell- and humoral-mediated immune responses to defense against the invading microorganisms. And it may have potential application in the minimizing the impact of fish diseases and enhancing the efficacy of antigen and DNA vaccines.