Figure 6
Screen of CpG-ODNs for hepcidin induction and its immunoprotection to bacterial infection. (A) The steady state level of hepcidin mRNA in liver upon various CpG-ODNs stimulation as determined by Q-PCR. The control fish (indicated as Control) received mock PBS. The sequences of the three known CpG-ODNs (2006, 1670 and R) and the candidate CpG-ODNs with Class A (A1–A5), Class B (B1–B5) and Class C (C1–C9) stimulatory motifs was shown in Supplementary Table S1. (B) Dose-dependent effect of ODN-C4 on the induction of hepcidin protein in serum as determined by ELISA, A450 represents absorbance (A450) value. The control fish (indicated as Control) received mock PBS. (C) Detection of iron concentration in serum of fish receiving ODN-C4 stimulation as determined by ICP-MS. Serum sample derived from the zebrafish without any treatment was the control group (indicated as Control), and serum sample derived from the mock PBS-injected zebrafish was the PBS group (indicated as PBS). (D) Examination of A. hydrophila (A.h) growth in M9 medium cultured in the presence of unstimulated serum (indicated as Control), 1.5 μg ODN-C4 stimulated serum (indicated as CpG stimulated) and stimulated serum neutralized with diluted (1:1,000) anti-hepcidin Ab (indicated as Antibody treated). (E) Survival rate of ODN-C4 stimulated fish within 5 days after A. hydrophila infection. The mock group received only ODN-C4 without bacterial challenge (indicated as Mock) and the control group received sterile PBS 24 h before the bacterial challenge (indicated as Control). All data are representative of at least three independent experiments. A significant difference was detected between each experimental and control group. *p < 0.05, **p < 0.01, ***p < 0.001. ns for not significant.
