세균 RNA 및 CpG-motif를 지닌 long double-stranded RNA가 어류 면역능에 미치는 영향 분석

Abstract

The recognition of pathogen-associated molecular patterns (PAMPs) by corresponding receptors trigger the activation of innate immune responses, and each PAMP-specific stimulating properties have been utilized for the development of immunostimulants or vaccine adjuvants. The innate immune system also distinct between live and dead microorganisms through the recognition of vita-PAMPs. Bacterial RNA is a representative vita-PAMP, and has been reported to be able to stimulate both innate and adaptive immunity in mammals. However, no information is available about the effect of vita-PAMPs on fish immunity. The immunostimulating effect of bacterial RNA on respiratory burst activity was investigated by measurements of the chemiluminescent responses of olive flounder kidney phagocytes in vitro. The results of this study suggest that bacterial RNA was shown to be immunomodulating on olive flounder leukocytes. In the present study, the immunostimulatory effect of bacterial RNA isolated from Escherichia coli was analyzed using juvenile olive flounder (Paralichthys olivaceus) as experimental fish. In addition, the immunostimulatory effect of bacterial RNA was compared with that of eukaryotic RNA isolated from Epithelioma papulosum cyprini (EPC) cells. The head-kidney leukocytes were isolated by Percoll density gradient centrifugation for the measurement of respiratory burst activity using chemiluminescence. NF-κB plays a key role in regulating the immune response to infection. Activity of NF-κB in EPC cells that treated with bacterial RNA was investigated using luciferase reporter system. As a results, the increase of luciferase activity was detected from supernatant of cell treated with bacterial RNA. Fish can recognize long dsRNAs via either the endosomal TLR3 or the plasma membrane TLR22. Unmethylated CpG dinucleotides in the DNA of various pathogens are recognized as a PAMP by TLR9 in vertebrates including fish. Recognition of CpG DNA by TLR9 triggers the recruitment of MyD88, which activates signal cascade that results in the production of various cytokines and chemokines related to antibacterial and antiviral immune responses. However, to the best of our knowledge, there has been no report concerning on the recognition of long dsRNAs containing unmethylated CpG motifs can exert not only long dsRNA-mediated but also CpG motifs-mediated immunomodulatory effects on hosts. Thus, in the present study, we compared the expression of TLR3, TLR22, TLR9 and typeⅠIFN responsive genes (Mx1 and ISG15) of olive flounder (Paralichthys olivaceus) in response to an intraperitoneal injection of in vitro transcribed long dsRNAs containing multiple CpG 1668 motifs. In the present study, the expression of TLR3 and TLR9 was not significantly increased in response to poly I:C, long dsRNA containing GFP or CpG motif sequences. On the other hand, the expression of TLR22 was conspicuously increased by the three molecules. These results suggest that TLR22 may be the main receptor of olive flounder for long dsRNAs administered through an i.p. route. Furthermore, no differences in the immune genes expression between long dsRNA-GFP and long dsRNA-CPG in this study suggests that fish could not recognize CpG motifs in long dsRNAs.