The immune responses triggered by CpG ODNs in shrimp Litopenaeus vannamei are associated with Lv Tolls

CpG oligodeoxynucleotides (ODNs) represent a kind of pathogen-associated molecular patterns (PAMPs) as well as a novel adjuvant that activate the innate immune system through interaction with Toll-like receptor 9 (TLR9) in mammals. In the present study, the synthetic oligodeoxynucleotides, CpG ODN 2395, was employed to investigate the interactive mode of CpG ODNs with three known Tolls ( Lv Toll1–3) from shrimp Litopenaeus vannamei. The mature peptides of extracellular domains of Lv Tolls ( Lv Toll-ECDs) were recombinant expressed and their binding activities to CpG ODN 2395 were further examined by ELISA. r Lv Toll1-ECD and r Lv Toll3-ECD exhibited affinity to CpG ODN 2395 in a dose-dependent manner when their concentrations ranged from 0.25 to 2.00 μmol/L, while r Lv Toll2-ECD did not show any binding activities to CpG ODN 2395 in tested concentrations. Additionally, after the stimulation of CpG ODN 2395, the luciferase activities of HEK293T cells transfected with Lv Toll1-mosaic or Lv Toll3-mosaic were significantly increased to 2.38-fold ( p < 0.01) and 1.56-fold ( p < 0.01), while that in the HEK293T cells transfected with Lv Toll2-mosaic declined to 0.41-fold. The TNF-α activities were significantly enhanced ( p < 0.01), and a significant increase ( p < 0.05) of the NO production was observed at 12 h post CpG ODN 2395 stimulation. Moreover, the induced TNF-α activities and increased NO production triggered by CpG ODN 2395 were abolished after the treatment of chloroquine (CQ). The uptake of CpG ODN 2395 by shrimp haemocytes was investigated using the laser scanning confocal microscope, and CpG ODN 2395 was observed to be internalized by the haemocytes and distributed in the cytoplasm with aggregated signals around the nucleuses. It suggested that the interactions of CpG ODNs with Lv Toll1 and Lv Toll3 as well as the mature of endosomes in the haemocytes of shrimp L. vannamei were indispensable for the triggering of immune responses by CpG ODNs, and the results provided a foundation for the application of CpG ODNs as the novel immunostimulants in aquaculture.