Adjuvants are used to increase the strength, quality, and duration of the immune response of vaccines. Neutrophils are the first immune cells that arrive at the injection site and can release DNA fibers together with granular proteins, so-called neutrophil extracellular traps (NETs), to entrap microbes in a sticky matrix of extracellular chromatin and microbicidal agents. Similar extracellular structures were also released by macrophages, mast cells, and eosinophils and are now generalized as “ETs.” Here we demonstrated that Alum adjuvant stimulation led to peritoneal cells swarming and ET release in vitro. Moreover, compared to antigen stimulation alone, ET release was significantly increased after stimulation with antigen-mixed adjuvants and in a time- and dose-dependent manner. In vivo, we were able to monitor and quantify the continuous changes of the ET release in the same fish by using the small animal in vivo imaging instrument at different times during the early stages after intraperitoneal immunization. The results showed that the fluorescence signal of ETs in the peritoneum increased from 0 to 12 h after injection and then gradually decreased. The fluorescence signals came from extracellular DNA fibers, which are sensitive to DNase I and confirmed by microscopy of peritoneal fluid ex vivo. In summary, this study introduced a new method for detecting ETs in the peritoneum of fish in vivo and indicated that ET formation is involved in the immune response at the early stage after intraperitoneal immunization to vaccines.
The peritoneal cavity plays an important role in the immune response, and intraperitoneal administration is an ideal vaccination route in fish. However, immune responses in the peritoneal cavity of teleost fish are still not completely characterized. This study characterized the morphology of peritoneal cavity cells (PerC cells) and their composition in flounder (Paralichthys olivaceus). Flow cytometric analysis of the resident PerC cells revealed two populations varying in granularity and size. One population, approximately 15.43% ± 1.8%, was smaller with a lower granularity, designated as lymphocytes. The other population of the cells, about 78.17% ± 3.52%, was larger with higher granularity and was designated as myeloid cells. The results of cytochemical staining and transmission electron microscopy indicated that peritoneal cavity in flounder normally contains a resident population of leukocytes dominated by granulocytes, macrophages, dendritic cells, and lymphocytes. The percentages of IgM+, CD4+, G-CSFR+, MHCII+, and CD83+ leukocytes among PerC cells determined by flow cytometry were 3.13% ± 0.4%, 2.83% ± 0.53%, 21.12% ± 1.44%, 27.11% ± 3.30%, and 19.64% ± 0.31%, respectively. Further, the changes in IgM+, CD4+, G-CSFR+, MHCII+, and CD83+ leukocytes in flounder after Vibrio anguillarum infection and immunization were compared. The composition changed rapidly after the infection or vaccination treatment and included two stages, a non-specific stage dominated by phagocytes and a specific immune stage dominated by lymphocytes. Due to the virulence effectors of bacteria, the infected group exhibited a more intense and complicated PerC cells immune response than that of the immunization group. Following our previous study, this is the first report on the morphology and composition of PerC cells and the early activation of PerC cells in flounder response to V. anguillarum infection and vaccination.
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