Vaccine Adjuvants Induce Formation of Intraperitoneal Extracellular Traps in Flounder (Paralichthys olivaceus)

Abstract

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.

Nanoparticles for stimulation of neutrophil extracellular trap‐mediated immunity

“…Numerous studies have indicated that NETosis is the initial immune response triggered by vaccines. Pure antigens alone often lack the potency for effective protection, prompting the exploration of various stimulants, known as adjuvants, to enhance vaccine efficacy [26]. The discovery of effective adjuvants is crucial due to the limitations posed by the absence of modern and efficient options, as extensively discussed in existing literature [27].…”

“…Alum, a collective term for aluminum oxides and oxyhydroxides commonly used in vaccines, is the most widely employed adjuvant globally, enhancing the effectiveness of vaccines such as diphtheria and others [31]. Aluminum salt adjuvants consist of microaggregates that are phagocytosed by macrophages or neutrophils when used in vaccination [26]. Remarkably, alum has been reported to induce fibrinous NETs within 3 h, emphasizing the speed of the immune response [26].…”

“…Aluminum salt adjuvants consist of microaggregates that are phagocytosed by macrophages or neutrophils when used in vaccination [26]. Remarkably, alum has been reported to induce fibrinous NETs within 3 h, emphasizing the speed of the immune response [26]. Previous studies indicated that alum induces NETs at the immunization site [32], but the detailed mechanism remained elusive for some time.…”

Transcriptome Analysis of Peritoneal Cells Reveals the Early Immune Response of Flounder (Paralichthys olivaceus) to Inactivated Vibrio anguillarum Immunization

“…In accordance with the specified time points and sampling strategies, the intestine, spleen, head kidney, and peripheral blood leukocytes (PBLs) were isolated to detect the expression of Th cell differentiation-related genes in various body compartments, including the blood, mucosae, and lymphoid organs. The isolation procedure for PoPerCs and PBLs was carried out as previously described [19,20]. All samples were promptly frozen in liquid nitrogen and stored at −80 • C until RNA isolation.…”