Mechanism Of Bacillus-produced Surfactin, A Novel Immunoadjuvant, To Activate Macrophages

Immunologic adjuvant can enhance or change the type of immune response when immuned with the antige beforehand or simultaneously. Many vaccines require adjuvants to be effective, so researching on adjuvant is extremely important for the development of vaccines.Surfactin, one kind of lipopeptide biosurfactants which was isolated from the culture of Bacillus amyloliquefaciens WH1, was found to be with characteristics of well security and strong immune adjuvant activity in previous studies. Injection or taking orally with surfactin could enhance the immune responses.Based on the previous studies, we explored the mechanism of surfactin by studying the effects of surfactin on macrophages, which was one kind of antigen-presenting cells. The mouse peritoneal macrophages were treated with 5 μg/m L dose of surfactin in vitro or 100 μg dose of surfactin in vivo, and then we used flow cytometry to detect the expression of cell costimulatory molecules, the production of ROS and apoptosis. We analyzed the source of intracellular ROS and the activation of nuclear transcription factor-κB with confocal microscopy. Besides, We used western blotting to detect the activation of protein kinase related to MAPKs and used ELISA to detect the concentration of inflammatory cytokines in cell culture supernatant. Real-time PCR was taken to detect the m RNA relative expression of gene related to inflammasome. The main results are as follows:(1) Surfactin could promote antigen presenting. We found that surfactin could increase the expression of cell surface markers(CD80, CD86, MHCI and MHCII) in different degrees. This result indicated that surfactin could activate antigen present cells, induce antigen present cells maturation, and then promote the immune response.(2) Surfactin could induce the expression of ROS. We found that surfactin could induce the expression of ROS which were mainly generated from the mitochondrial pathway. Besides, surfactin could activate macrophages, which may cause respiratory burst, and then produce high levels of ROS.(3) Surfactin could activate MAPKs signal transduction pathways and nuclear transcription factor-κB. The results showed that surfactin could activate nuclear transcription factor-κB by ROS. Further studies showed that surfactin could activate ERK, p38 MAPK and JNK signal transduction pathways, which indicated that surfactin may activate nuclear transcription factor-κB by MAPKs signal transduction pathways, and then regulate cell proliferation, differentiation, apoptosis and other physiological and biochemical processes.(4) Surfactin promoted the formation of inflammasomes. We found that the concentration of TNF-α and IL-1β in cell culture supernatant was increased after being treated with surfactin. In addition, we found that the m RNA relative expression of inflammasome, IL-1β, IL-18 and IL-6 was increased in different degrees after being treated with surfactin, which indicated that surfactin had involved in immune regulation through the release of cytokines. The mechanism of activation of the inflammasome remains to be further study.According to the results, we speculated that surfactin-induced ROS as a second messenger activated MAPKs signal transduction pathway, and then activated nuclear transcription factor-κB, regulated cell proliferation, differentiation, apoptosis and other physiological and biochemical processes, activated inflammasome, and then induced immune responses.