Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Hamamatsu, Japan.
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Microbiol Immunol. 2024 Jul;68(7):224-236. doi: 10.1111/1348-0421.13156. Epub 2024 May 26.
Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesicles in Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharide of native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patterns in lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.
病原菌在上皮细胞上形成生物膜，大多数细菌生物膜表现出增加的膜泡（MVs）的产生，在革兰氏阴性菌中也称为外膜泡。许多研究已经研究了在浮游条件下释放的 MV；然而，生物膜释放的 MV 对免疫反应的影响尚不清楚。本研究旨在研究铜绿假单胞菌 PAO1 的浮游和生物膜培养物中获得的 MV 的特征和免疫调节活性。通过测量促炎细胞因子的 mRNA 表达，研究了巨噬细胞对浮游衍生 MV（p-MVs）和生物膜衍生 MV（b-MVs）的先天免疫反应。结果表明，b-MVs 诱导的促炎细胞因子（包括 Il1b、Il6 和 Il12p40）的 mRNA 表达高于 p-MVs。两种类型的 MV 之间 TLR4 的 mRNA 表达水平不同，但 TLR2 不同。多粘菌素 B 显著中和了 b-MV 介导的细胞因子诱导，表明天然 b-MVs 的脂多糖是免疫反应的来源。此外，热处理或匀浆化的 b-MVs 诱导包括 Tnfa、Il1b、Il6 和 Il12p40 在内的细胞因子的 mRNA 表达。MV 的热处理导致 TLR2 而不是 TLR4 的表达增加，表明 TLR2 配体在检测裂解 MV 中的病原体相关分子模式中起作用。总之，我们的数据表明铜绿假单胞菌生物膜中产生了有效的免疫调节 MV，这种行为可能是细菌感染宿主细胞的一种策略。此外，我们的发现将有助于使用 MV 开发新型疫苗。
