Nikapitiya Chamilani, Wasana Withanage Prasadini, Jayathilaka E H T Thulshan, Jayasinghe J N C, Lee Jehee, De Zoysa Mahanama
College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 34134, Republic of Korea.
Department of Marine Life Sciences & Center for Genomic Selection in Korean Aquaculture, Jeju National University, Jeju 63243, Republic of Korea; Marine Life Research Institute, Jeju National University, Jeju 63333, Republic of Korea.
Fish Shellfish Immunol. 2025 Jan;156:110034. doi: 10.1016/j.fsi.2024.110034. Epub 2024 Nov 19.
Exosomes are released from multiple cell types as part of their normal physiology as well as during acquired abnormalities. In this study, we investigated the effect of pathogenic Edwardsiella piscicida infection on olive flounder (Paralichthys olivaceus) exosomes at morphometric, physicochemical, and molecular levels. Unique cup-shaped exosomes were isolated from the plasma of non-infected (PBS-Exo) and E. piscicida experimentally challenged (Ep-Exo) olive flounder using ultracentrifugation. The average particle size, concentration, and zeta potential were 150.9 ± 6.9 nm, 5.67 × 10 particles/mL, and -25.6 ± 1.36 mV for PBS-Exo while 138.7 ± 1.9 nm, 1.22 × 10 particles/mL, and -35 ± 1.82 mV for Ep-Exo, respectively. Expression of tetraspanin markers (CD81, CD9, and CD63) confirmed the presence of olive flounder exosomes. Differentially expressed (DE) known (9) and novel (29) miRNAs (log fold change ≥1; p < 0.05) were identified in the Ep-Exo that could be potential as diagnostic biomarkers for the infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the predicted target genes of the DE miRNAs were highly enriched in metabolic and immune roles. Both PBS-Exo and Ep-Exo were non-toxic in vitro (up to 100 μg/mL) and in vivo (up to 400 μg/mL). Compared to the vehicle, PBS-Exo at 50 μg/mL induced Nf-kB (>1.50-fold) while at 100 μg/mL, Il8, Il10, Nf-kB, P53, and Inf were induced (>1.50-fold) in fathead minnow cells (FHMs). This suggests that the PBS-Exo contains molecules that moderately stimulate gene expression. In the future, validating the exact olive flounder immune response target genes that interact with DE miRNAs in Ep-Exo will be crucial for investigating the host-pathogen interactions, immune defense mechanisms, and therapeutic targets for olive flounder against E. piscicida infection.
外泌体作为多种细胞类型正常生理过程的一部分以及在获得性异常期间被释放。在本研究中，我们在形态计量学、物理化学和分子水平上研究了致病性杀鱼爱德华氏菌感染对牙鲆（Paralichthys olivaceus）外泌体的影响。使用超速离心法从未感染（PBS-Exo）和经杀鱼爱德华氏菌实验性攻毒（Ep-Exo）的牙鲆血浆中分离出独特的杯状外泌体。PBS-Exo的平均粒径、浓度和zeta电位分别为150.9±6.9nm、5.67×10颗粒/mL和-25.6±1.36mV，而Ep-Exo的分别为138.7±1.9nm、1.22×10颗粒/mL和-35±1.82mV。四跨膜蛋白标志物（CD81、CD9和CD63）的表达证实了牙鲆外泌体的存在。在Ep-Exo中鉴定出差异表达（DE）的已知（9个）和新的（29个）miRNA（对数倍数变化≥1；p<0.05），它们可能作为感染的诊断生物标志物。基因本体论和京都基因与基因组百科全书通路分析表明，DE miRNA的预测靶基因在代谢和免疫作用方面高度富集。PBS-Exo和Ep-Exo在体外（高达100μg/mL）和体内（高达400μg/mL）均无毒。与载体相比,50μg/mL的PBS-Exo在黑头呆鱼细胞（FHM）中诱导Nf-kB（>1.50倍），而在100μg/mL时，诱导Il8、Il10、Nf-kB、P53和Inf（>1.50倍）。这表明PBS-Exo含有适度刺激基因表达的分子。未来，验证Ep-Exo中与DE miRNA相互作用的确切牙鲆免疫反应靶基因对于研究牙鲆针对杀鱼爱德华氏菌感染的宿主-病原体相互作用、免疫防御机制和治疗靶点至关重要。
