NETosis-Like Response Triggered by Extracellular Vesicle (EV)-Delivered Viral Nucleic Acid, a Novel Cellular Immune Mechanism in Crustacean.

As a novel identified manner of cell death, NETosis is widely regarded as an effective approach to resist pathogen infection but mainly focused on vertebrates with systematic cell typing. Besides, the role of extracellular vesicles (EVs), which are essential tools for intercellular information exchange, in regulating NETosis during pathogen infection has yet to be addressed. Here, we found that viral mRNA wsv271 could be packaged by EVs secreted by haemocytes during WSSV infection in mud crab, and delivered to the neutrophil-like cells, followed by translation into viral protein, and then interacted with the TIR domain of Toll4 to recruit MyD88, so as to activate P38-MAPK signal pathway and further facilitate PAD4 phosphorylation and nuclear translocation to mediate histone-H3 citrullination, which eventually activated NETosis-like response in haemocytes to suppress the spread of viral infection. Therefore, our research not only identified neutrophil-like cells from the haemocytes of a crustacean based on single-cell transcriptomics but also revealed a novel NETosis induction mechanism mediated by EVs-derived viral nucleic acid delivery.