Edwardsiella tarda triggers the pyroptosis of the macrophage of Japanese flounder (Paralichthys olivaceus)

Abstract

Pyroptosis is a caspase-1-dependent form of programmed cell death and is characterized by cell lysis and inflammatory cytokine release, which can remove replication niches by releasing intracellular pathogens and facilitating the direct killing of intracellular pathogens by neutrophils or other cells. A method for separating and identifying macrophages of Japanese flounder was described in this study. Upon the stimulation with Edwardsiella tarda, significant swelling with characteristic large bubbles from the membrane was observed in macrophages, which was also positive for Annexin-V and propidium iodide (PI) stain. DNA fragmentation of the macrophage was detected by TUNEL and DNA ladder assays. LDH release assay showed that E. tarda infection impaired cell membrane integrity. The expression levels of pyroptosis-related genes and inflammatory cytokines were substantially up-regulated after E. tarda infection by qRT-PCR, strongly indicating that E. tarda triggered the pyroptosis in the macrophages which played a vital role in the immune response of Japanese flounder after E. tarda invasion. Additionally, treatment of VX-765, a small-molecule inhibitor of caspase-1, significantly reduced macrophages pyroptosis caused by E. tarda, implying that pyroptosis was associated with caspase-1. This study could improve our understanding of the immune mechanism underlying the resistance of teleosts to pathogen invasion.