IBV Regulated Secondary Pyroptosis Via ROS/Caspase-8 In Chicken Macrophages

Infectious bronchitis virus(IBV)is a single-stranded positive RNA virus with a genome of 27.6 kb,belonging to gamma coronavirus.IBV causes highly contagious and acute respiratory diseases in poultry,causing serious economic losses to the poultry industry.In-depth exploration of the pathogenic mechanism of IBV is conducive to the development of targeted drugs.Pyroptosis is a gasdermins(GSDMs)protein-dependent inflammatory cell programmed death(PCD),which is an important mechanism against viral infection.Studies have shown that the interaction between IBV and host cells can cause apoptosis and autophagy.The link between IBV infection and pyroptosis has not been reported in detail.The purposes of this study were to investigate: The molecular mechanism of IBV infection-induced pyroptosis in chicken macrophages;The effect of Caspase-8 on IBV-induced pyroptosis;The relationship between IBV-induced ROS accumulation and cell pyroptosis.The MTT assay,lactate dehydrogenase(LDH)release assay,and propidium iodide(PI)staining revealed that IBV infection reduced cell viability,increased LDH release,and increased the rate of PI-positive cells.The observation under an inverted microscope,scanning electron microscope(SEM),and transmission electron microscope(TEM)revealed that IBV infection resulted in pyroptosis characteristics in the cell morphology and ultrastructure.The RT-q PCR and Western Blot results revealed that IBV infection could activate the Caspase-3/DFNA5 pathway;RNA interference was used to down-regulate DFNA5,which reduced the cell death and LDH release of infected cells.Therefore,we speculated that IBV infection mediated HD11 cell pyroptosis via Caspase-3/DFNA5.Through JC-1 staining and detection of apoptosis-related genes Caspase-8/9/3,Bid,Bax,Bak,Cyt C,and Parp by RT-q PCR as well as detection of Caspase-8/9/3,Bax,and Cyt C by Western Blot,we found that IBV infection caused mitochondrial membrane potential decrease and up-regulated the m RNA and protein expression levels of apoptosis-related genes.Pretreatment of infected cells with Caspase-8 inhibitors revealed a marked reduction in cell death and inhibition of activation of apoptotic key molecules and DFNA5,suggesting that Caspase-8 is a crucial molecule linking IBV with pyroptosis secondary to intrinsic apoptosis.In addition,we found that Caspase-8 inhibitors significantly reduced IL-1? transcription level and IL-1? secretion through RT-q PCR and ELISA assay.The above results indicated that Caspase-8 activation could promote secondary pyroptosis induced by IBV infection.The detection of intracellular reactive oxygen species(ROS)with the DCFH-DA probe revealed that IBV infection-induced intracellular ROS accumulation.Treatment of virus-infected cells with ROS inhibitor NAC revealed that after ROS inhibition,cell swelling and death were alleviated,LDH release was reduced,m RNA levels of intrinsic apoptosis-related genes and DFNAF5 were significantly reduced,and the expression levels of some key proteins,such including DFNAF5,cleaved Caspase-3/8,and Bax,were reduced.The above results suggested that IBV might trigger Caspase-3/DFNA5-mediated pyroptosis through ROS-activated intrinsic apoptosis.In summary,IBV infection causes pyroptosis via the ROS-mediated Caspase-8/Caspase-3/DFNA5 pathway,and the pathogenic mechanism described above provides a theoretical basis for the development of targeted antiviral drugs.