The Main Functional Study Of Excretory Secretions Of Trichinella Spiralis Adult Worms Acted On Neutrophils

As the essential natural immune cells against foreign pathogens,neutrophils(Polymorphonuclear,PMN)can release extracellular traps composed of DNA,histone H3,myeloperoxidase(MPO)and elastase etc.,in addition to the traditional model of phagocytosis and degranulation.Neutrophil extracellular traps(NETs)plays an essential role in capturing and killing pathogens in vivo.However,there is no systematic study on the mechanism of Trichinella spiralis(T.spiralis)escape from neutrophil capture and killing.Trichinosis is a zoonotic parasitic disease caused by T.spiralis that seriously endangers human health.Current studies have confirmed that T.spiralis excretory secretion(ES)has the nuclease(DNase)activity that degrades ETs to escape capture.However,this experiment was conducted to investigate the effects of T.spiralis adult ES(Ad3 ES)on neutrophil extracellular trap formation,reactive oxygen species(ROS)production and cytokine production under the condition of adding a nuclease inhibitor(Aurintricarboxylic acid,ATA)to block nuclease activity.First,mouse bone marrow neutrophils(BMDNs)were extracted,by nuclear staining microscopy more than 90% of the cells with the horseshoe-shaped nucleus in the same field of view.Furthermore,the cells that could be labeled with neutrophil surface markers were detected by flow cytometry with a purity of 83.66%,which met the requirements of subsequent experiments.As it is well-known that PMA used as a commonly used agonist for the induction of NETs,so PMA-induced neutrophils release NETs consisting of H3,MPO,and elastase by laser confocal microscopy using immunofluorescence techniques.To verify the accuracy of this result,the Pico Green method was used to quantify the DNA content in the cell culture supernatant.The results showed that with the increase of PMA dose and induction time,the DNA content also gradually increased.Thus,it verified that the extracted PMN had normal function.Secondly,investigate the inhibitory effect of Ad3 ES on the release of NETs from PMN.When ATA inhibited the nuclease activity in Ad3 ES,the results showed that the PMN pre-stimulated by Ad3 ES could not release NETs after induction with PMA,Ad3 ES can effectively inhibit the release of NETs from PMN.The mechanism by which Ad3 ES inhibits the production of NETs was subsequently investigated.Whether the inhibition of PMN-released NETs was dependent on ROS or cell death.It was found that Ad3 ES could effectively inhibit ROS production,thereby interfering with PMNreleased NETs,and LDH assay results also showed that the inhibitory effect of Ad3 ES on NETs is not achieved by promoting PMN death.Finally,investigate the effects of Ad3 ES on neutrophil phagocytic function and secreted cytokines.The p ET22b-e GFP plasmid was constructed according to the p EGFP-N1 plasmid available in the laboratory.And E.coli was used to express an enhanced green fluorescent protein.The results showed that Ad3 ES could promote the phagocytosis of PMN,promote the expression of cytokines IL-1?,IL-10,and TNF-?,decrease the expression of IL-4,and had no significant effect on the expression of IFN-?.These results indicate Ad3 ES can regulate the immune function of neutrophils by affecting the ability of PMN to express cytokines.