Study On The Relationship Between Immune Escape And The Degradation And Inhibition Of Neutrophil Extracellular Traps By ES Of Trichinella Spiralis

Since the discovery of neutrophil extracellular traps(NETs),more and m ore researchers are paying attention to this field.A variety of species and compou nds have been found to induce neutrophil to release NETs,including Phorbol-12-m yristate-13-acetate(PMA),lipopolysaccharide(LPS),pro-inflammatory factors,endot helial cells,etc.,Bacteria such as Candida albicans and Staphylococcus aureus,vir uses such as dengue virus and human immunodeficiency virus,various parasites su ch as Leishmania,Eimeria and Plasmodium,and even laccase in plant nut shell s can also stimulate neutrophils to produce NETs.But only parasitic nematod es have not been reported to cause neutrophil to release NETs.As a typical parasitic nematode,Trichinella was used in this study to co-culture with mouse bone marrow neutrophils(PMN)to see if parasitic nematodes can also induce neutrophil to produce NETs.First,we extracted mouse bone marrow neutrophils to verify their purity and function.We used flow cytometry and nuclear staining to verify the purity of PMN.The results showed that PMN reached 83.66%,More than 90% of cells with horseshoe-shaped nucleus under the microscope,so the purity of PMN meets the requirements of subsequent experiments.PMA and LPS are recognized as typical stimulators of neutrophil to release NETs in vitro and can be used to verify the function of extracted PMN.Neutrophils,when stimulated by PMA or LPS,release a fibrous network composed of a DNA backbone and granule proteins to the outside of the cell.The granule proteins include histone H3,myeloperoxidase(MPO),elastase,etc.Therefore,after stimulating PMN with PMA or LPS,we used immunofluorescence to detect granule proteins on NETs,and PicoGreen to quantify the DNA content in cell culture supernatants.The results showed that PMN could release NETs after stimulation with PMA or LPS.There were three granule proteins of H3,MPO and elastase in NETs structure,and the DNA content in cell culture supernatant increased with the induction time and dose of PMA and LPS,which indicates that the PMN we extracted has normal cell function.Next,we co-cultured Trichinella spiralis adults(Ad3)with PMN and stained with Hoechst 33342/SytoxGreen.Under laser confocal microscopy,Ad3 was able to induce PMN to release NETs.It is well known that parasites have their own strategies to evade the host's immune response.What strategy does Trichinella use when facing NETs released by neutrophils? Previous studies in our laboratory have found that Trichinella excretion and secretion(ES)has nuclease activity,and the skeletal structure of NETs is DNA.Therefore,in this experiment,we collected Ad3 ES and added it to the medium in which PMA induced PMN to release NETs.It was found that PMA-induced release of NETs was degraded by Ad3 ES,and the results of agarose gel electrophoresis of Ad3 ES incubate with substrate DNA also showed that Ad3 ES had nuclease activity.Using 1D zymogram and protein profile,we identified a DNase II family protein in Ad3 ES,and detected its nuclease activity after mammalian cell protein expression,and we found that it does not have nuclease activity.Therefor proteins that exert nuclease activity in Ad3 ES require further investigation.Subsequently,when we used ATA,a nuclease inhibitor,to inhibit the nuclease activity of Ad3 ES,and then pretreated PMN with ES,it was found that PMN pretreated with Ad3 ES could not release NETs after PMA stimulation,indicating Ad3 ES can suppress NETs generation.In the process of PMA-induced PMN to produce NETs,neutrophil ROS production and respiratory burst are the key points.Therefore,we examined the effect of Ad3 ES on ROS production,and found that Ad3 ES can inhibit ROS production.The results of LDH assay indicate that the inhibitory effect of Ad3 ES on NETs is not achieved by promoting the death of PMN.Next,we examined the effects of Ad3 ES on other functions of neutrophils.The results showed that Ad3 ES can promote the ability of PMN to phagocytose bacteria,and ES also have a greater effect on the production of cytokines.The expression of pro-inflammatory cytokine IL-1? was decreased,and the expression of IL-10 was increased.The expression of tumor necrosis factor TNF-? was also significantly increased,indicating that Ad3 ES can regulate the immune response of neutrophils.