The Formation And Killing Effects Of Mouse Macrophages Extracellular Traps On Giardia Lamblia

Giardia duodenalis,also known as G.lamblia(simply named Giardia),is an important zoonotic protozoan that is parasitic in the host duodenum,mainly causes diarrhea.Giardia can be transmitted through water sources.The quality of urban drinking water supply in China have listed Giardia testing as a mandatory item.At present,the commonly used drugs for the treatment of giardiasis are metronidazole,tinidazole,etc.,but the side effects of the drug are large and the Giardia has developed drug resistance.There is currently no vaccine against giardiasis.Therefore,giardiasis is still a protozoan disease that seriously endangers the health of humans and animals.Innate immunity is the first line of defense against pathogens.The role of host natural immunity in the fight against Giardia infection is still not fully understood.The further study of the host innate immunity defense Giardia will provide a theoretical basis for the prevention and treatment of this disease.Extracellular Traps(ETs)are important innate immune way of the body against pathogens.When neutrophils,macrophages,etc.are stimulated by external pathogens or certain chemicals,a fibrous network structure mainly containing DNA as a skeleton and attached with granule proteins is released to the outside of the cells,and the structure can be used for various cells in pathogenic microorganisms defense.Giardia is an extracellular parasitic protozoan.It is unclear whether Giardia can induce macrophages ETs(METs)and whether the METs have capture and killing effects on Giardia.This study investigated the formation and killing effects of METs induced by Giardia,in order to determine the role of extracellular traps in the defense against Giardia.The main research contents and results are as follows:Giardia trophozoites induce ETs formation of macrophages and its components identification.Firstly,mouse primary macrophages were isolated and the purity was determined by detecting macrophage-specific CD11 b protein.In order to determine the ability of these primary peritoneal macrophages to release extracellular traps,macrophages were treated with PMA,Zymosan and LPS,and the release of extracellular DNA was observed by laser confocal microscopy after staining with nucleic acid dyes Hoechst 33342 and Sytox Orange.The results showed that the purity of the primary peritoneal macrophages of the isolated mouse was up to 95%.PMA,Zymosan and LPS stimulated macrophages could release extracellular reticular DNA.This indicates that the primary peritoneal macrophages have the ability to release ETs.After the Giardia trophozoites(live and dead)stimulated macrophages,the extracellular network was observed by scanning electron microscopy.The release and composition of extracellular reticular DNA were observed by laser confocal microscopy after Sytox Orange,histone 3 antibody and myeloperoxidase antibody treated respectively.The results showed that Giardia trophozoites(live and dead)induced extracellular network formation of macrophages,and the main components were DNA,histone 3 and myeloperoxidase.This indicates that Giardia trophozoites can induce macrophage production of ETs.The role of p38,ERK pathway,MPO,Ca2+(SOCE)and NADPH oxidase in the release of METs induced by Giardia.Giardia trophozoites stimulated p38 inhibitor SB202190,ERK inhibitor UO126 pretreated macrophages and normal macrophages,phosphorylation of p38 and ERK was detected by western blot.Giardia trophozoites stimulated p38,ERK,MPO,Ca2+(SOCE),NADPH oxidase inhibitor-pretreated macrophages and normal macrophages.Different samples were stained with nucleic acid dye Sytox Green and detected by fluorescence microplate reader for DNA release detection.The results indicated that Giardia trophozoites can induce phosphorylation of p38 and ERK proteins in macrophages,and SB202190 and UO126p38 inhibited Giardia induced phosphorylation of p38 and ERK in macrophages.The extracellular DNA release induced by Giardia trophozoites was significantly decreased after treatment with p38,ERK,MPO,Ca2+(SOCE)and NADPH oxidase inhibitors(P<0.05).This indicates that p38,ERK pathway and MPO,SOCE and NADPH oxidase play a certain role in the formation of METs induced by Giardia.Determination of intracellular reactive oxygen species production and extracellular lactate dehydrogenase release during Giardia trophozoites induced METs.To determine whether METs caused by Giardia are dependent on the outbreak,of reactive oxygen species,fluorescent probes DCFH-DA were used for the detection.In order to study whether macrophages were necrotic during the production of METs induced by Giardia trophozoites,lactate dehydrogenase released from macrophages were detected.The results showed that Giardia stimulation increased intracellular reactive oxygen species production(P<0.05).There was no significant difference in lactate dehydrogenase levels between the Giardia stimulation group and the normal cell group(P>0.05).This indicates that the cells did not undergo necrosis during the formation of METs induced by Giardia.In vitro killing effects of METs on Giardia trophozoites.In order to exclude the interference of macrophage phagocytosis.After adding cytochalasin D to block macrophage phagocytosis,the extracellular DNA induced by Giardia was detected by indirect fluorescence method.The effect of METs capture on Giardia trophozoites was detected by trypan blue staining.Ultrastructural changes of morphological changes were observed by scanning electron microscopy.The results showed that cytochalasin D did not affect the release of METs induced by Giardia trophozoites.The trophozoite survival rate decreased by 68.25% compared with the control group after METs treatment for 4 hours.The main lesion of the parasite body was the cell membrane destruction on the surface of the abdominal suction cup.Other surfaces showed varying degrees of shrinkage.It indicates that METs have a certain killing effects on Giardia trophozoites.In summary,this study determined that Giardia can induce the release of ETs from macrophages,and these METs have a certain killing effects on Giardia trophozoites.It indicates that the extracellular trap is an innate immune response in Giardia defense.