A Preliminary Study On The Molecular Mechanism Of Capturing And Killing Listeria Monocytogenes Through Extracellular Traps In Microglia

Extracellular traps was first discovered in neutrophils, it is an unique way ofimmune system defense against pathogen. while innate immune cells are stimulated bypathoges or some chemical substances such as PMA (phorbol-12-myristate-13-acet ate),they release ETs, a concentrated chromatin folds fibrous structure and decoration withparticles derived peptides and some intracellular protein. In addition to neutrophils,mastcells, eosinophils and macrophages can produce ETs. Extracellular traps released bymacrophages was called METs. NETs is a complex process, the activation of NADPHoxidase and the induction of Raf-MEK-ERK pathway are the most important pathway.People have done a lot of researches for NETs, however there is little known about thethe mechanism of METs.Microglial cells is resident macrophage in the central nervous system (CNS).There are a huge number of microglia cells in CNS and account for twelve percent ofthe total cells in CNS. Microglia plays a monitoring nervous system role in thebrain,having the ability to defend many pathogens invasion. However,L.monocytogenes is a kind of bacteria which can cross the blood-brain barrier andreproduction in brain.Prior to this study, we found that the human macrophage cell line THP-1andmurine macrophage cell line RAW264.7infected by Lm could produce METs,whether Lm could cause microglia（nervous system macrophages）to produce METshas not been reported at home and abroad. Microglial cell line BV-2havingmorphological and functional characteristics of microglial cells, and has been provento be effective microglia main alternative cell lines. This paper, we use BV-2cells asa model cell to study whether Lm and PMA could induced BV-2to produceextracellular trap and do a preliminary study of related mechanisms.First, we use fluorescence microscopy show the composition of METs whichreleased by BV-2with PMA and Lm (ATCC19111) infection. In different amount of bacteria and time points, BV-2extracellular DNA absorbance values was detected byfluorescent elisa. The bactericidal efficiency of METs produced by BV-2was detectedby colony counts and live/dead stain method. We conclude that PMA and Lm couldinduce BV-2to produce extracellular trap which contain myeloperoxidase(MPO),elastases and histion3. The production of this METs is positively related to the infectiontime and the amount of bacteria, then the METs produced by BV-2which infection ofLm has the ability to kill the bacterial.Secondly, we use the immunoblotting and extracellular DNA quantificationmethods to verify the relationship between the BV-2extracellular trap andRaf-MEK-ERK path way and NADPH oxidase. Using the HE staining,immunofluorescence technology and extracellular DNA quantitative to verify whetherthe METs is exist in rats’ brain. The results show that BV-2release METs isconnection with ERK pathway, NADPH oxidase and ROS induced by Lm. METsexist in rats’ brain which infected with Lm, and the amount of DNA was increasedsignificantly.This study explores molecular mechanisms that how Lm and PMA activatemicroglia to produce extracellular traps. Providing a theoretical basis for the study ofmacrophages and extracellular traps. And this will lay a foundation for the scientificresearch of Lm infections neurological disease and clinical applic.