Characterization Of Murine Macrophage Extracellular Traps And Identification Of Active Deoxyribonucleases In Trichinella Spiralis ES Products

Neutrophil extracellular traps (NETs) is an important innate immune defencemechanism which was discovered only a few years ago. After being stimulated bysome pathogenic microbes or chemical regents, neutrophil release extracellularfibrous structure which iscomposed of DNA and granular proteins. This structurecould ensnare and kill varied kinds of invading microbes. This new mechanism wasalso reported to exist in eosinophil and mast cells. Two models describing the releaseof extracellular traps (ETs) have been proposed: extrusion of mixed nuclear DNA withgranular proteins in neutrophil and mast cells, and catapult-like releasing ofmitochondrial DNA in eosinophil. Nuclear originated ETs releasing often need2-3hours and lead to cell death, while mitochondrial originated ETs was released byintact cells and occurred in several seconds. Cell death induced by ETs formationshows characters different from apoptosis or necrosis, which was designated to be anew cell death pathway named NETosis. Reported ETs-inducing microbes includebacteria, fungi and protozoa, but whether pathogenic parasitic nematodes also induceETs is still unknown. Besides neutrophil, eosinophil and mast cells, the ETsmechanism exists in macrophages is still less known and need to be furtherinvestigated.Trichinella spiralis is a typical pathogenic nematode. Ingestion of uncooked orhalf-cooked T. spiralis contaminated meat lead to human or animal trichinellosis. Thelarvae could parasitize in host’s muscle for a long time. A characteristic of T. spiralisinfection is inefficiency of host innate immune system. For example, T. spiralisinvading to intestine cells leads to innate immune cells assembling, but lacks offollowing inflammatory reaction. The excretion/secretion (ES) product of T. spiralis is believed to play a key role in immune escape process of T. spiralis. The ES productcontains varied kinds of proteins and enzymes, which could directly act on host’simmune cells to regulate immune response. Our previously work has identified28devolopmental stage-specific DNaseⅡgenes from Ts cDNA library by immunologicalscreening. Immunofluorescence experiments indicated that these DNaseⅡ locateincuticle of T. spiralis new born larvae, cord cells of adult worm and stichosome ofmuscle larvae, respectively. All of the three positions are main secretory organs of T.spiralis in different developmental stages. Because that the main function of DNaseⅡis to degrade DNA, we deduced Ts DNaseⅡ may help in degradation of ETs formedby innate immune cells.In this study, we used murine macrophages cell line to investigate whether ETsmechanism also exist in macrophages. Firstly, E. coli and C. albicans infectedmacrophages were observed to release extracellular fiber-like structure by laserconfocal microscopy (LCM) and scanning electron microscope (SEM). Thesestructures contain DNA component and are able to ensnare bacteria and fungi, whichexhibited the same features with NETs. So they were named “MacrophageExtracellular Traps (METs)”. Non-pathogen stimulus displayed different abilities toinduce METs formation. Hemolysin and zymosan were efficient regents to stimulatemacrophage releasing METs, while PMA, LPS and hydrogen peroxide, which arepowerful stimulus of NETs, displayed limited METs inducing activity. Thisphenomenon indicates macrophage may have different ETs formation mechanismcompared with other innate immune cells. We also found that the quantity of METsformation was depended on the concentration of stimulus, and that the formation ofMETs occurred in a short time (less than15min) after incubation with pathogens.Secondly, we investigated the characteristic of METs. Immunofluorescence ofMETs shown some anti-microbe enzymes co-localized with extracellular DNA,including histone, tartrate-resistant acid phosphatase (TRAP), lysozyme andmyeloperoxidase (MPO). Interestingly, not all METs contain histone and TRAP,indicating there may be more than one type of METs existing in murine macrophages.By PCR and fluorescence in situ hybridization (FISH) experiments, we observed bothnuclear and mitochondrial DNA existing in METs. We also observed both cell death-associated METs and living cell released METs coexisting in macrophage. Thisphenomenon indicates that macrophage could release METs in either nuclear DNApathway or mitochondrial pathway. We also evaluated the antimicrobial activity ofMETs by modified plate assay. Result showed METs had limited capability to kill E.coli and C. albicans. In order to corroborate this finding, we conducted in situLive/Dead fluorescence stain of METs ensnared microbes. Fluorescence microscopeobservation revealed living cell released METs didn’t kill microbes at all, while celldeath associated METs could kill only very few E. coli and limite several C. albicansto germinated. Moreover, we investigated the relationship between METs and ROS,and whether METs lead to cell death in apoptosis pathway. We found neither ROSproduction nor apoptosis behavior occurred in METs formation process.Thirdly, we investigated whether METs can response to pathogenic nematode T.spiralis. LCM and SEM observation revealed living T.spiralis muscle larvae ornewborn larvae did not induce METs. But macrophage can produce METs to ensnareand kill T. spiralis in present of nuclease inhabitor aurintricarboxylic acid (ATA).Interestingly, the less the T.spiralis was added to macrophages cultures, the longgertime needed to induce METs production. So we deduced that METs may also inducedby ES component. Our following experiment confirmed this deduction. We also foundanti-T. spiralis serum cannot block this escape mechanism. T. spiralis was notensnared by METs in present of anti-T. spiralis antibodies, but in this case, weobserved phagocytosis of newborn larvae by macrophage. METs were more effectiveto kill newborn larvae than phagocytosis.Lastly, we investigated the active nuclease component in T. spiralis ES products.By DNA substrate cleavage and intermediate product terminal group analyse, wefound nuclease activity existed in ES products and display DNaseⅡ characteristic.We expressed the known DNaseⅡfrom differentmental develop stages (P43andT3223) in E. coli and analyse their activity. Unfortunately, recombinant muscle larvae(P43) and adult worm (T3223) specific DNaseⅡ showed no activity. In order tocorroborate this result, we purified native P43and T3223protein from correspondingES product by using ion exchange chromatography, gel filtration chromatography andimmunoaffinity chromatography. Activity analysis of these two native proteins suggested P43was inactive, while T3223possess activity. In order to identify theactive DNaseⅡ component, we performed SDS-PAGE zymography analysis of ESproducts. Result showed the DNaseⅡ activity in ES origined from several proteinswith different molecular weights. Because it’s difficult to collect enough ES fordirectly purifying active components, we established an2D nuclease zymographycombined NanoLC-ESI-MS/MS method to identify them. Our work provided a newway to identify nuclease from a small quantity samples, and the identified activenuclease was the candidates for further investigation of T. spiralis ETs-escapemechanism.