Aging Related ATG5 Defect Impairs Neutrophil Extracellular Traps Formation

Background: Neutrophil is the frontline of the innate immune system.In addition to the classical chemotaxis,phagocytosis and clearance,there is another extremely important mechanism to eliminate pathogens in neutrophils,which is known as neutrophil extracellular traps(NETs).NETs is a complex three-dimensional decondensed structure with DNA backbones and histones,proteases,as well as antimicrobial peptides decorated.We now have a profound knowledge of how NETs are induced and formed.Still there is no definite answer to questions like whether there are other physical,chemical or biological substances capable of inducing NETs and what the critical component is during the process.The morbidity and mortality of the elderly population are much higher than that of the young people.This phenomenon is most prominent in infectious diseases,which is believed to be closely related to immunosenescence,the negative impact of aging on the immune system.As an important component of the innate immunity,functions of neutrophils are changed greatly by aging,such as the impirment of chemotaxis,the disability of eliminating bacteria,and the change in the expression of inflammatory factors and anti-apoptotic activity.Currently,the influence of aging on the formation of NETs is undetermined and the mechanisms are not clear.Besides,if aging is going to affect the the process of NETosis,will it affect the other common form of cell death,apoptosis? Is there any like between NETosis and apoptosis? These are the focuses of the present research.Section 1: TLR2 ligands-induced NETosis is regulated by ROS production and autophagyObjective: PMA has been extensively utilized to activate NETosis.Other treatments,such as LPS,IL-8,and f MLP(N-Formylmethionyl-leucyl-phenylalanine)are also commonly used.We first examined whether NETosis can be effectively elicited by TLR2 ligands in vitro and whether TLR2-My D88 is necessary during the process.Next,we will test whether TLR2 and TLR4 play a synergistic role in inducing NETs formation.The role of ROS and autophagy in TLR2 ligands-induced NETosis will also be examined.Methods: Several representative components from diverse categories of cell-wall components were selected to stimulate PMNs isolated from the peritoneal cavity with casein solution.The abilities of those ligands used to induce NETosis were demonstrated by fluorescence quantification and confocal microscopy.TLR2 knock-out(KO)and My D88 KO mice were utilized to determine the necessity of TLR2-My D88 pathway in NETosis induced by ligands above.Low doses of LPS and PGN were used alone or together to test the synergistic role of TLR2 and TLR4 in NETs formation.Furthermore,we used gp91 phox KO mice,enhancer and inhibitor of autophagy to examine the role of ROS and autophagy in TLR2 ligandsinduced NETosis.Results: The ligands used were all able to induce NETosis,as demonstrated by fluorescence quantification and confocal microscopy.PMNs from TLR2 KO and My D88 KO mice were not able to generate NETs under stimulation.The combination of low-dose LPS and PGN induced much higher NETosis than either one alone,indicating a synergistic effect.The reduction in ROS in gp91 KO PMNs was accompanied by a decrease in NETosis.Inhibition of mammalian target of rapamycin(m TOR)to augment autophagic activity enhanced PGNinduced NETs formation.Conclusion: The TLR2 ligands used are able to induce NETosis.TLR2 and My D88 are both necessary for effective NETs formation.There is a synergistic effect between TLR2 and TLR4 in inducing NETosis.Autophagy and ROS generated by NADPH oxidase were involved in NETosis induced by TLR2 ligands.Section 2: Atg5 related defect in autophagy is responsible for the diminished NETosis in aged miceObjective: The innate immune system becomes dysregulated in many aspects with aging.To compare the differences in NETosis between aged and young mice.And to elucidate the mechanisms(altered autophagic activity,impaired chemotaxis,decreased ROS production,etc.)by which NETs formation was reduced in aged mice.Methods: NETs formation in the peritoneal lavage was detected and analyzed 6 h after CLP.The quantity of PMNs in the peritoneal lavage and bone marrow from both young and aged mice were analyzed.The surface expression of TLR2 and NADPH dependent ROS production in PMNs were measured by flow cytometry.Neutrophils from young and aged mice were stimulated with PGN for 2 h,4 h,and 6 h.The autophagic activity,represented by the expression of LC3 B,was monitored.Next,to define which step the difference in autophagic level between aged and young mice comes in,we used transcriptome data analysis plus protein level verification.Results: There were fewer NETs in the peritoneal lavage from aged mice compared with that from young counterparts.The proportion of Ly6G+CD11b+ PMNs in the peritoneal lavage was lower in aged mice as compared with that in young mice,despite that there was no difference in the PMNs counts in the bone marrows.There was no significant difference in the PMN surface expression of TLR2 between aged and young mice before and following PGN stimulation.However,we found that ROS production is lower in neutrophils from aged mice.The level of LC3 B in the aged group was substantially lower than that in the young group following PGN stimulation.Meanwhile,the use of rapamycin greatly restored the ability of PMNs from aged mice to release NETs.The level of Atg5 in aged mice was significantly lower than that in young ones.Besides,a relatively higher level of m TOR in aged mice was found.Conclusion: NETosis is diminished after TLR2 ligand stimulation in aged mice.The decreased ability of PMN to form NETs is associated with defected autophagic activity in aged mice.Atg5 maybe the key molecular leading a defected autophagic activity after PGN treatment.Other factors,such as dcreased chamotaxis,less ROS production and elevated m TOR may also participate in reduced NETosis.Section 3: Defect in autophagic activity leads to a shift from undergoing NETosis to apoptosis in aged miceObjective: To investigate whether lower autophagic activity leads to an increased level of apoptosis in PMNs from aged mice.Methods: The proportion of apoptotic PMNs(Annexin-V+,7-AAD-)was measured by flow cytometry at 6 h and 12 h after PGN stimulation or left untreated.Next,two inhibitors of autophagy(wortmannin and bafilomycin A1)were used to treat PMNs from young and aged mice in combination with PGN stimulation.Confocal microscopy was used to directly visualize the effects of autophagy inhibitors on both NETosis and apoptosis.Results: In the basal group(untreated for 6 h),or after PGN stimulation,the percentage of apoptotic PMNs was higher in the aged group than that in the young group.Either the addition of wortmannin or bafilomycin A1 to PGN increased the percentage of apoptotic cells.Confocal microscopy showed that the formation of NETs was reduced,while apoptotic cell death increased when autophagy inhibitor wortmannin or bafilomycin A1 was applied.Conclusion:The decrease in NETosis,and the increase in apoptosis we see here in aged mice may be regulated by autophagic activity.