The Mechanisms Of Low-density Granulocytes Formation In Mycobacterium Tuberculosis Infection

Objective:To investigate the mechanisms of the transformation of normal density granulocytes?NDGs?from peripheral blood to low density granulocytes?LDGs?during Mycobacterium tuberculosis infection.Methods:Peripheral blood of TB patients was collected and separated by Ficoll density gradient centrifugation.LDGs were isolated from the peripheral blood mononuclear cell?PBMC?layer by negative sorting using immunomagnetic beads.NDGs were purified from the erythrocyte layer by lysing red cells.The neutrophil extracellular traps?NETs?structure of LDGs and NDGs was shown by immunofluorescence staining in the condition of no stimulation or stimulation with PMA.Exploring the relationship between LDGs production and NETs releases:?1?Neutrophils were treated with PMA to induce NETs formation.NETs were observed by immunofluoescence technique,LDGs formation was detected by flow cytometry.?2?Before Mtb infection,pretreated neutrophils with Zn²+chelating agent TPEN to down-regulate the production of NETs,and PMA to up-regulation of NETs,further explored the effects of NETs release on the formation of LDGs.Exploring the relationship between neutrophil reactive oxygen species production?ROS?and the LDGs formation:?1?After Mtb infection,neutrophil ROS production were detected by flow cytometry.?2?Before Mtb infection,neutrophils were pretreated with ROS inhibitors NAC and DPI to down-regulate ROS levels;and exogenous ROS H₂O₂ preconditioning neutrophils to up-regulated ROS levels,further explored the relationship between ROS production and LDGs production.To investigate whether ROS regulates the production of LDGs by regulating NETs release:inhibiting the production of NETs by the PAD4 inhibitor CL-Amidine without changing the level of neutrophil ROS,after Mtb infection,LDGs formation were detected by flow cytometry,NETs structure was shown by immuno-fluorescence staining,explored whether ROS involved in regulation of LDGs production by regulating NETs production.Exploring the relationship between neutrophil degranulation and LDGs production:?1?Flow cytometry was used to detect the expression of NDGs and LDGs degranulation phenotype?CD11b,CD66b?in tuberculosis patients,and to explore whether the LDGs formation was accompanied by neutrophil degranulation.?2?Treating collcted healthy human neutrophils with degranulation stimulant FMLP,and investigate the effect of neutrophil degranulation on LDGs formation.?3?Before stimulated with Mtb and FMLP,neutrophils were pretreated with degranulation inhibitor PTX inhibit the production of degranulation,and detected LDGs formation and degranulation phenotype?4?Uing TPEN inhibit NETs production,and treated neutrophil with FMLP to promote the degranulation of neutrophils,detect the formation of LDGs,and explore whether the production of degranulation-related LDGs is independent of the NETs pathway.Results:1.In the natural state,TB-LDGs spontaneously release higher levels of NETs,which is significantly higher than TB-NDGs.After PMA stimulation,TB-NDGs can release a large amount of NETs,which is significantly higher than TB-LDGs.The process of neutrophils convert to LDGs after Mtb infection is accompanied by the release of a large number of NETs;the regulation of NETs can change the production of LDGs.2.Down-regulation of neutrophil ROS levels by NAC and DPI can significantly inhibit the production of LDGs.Up-regulation of ROS levels by H₂O₂ can further promote the production of LDGs based on Mtb infection.Inhibiting the production of NETs by the PAD4 inhibitor CL-Amidine without changing neutrophil ROS level can aslo inhibit LDGs formation.3.TB-LDGs have a higher level of degranulation phenotype?CD11b,CD66b?than TB-NDGs.The degranulation stimulant FMLP stimulated neutrophils to significantly up-regulate the production of LDGs,but had no significant effect on the NETs production.The inhibition of neutrophil degranulation by the degranulation inhibitor PTX significantly inhibited the production of LDGs induced by Mtb/FMLP.The inhibition of the NETs formation had no significant effect on the expression of degranulation and the production of degranulation-related LDGs.Conclusion:The production of tuberculosis-related low-density granulocytes is related to the release of NETs and neutrophil degranulation.