The Characteristics And Mechanisms Of Low-Density Granulocytes In The Condition Of Tuberculosis

Objective:1、 To investigate the frequency of LDGs in the peripheral blood of patients with pulmonary tuberculosis, and explore the relationship between LDGs frequency and the severity of tuberculosis.2、 To demonstrate the molecular phenotypes and markers involving cell apoptosis, necrosis and ROS generation.3、 To verify the correlation between Mtb infection and LDGs generation, and investigate the origin of LDGs following the infection of M.tuberculosis.4、 To explore the possible mechanisms of LDGs formation via in vitro experiments.5、 To investigated the phagocytosis and bactericidal effects of LDGs against M.tuberculosis.Methods:1 、 Peripheral blood were collected from TB patients and healthy controls.According to the results of sputum acid-fast bacillus staining and received treatment,TB patients were further divided into acid-fast staining positive and negative groups,or newly diagnosed patients and patients received treatment. The percentages of LDGs in PBMC fraction were detected by flow cytometry.2、The expression levels of phenotypic markers including CD15, CD16, CD33,CD66 b and CD62 L,the levels of intracellular ROS and apoptosis ratios of LDGs and autologous NDGs were detected by flow cytometry.3、The induction of neutrophils into LDGs in vitro:(1) Peripheral blood samples were collected from healthy volunteers and challenged with M.tuberculosis H37 Rv with different infection proportion,we analysis the percentages of LDGs in PBMC fraction by flow cytometry in different infection points;(2)Peripheral blood samples were challenged with different virulence mycobacteria we analysis the percentages of LDGs in PBMC fraction by flow cytometry;(3)we isolate and purify neutrophils in healthy human peripheral blood,challenged with M.tuberculosis H37 Rv with differentinfection proportion,we separate LDGs and NDGs by Ficoll density gradient centrifugation after 4h,then calculate LDGs accounted for the proportion of neutrophils by Neubauer hemocytometer;4、To explore the LDGs sources from the way of apoptosis : we use H37 Rv challenge with healthy human peripheral blood after employing apoptosis inhibitor,and analysis the relationship between apoptosis and LDGs.5、To explore the LDGs sources from the way of ROS : we use ROS promoter(PMA) and ROS inhibitors(NAC), and analysis the relationship between apoptosis and LDGs.6、LDGs functions:(1) We contrast LDGs of TB patients(or LDGs by induced in vitro)and NDGs to phagocytosis H37Rv-GFP by flow cytometry.(2) LDGs and NDGs bactericidal capacity of H37 Rv in vitro: H37 Rv was incubated with or without cells for indicated times and the colony forming units were monitored over times.Results:1、The PBMCs of subjects with TB contained significantly elevated percentages of LDGs compared with the PBMCs of control subjects and the percentages of LDGs in the PBMCs with positive AFB( acid-fast bacillus) were significantly elevated compared with those of patients with negative AFB, the percentages of LDGs in the PBMCs of PTB patients after 2 weeks of anti-tuberculous therapy were significantly lower than those in patients who had not received. After 6-months of anti-tuberculous therapy, the percentage of LDGs in PTB patients recovered to the levels of those in healthy volunteers.2、 When compared to NDGs from TB patients and health control, the mean fluorescence intensities(MFI) of membranal CD15, CD33,CD66 b,ROS and apoptosis were significantly enhanced and CD62 L were significantly reduced on LDGs from patients with TB.3、Mycobacterial infection can significantly raise LDGs levels in peripheral blood. Mycobacterium induced LDGs ability to generate with time and dose dependence of infection, and associated with the virulence and vitality of mycobacteria.Mtb infect mature neutrophils can also induce the generation of LDGs.4、 Neutrophil apoptosis levels has no direct correlation with LDGs.5、 PMA can increase ROS levels in neutrophils and significantly increase the generation of LDG level, on the contrary, the NAC inhibite neutrophil ROS levels were significantly lower the generation of LDG level after Mtb infection6 、 When compared to NDGs, LDGs displayed significantly increased phagocytosis of H37 Rv and the bacterial load in LDGs was not statistically significant.Conclusion:1、The frequency of LDGs in the PBMC fraction was significantly elevated in tuberculosis patients and correlated with the severity of tuberculosis.2 、 LDG is a group of activated neutrophils that have degranulated. When compared to NDGs, LDGs displayed significantly increased phagocytosis but comparable bactericidal effects on M.tuberculosis.3、 In vitro infection with mycobacteria can induce the generation of LDG in peripheral blood from health subjects. M.tuberculosis infection can also converts purified mature NDGs to LDGs, which suggested that in situ activation contributes to the generation of LDGs in the condition of M.tuberculosis infection.4、The frequency of LDGs was positively correlated with the levels of ROS. The generation of LDGs in the condition of mycobacterial infection can be regulated by modulating the level of ROS.