The Impact And Related Mechanisms Of Redox State On The Formation And Development Of Tuberculous Granulomas

Objective:To investigate the impact of redox state on the formation and development of tuberculous granulomas,and try to reveal the related mechanisms through several aspects including the polarization of monocytes and macrophages,the levels of cytokines,the production of reactive oxygen species?ROS?,the differentiation,activation and chemotaxis of lymphocytes.Method:1 Human PBMC was used to construct the in vitro model of tuberculous granuloma followed by the infection with BCG.Fresh PBMC was pretreated with NAC and BSO for 2h,then infected with attenuated vaccine strain of Mycobacterium bovis Bacille Calmette Guerin?BCG?at the MOI of 1:10?BCG:PBMC?.The formation and development of tuberculous granulomas at different time points was monitored,counted and recorded by imaging.2 Respectively from the following aspects to explore the mechanism of granuloma formation in vitro in different redox state:?1?Flow cytometry detection the expression of polarized molecules such as CD40?CD64?CD163 and CD206 in monocytes and macrophages,in order to understand the impact of redox state on the polarization phenotypic of monocytes and macrophages.?2?The cytokines levels in the cell supernatant of different treatment group was detected by ELISA,including the expression of TNF-?,IFN-?and IL-10.The expression of TNF-?in the monocytes was detected by flow cytometry,in order to understand the effects of production cytokine on the model of different redox state.?3?Flow cytometry was used to detect the production of ROS in monocytes,in order to understand the effects of the production of reactive oxygen in the different redox states.?4?In order to observe the granuloma formation in vitro,we increase or cut the generation levels of ROS by analog or inhibitor,to further confirm the ROS levels affect granuloma formation.?5?Flow cytometry was used to detected the expression of CD4 and CD8 on the T lymphocytes,in order to investigate the effects of the differentiation of T cells in different redox states.Deteced the Synthesis of IFN-??IL-4 in tracellular of CD4⁺T lymphocytes and the expression of CD69 in the T lymphocyte,in order to investigate the effects of the T lymphocytes activation in different redox states.?6?Under the use of BCG as the source of chemotaxis,we use the Transwell chamber method to detect the effects of the lymphocytes and chemotaxis to BCG in different redox states.With analysis for lymphocytes,observation the chemotaxis activity of lymphocytes under the infected monocytes.3 To investigate the effect of the changes redox state on the formation of granuloma in animals experiments,the mice was pretreated by intraperitoneal injection with NAC,then Mycobacterium tuberculosis?MTB?H37Rv was used to infect C57BL/6 mice pretreated with NAC and C57BL/6 mice respectively by atomization inhalation,to establish a mouse model of tuberculosis,then the mice were sacrificed at different time points after infection respectively,observed and photographed in lung and granuloma formation,at the same time,some lung tissues were fixed in paraformaldehyde and paraffin sections were used to observe the pathological changes of lung tissue by HE staining.Results:1 After BCG infection in each groups of PBMC,from the second day begin to form cell cluster,and the third days to form a typical granuloma structure.After the third day,compared with the untreated control group,the PBMC group treated with NAC significantly increased in the formation of granulomas in number after BCG infection?38.25±0.85/LPF vs 54.00±1.08/LPF,P<0.05?.On the contrary,the number of granulomas formed by PBMC in the BSO pretreated group was significantly lower than that in the untreated control group after BCG infection?38.25±0.85/LPF vs 26.50±0.65/LPF,P<0.05?.With the extension of infection time,the volume of granuloma increased gradually,but the number of granulomas was not significantly changed longer in each group.2.Effects of redox state on the polarized phenotype of monocytes and macrophages:?1?Compared with the control group,the polarized phenotype of monocytes and macrophages tended to toward the M1 after BCG infection 24h later.?2?Compared with BCG infection group with no pretreated,the group of NAC pretreatment can significantly cut the expression of M1 polarization phenotype in monocytes and macrophages,the expression of M2 polarized molecular had no significant effect.?3?the group of BSO pretreatment can significantly raise the expression of M2 polarization phenotype in monocytes and macrophages,the expression of M1 polarized molecular had no significant effect.3.Effects of redox state on the TNF-?expression in monocytes:Compared with the normal control group,the expression of TNF-?in the CD14⁺monocytes of BCG infected group was significantly higher,but NAC and BSO pretreatment had no significant effect on the level of TNF-?in monocytes.4.The effects of redox state on the level of ROS produced by monocytes and its influence on the formation of tuberculous granuloma:?1?Compared with the normal control group,the level of ROS in the BCG infection group was increased;Compared with untreated group,the level of ROS in NAC pretreated group was significantly lower after BCG infection,and BSO pretreatment could significantly increase the level of ROS in monocytes after BCG infection.?2?Using NADPH oxidase inhibitor DPI to block the formation of intracellular ROS can significantly promote the formation of granuloma induced by BCG infection,and the level of ROS raised by H₂O₂ can significantly inhibit the formation of granuloma.?3?After NAC pretreatment,then exogenous give H₂O₂ in order to increase the level of ROS,can partial offset the role of NAC promote the formation of granuloma,but relatively simple H₂O₂ treatment group had a significantly increased the number of granuloma formation.5 The effects of redox state on the differentiation of T lymphocytes:Compared with the isolated infection group,NAC and BSO pretreatment had no significant effect on the ratio of CD3⁺CD4⁺T cells and CD3⁺CD8⁺T cells in PBMC,and it also had no significant effect on the ratio of Th1/Th2 in CD3⁺CD4⁺T cells.6 Effects of redox state on the activation of T lymphocyte:Compared with the isolated infection group,there was no significant difference in the expression of CD69 in the group of NCA/BSO pretreated.7 The effect of redox state on the level of cytokines in cell culture:Compared with the normal control group,the expression levels of TNF-?,IFN-?and IL-10 were significantly higher in the BCG infection group;Compared with the single infection group,in NAC pretreatment group the expression of TNF-?,IFN-?were decreased,but there was no difference in the expression of IL-10;There was no significant difference in the expression of the three cytokines in the BSO pretreatment group.8 The effects of redox state on the chemotaxis activity of cells:?1?Based on PBMC cell to be analysed and BCG as chemotactic source,compared with the isolated infection group,the group of NAC pretreatment could significantly inhibited the chemotactic activity of monocytes and lymphocytes;but BSO pretreatment had no significant effect on the chemotactic activity of monocytes and lymphocytes.?2?To be analysed for purification lymphocyte cells,the ability of infected monocyte chemotactic lymphocyte was stronger than that of BCG and uninfected monocyte.9 Each groups of mice after MTB infection with 8 weeks,lung nodular granulomas are visible.Compared with mice not treatment,NAC treatment group pulmonary granulomas is not only the rise in the numbers,but also the volume are increased.The HE staining of lung tissue show that the NAC treatment group pulmonary inflammatory cells infiltrating significantly increased than simple infection group,and granulomatous inflammation cell aggregation was also significantly increased,lung tissue pathology change is more serious.Conclusion:1 Redox state can significantly affect the granuloma formation,antioxidants NAC can promote the formation of granuloma induced by MTB in vitro,and the main manifestations are increase the number of granulomas;It also can promote the lung tissue formation of granuloma and focal inflammatory cell infiltration in mice mode of tuberculosis,but it also can aggravate the pathological damage of lung tissue.2 The mechanism that NAC can promotes the formation of granuloma may be correlation with down-regulation of ROS levels in monocytes.It has no significant correlation with the polarization of Monocyte and macrophages,the production of cytokine,the differentiation and activation of lymphocytes.