| Background and purpose: Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis,which poses a serious threat to global health.The main challenge of tuberculosis control is the lack of biomarkers to distinguish the status of different infections and evaluate the severity of the disease;Lack of new drug targets and therapeutic drugs to solve the problem of drug resistance of tuberculosis.Analyzing the status of immune cells is helpful to understand the influence of Mycobacterium tuberculosis infection on host defense.In different infection types and immune microenvironment,the immune mechanism mediated by immune cells is also different.It is very important to fully understand how the immune system integrates signals from different cell types and coordinates the response,and how the individual variation of these cell types leads to the difference of infection results.Transcriptome sequencing can reveal the differences of gene expression profiles,but it cannot explain the characteristics of specific subsets in cells.Single cell sequencing can be used to analyze the transcription profile of a single cell and reveal the identity of cells in complex heterogeneous immune groups.Some subsets of cells are considered as markers to control the host’s immune response to diseases or different disease states.For example,CD4+T and CD8+T cells play a key role in the development of immune response.When exposed to antigen for a long time,T cells gradually lose their effector function and show an irrecoverable exhaustion phenotype,that is,T cell exhaustion.This state of exhaustion may affect the clinical manifestations of Mycobacterium tuberculosis infection,such as the severity and prognosis of the disease.After the infection of Mycobacterium tuberculosis,the metabolic activity of exhausted cells has changed,and the metabolic system plays an important role in maintaining the balance in the host.The metabolic disorder of the host may be beneficial to the development of tuberculosis.Therefore,using single cell sequencing technology combined with metabonomics can explain the dynamic changes,properties and functions of cell subsets in the process of tuberculosis infection,so as to better understand the diagnosis and treatment of tuberculosis.Methods: Firstly,by sorting out the single cell sequencing data of peripheral blood mononuclear cells infected by Mycobacterium tuberculosis in the database,the cell communication differences of peripheral blood mononuclear cells in different infection types were integrated and analyzed,and various anti-tuberculosis drugs were predicted at the same time.Secondly,the characteristics and changes of subsets of CD4+T cells and CD8+T cells isolated from peripheral blood mononuclear cells of healthy individuals and tuberculosis patients were determined by single cell sequencing analysis.Through enrichment analysis and other methods,the exhausted subsets of CD8+T cells were elaborated,and the central node genes related to exhausted cell subsets were screened out,and the screened exhaustion marker genes were verified by molecular biology.In addition,the non-targeted metabonomics based on LC-MS was used to analyze the exhausted CD8+T cells after tuberculosis infection,and the differential metabolites were obtained,so as to pay attention to the metabolic changes of exhausted cells.Finally,combined with network pharmacology,molecular docking,molecular dynamics simulation and biological experiments,the anti-tuberculosis target and mechanism of naringin were determined.Results: First of all,the distribution and changes of various cells in peripheral blood mononuclear cells under different infection conditions were determined,including T cells,B cells,NK cells and monocytes,and the characteristics of cell communication among cell subsets under different conditions were determined.It was found that the cell communication intensity after infection was stronger than that before infection,especially in the state of latent infection.Signal pathways such as TNF,ITGB2 and CLEC can be used as markers to distinguish the infection status inside and outside the lung,while CD6,ALCAM and COLLAGEN can be used to distinguish latent infection from active tuberculosis.At the same time,drugs that may have therapeutic value under different infection conditions were screened,and it was speculated that naringin,betulinic acid,ofloxacin,neomycin and Rottler Lin had certain significance in the treatment of tuberculosis.Secondly,seven CD8+T cell subsets and eight CD4+T cell subsets isolated from peripheral blood were identified,and the changes and characteristics of transcriptome landscape of each subset were expounded,especially the exhausted cell subsets.It was found that exhausted CD4+T cells and CD8+T cells had different marker genes and pathways.CD8-EX3 subgroup in CD8+T exhausted cells was identified as a specific subgroup,and the identified gene module further identified the key factors affecting CD8+T cell exhaustion,which provided a potential marker for the prognosis of tuberculosis infection.Two genes,HIFX and VIM,were screened and verified as exhaustion markers in CD4+T cells,and four genes,ITM2 C,RGS1,IRF1 and SATB1,were screened and verified as candidates for exhaustion markers in CD8+T cells.In addition,the metabolic changes of CD8+Texhausted characteristic cells were expounded by using the non-targeted metabolomics technology based on LC-MS.There were 100 differential metabolites in positive ion mode and 163 differential metabolites in negative ion mode,and tetrahydrocurcumin,glutathione,vardenafil and so on were selected as exhaustion-related metabolites,and purine metabolism and folic acid synthesis were used as metabolic pathways for further exploration.Finally,combined with network pharmacology,molecular docking and molecular dynamics simulation,the possible anti-tuberculosis targets of naringin were determined from the protein level,such as PTGS2,CASP3,ESR1 and PPARG,which could affect IL-17,arachidonic acid,cell death and inflammatory response.At the same time,the possibility of naringin as an anti-tuberculosis drug was confirmed by the minimum inhibitory concentration experiment.The above contents will help to understand the changes and characteristics of immune cells in Mycobacterium tuberculosis infection more comprehensively and formulate effective intervention strategies,which will provide new scientific basis and methods for clinical research.Conclusion: 1.In this paper,the proportion of cell subsets in peripheral blood mononuclear cells infected by Mycobacterium tuberculosis in different states was clarified,and it was found that the cell communication mode was generally stronger than that in healthy state,and signal pathways such as ITGB2 and CLEC could be used to distinguish the infection types inside and outside the lung,and signal pathways such as CD6 and ALCAM could be used to distinguish the latent patients from the active patients.2.In this paper,the composition,changes and characteristics of the subsets of CD8+T cells and CD4+T cells in active pulmonary tuberculosis were defined,and HIFX and VIM were identified as exhaustion marker genes of CD4+T cells,while ITM2 C,RGS1,IRF1 and SATB1 were identified as exhaustion marker genes of CD8+T cells.3.In this paper,the metabolic changes of exhausted cells after infection were defined,and 263 different metabolites were found in the positive and negative ion mode,among which tetrahydrocurcumin,glutathione and vardenafil changed significantly,and were enriched in the pathways of cofactor biosynthesis,purine metabolism and folic acid synthesis.4.In this paper,the mechanism of naringin in the treatment of tuberculosis at the protein level was clarified,and it was found that naringin can play an anti-tuberculosis role through multiple targets such as PTGS2,ESR1 and PPARG,as well as IL-17,arachidonic acid and inflammatory response. |
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