Study On The Immune Regulation Of Tuberculosis By Wnt/β-catenin Signaling Pathway And Drug Resistance Of Tuberculosis

Background&Objective:Tuberculosis（TB）is an infectious disease caused by Mycobacterium tuberculosis（MTB）,WHO estimated 10.1 million new TB cases in the world in its2018 global TB report,which is still a major public health problem worldwide.MTB can induce complex immune responses and regulation processes after infecting the host.Elucidation of the host’s immune regulation mechanism after MTB infection is the precursor and key to the development of therapeutic tuberculosis vaccine.Many studies have demonstrated that T cells can play a key role in controlling MTB infection.The Wnt/β-catenin classical signaling pathway is thought to regulate the developmental and homeostatic processes of the individual,and the key signaling pathways of the host’s immune response to microbial pathogens,which have been shown to be involved in the regulation of tuberculosis,including macrophages after MTB infection.Regulation of inflammatory response,apoptosis and necrosis.To demonstrate the immunoregulatory effect of Wnt/β-catenin signaling pathway on tuberculosis and drug resistance in tuberculosis,We purchased theβ-catenin gene Mouse(B6.129-Ctnnb1^tm2Kem/Knw J)were knocked out,andβ-catenin knockout mice of CD4⁺T cells were constructed by breeding and breeding,and SPF wild type C57BL/6 mice were used as a control group.MTB airway nasal infection,genetic,protein,bacterial and pathological studies 28 days after infection.The clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were used to determine the MIC values of cyclases of 177 clinical isolates,and the mutation sites and gene expression levels of Ald,Alr and ddl A genes were analyzed.Methods:1.Breeding mice with conditional knockout ofβ-catenin gene in CD4⁺T cells,hybridize withβ4-catenin^-/-and CD4-Cre transgenic mice,and use the principle of Cre-lox system to breed CD4⁺T cells were conditionally knocked out of theβ-catenin gene(CD4-Cre;β-catenin^-/-)and transgenic mice were identified.2.C57BL/6 mice were infected intranasally with H37Rv,including wild-type mice andβ-catenin knockout mice,and continued to infect for 4 weeks.After the end of the4th week,the mice were sacrificed by cervical dislocation.3.Take lung tissue and spleen tissue specimens:（1）The percentage of T cells was detected by flow cytometry,and the effect of CD4⁺T cell conditional knockoutβ-catenin gene on T cell proliferation was studied.（2）The levels of inflammatory factors（TNF-α,IFN-γ）were detected by flow cytometry,and the effect of CD4⁺T cell conditional knockoutβ-catenin gene on inflammatory response in mice infected with MTB was studied.（3）The levels of inflammatory factors（TNF-α,IFN-γ）andβ-catenin downstream transcription factor TCF-1 were detected by Real-time PCR,and the tissue homogenate was plated,and CFU was performed after 2-3 weeks of culture.Count,detect the amount of tissue bacteria,and study whetherβ-catenin has a protective effect in the host infected with MTB.4.The clinical isolates of Mycobacterium tuberculosis from tuberculosis patients sputum were collected,and the MIC values of cycloserine of 177 clinical isolates were determined using 7H9 liquid medium.5.Screening for strains resistant to cycloserine and sensitive,extracting genomic DNA and RNA,and analyzing the mutation sites and gene expression of Ald,Alr and ddl A genes.Results:1.The mice that conditionally knocked out theβ-catenin gene in CD4⁺T cells were extracted from the genomic DNA by mouse tail,and the genes for conditional knockout ofβ-catenin mice by CD4⁺T cells were identified by PCR.The phenotype is:ctnnb homozygous（-/-）and CD4-Cre positive expression,wherein the ctnnb homozygote has a band size of 300 bp,the heterozygous band size is 300 bp and 223bp,and the wild type band size is 223 bp.2.CD4⁺T cells conditionally knocked outβ-catenin and resulted in a decrease in the percentage of CD3⁺T and CD3⁺CD4⁺T cells in spleen and lung tissues.3.Flow cytometry was used to stain single cells of lung and spleen of H37Rv infected mice.It was found thatβ-catenin^-/-infected mice showed significantly lower expression of protective cytokines in lung and spleen tissues thanβ-catenin^+/+control group.4.Compared withβ-catenin^+/+control mice,β-catenin^-/-mice infected with H37Rv showed lower levels of protective inflammatory factors and downstream transcription factors in lung tissue,accompanied by increased bacterial load in lung and spleen tissues.5.Pathology:Gene knockout mice infected with H37Rv can observe that the pathological inflammatory degree of lung tissue is heavier than that of wild-type mice.6.cycloserine-resistance rates were as low as 4.28%in 140 strains of drug-resistant M.tuberculosis,we preliminarily defined the MICs of MTB≥32μg/ml as cycloserine-resistant.7.The mutations of Ald,Alr and ddl A loci were not different in the cycloserine-resistant strains and sensitive strains.The gene expression of the cycloserine-resistant strains at the Alr locus was significantly higher than that of the sensitive strains.Conclusion:1.β-catenin has been proved to regulate the number of TB-specific activated T cells in gene knockout mice infected with tuberculosis in animal experiments.2.Animal experiments ofβ-catenin^-/-infected mice prove thatβ-catenin may play an immune protective effect in the host of tuberculosis.3.The drug ressitance rate of cycloserine is low in MDR-TB,suggesting that cycloserine can be an effective and reliable clinical treatment for MDR-TB.4.The cycloserine-resistance mutation in MTB has not been found,yet the overexpression of Alr gene found to be highly related to the serine-resistance of MTB,providing a possible explanation of MTB resistance mechanism.