Expression Characteristics Of BATF2 In Patients With Tuberculosis And The Anti-tuberculosis Mechanism Of It In Macrophages

Objective Tuberculosis(TB)is a contagious disease caused by Mycobacterium tuberculosis(Mtb).In 2020,it caused about 1.5 million deaths worldwide.At present,about 1/4 of the world’s population is infected with Mtb.The latest World Health Organization Tuberculosis Report identified 30 countries with high burden of TB,and China is one of them.Previous studies in our research group showed that the proportion of CD4~+T cells and Wnt/β-Catenin pathway related genes,such as TCF-7,β-catenin,cyc D2,in peripheral blood cells of patients with severe cavitary pulmonary TB were significantly reduced,whereas the expression of BATF2 in peripheral blood cells of TB patients was increased.The aim of this study was to explore the effects of BATF2 on macrophage proliferation,apoptosis,inflammation,antigen-presenting function and the expression ofβ-catenin,effects on Mtb phagocytosis and early inhibition.Screening for interacting proteins with BATF2,the target genes of BATF2,and preliminarily exploring their function in TB.To providing new ideas for the prevention and control of TB and vaccine research.Methods Our prior research with gene expression omnibu database analysis and real-time fluorescence quantitative polymerase chain reaction(q PCR)analysis has indicated that BATF2 was highly expressed in TB patients.To explore the function of BATF2 in macrophages infected with Mtb,BATF2 gene overexpression or knockdown lentivirus vectors were constructed and used to infect THP-1 cells to construct a stable cell lines of BATF2 gene overexpression or knockdown THP-1 cells.CCK-8 and Edu-555 cell proliferation detection kit were used to detect the proliferation of BATF2 gene overexpression or knockdown THP-1 cells,and Annexin V-FITC/PI apoptosis detection kit was used to detect its apoptosis.Immunoblotting was used to detect the expression ofβ-catenin in BATF2 gene overexpression or knockdown THP-1 cell-derived macrophages before and after Mtb infection.The pro-inflammatory factor genes(IL1B,IL6,TNF,i NOS,IL12B,IFNG)and anti-inflammatory factor gene IL10were detected by q PCR.Intracellular CFU assay was used to detect the phagocytosis and early inhibition of macrophages derived from BATF2 gene overexpression or knockdown THP-1 cells against Mtb.The expression of antigen presentation related genes(HLA-A,CIITA,HLA-DRA)were detected by q PCR.BATF2 lacks the complete DNA binding domain and must form a transcription complex with other proteins to regulate downstream target genes.To further explore the specific molecular mechanisms of BATF2,the proteins interacting with BATF2were screened in HEK 293T cells by co-immunoprecipitation(Co-IP)-mass spectrometry(MS),and verified by CO-IP and laser scanning confocal microscope(LSCM).IRF4,which may interact with BATF2,was verified in HEK 293T cells by Co-IP.The target genes regulated by BATF2 were screened by chromatin immunoprecipitation(Ch IP)-next generation sequencing(NGS/seq)technology in HEK 293T cells,and verified by Ch IP-q PCR and dual luciferase reporter gene experiment.Real time q PCR was used to detect whether the change of BATF2expression in THP-1 cells would affect the expression of downstream target genes.Then,the overexpression or knockdown lentivirus targeting the downstream target gene of BATF2 was constructed to infect THP-1 cells.The effects of the expression of downstream target genes of BATF2 on the proliferation and apoptosis in THP-1 cells and the expression ofβ-catenin,pro-/anti-inflammatory factor gene,and phagocytosis,early inhibition,antigen presentation ablity for Mtb of THP-1 cell-derived macrophages were also explored.Results In this study,the stable strain of BATF2 gene overexpression or knockdown THP-1 cells was successfully constructed.BATF2 gene overexpression can significantly reduce the proliferation of THP-1 cells,while its gene knockdown can significantly increase the proliferation;BATF2 gene overexpression can significantly increase the apoptosis levels of THP-1 cells,while its gene knockdown has no significant effect on the apoptosis levels.Overexpression or knockdown of BATF2 gene can significantly reduce or increase the phagocytosis and early intracellular inhibition of THP-1 cells differentiated macrophages to Mtb H37Rv,respectively.Overexpression or knockdown of BATF2 gene can significantly increase or reduce the pro-inflammatory genes(IL1B,IL6,TNFα,IL12B and IFNG)and anti-inflammatory factor gene IL10 of THP-1 cells differentiated macrophages after Mtb infection,respectively.Upon Mtb infection,BATF2 gene overexpression or knockdown can significantly reduce or increase the expression of MHC class II related genes in THP-1cells differentiated macrophages,respectively.Co-IP and LSCM confirmed that BATF2 interacted with IRF4.Through Co-IP-MS,Co-IP and LSCM,SINHCAF interacted with BATF2 was found.The results of Ch IP-seq and Ch IP-q QPR showed that BATF2 could be enriched in the promoters of TTC23,PRKAA2,TTC23 and RAPGEF4.The dual luciferase reporter gene experiment showed that the promoters of LAMC1 and TTC23 genes had the function of driving subsequent gene expression.The results of Ch IP-q QPR showed that IRF4 and SINHCAF could also be enriched in the promoters of LAMC1 and TTC23 genes.The m RNA expression of LAMC1 and TTC23 genes is positively regulated by BATF2.Gene knockdown of LAMC1 could significantly increase the proliferation of THP-1 cells,while gene overexpression or knockdown of TTC23 could significantly reduce or increase the proliferation,respectively;LAMC1 knockdown can significantly reduce the proportion of early apoptotic cells and total apoptotic or dead cells of THP-1 cells.TTC23 overexpression or knockdown can significantly increase or reduce the proportion of those in THP-1 cells,respectively.LAMC1 gene knockdown can significantly reduce the overexpression ofβ-catenin in THP-1 derived macrophages before and after Mtb infection,and overexpression or knockdown of TTC23 can significantly reduce or increase the expression ofβ-catenin,respectively.Overexpression or knockdown of TTC23 can significantly reduce or increase the phagocytosis and early intracellular inhibition of THP-1 derived macrophages for Mtb,while LAMC1 has no effect on those.TTC23 and LAMC1 overexpression or knockdown can significantly increase or reduce the m RNA expression of detected pro-inflammatory factors IL1B,IL6,IFNG and anti-inflammatory factor IL10 in THP-1derived macrophages after Mtb infection.TTC23 overexpression or knockdown can significantly reduce or increase the m RNA expression of MHC-Ⅱrelated genes in THP-1 derived macrophages after Mtb infectionConclusion The highly expressed BATF2 in TB patients may interact with IRF4and SINHCAF in THP-1 cells to regulate LAMC1 and TTC23,followed by cell proliferation inhibition,β-catenin expression inhibition,apoptosis promotion and inflammation promotion,it will also increase the production of anti-inflammatory factor meanwhile.And BATF2 interacting with IRF4 and SINHCAF impairs the phagocytosis,early elimination and antigens presention to CD4~+T cells ability of macrophages response to Mtb infection through TTC23.