Role Of Vitamin D-Induced Autophagy In Macrophage Against Mycobacterium Tuberculosis

Objective:The interaction of macrophages (macrophage, Mφ) and Mycobacterium tuberculosis(Mtb) affects the development of Tuberculosis(TB) in the body.Improving the bactericidal capacity of Mφ agaist Mtb become an important strategy to treat TB and reduce the incidence. Vitamin D play a key regulatory role for induction of autophagy in Mcp and maturation of autophagy lysosomes by binding to the Vitamin D receptor(VDR). This study was undertaken to investigate the role of vitamin D-induced autophagy in Mcp against Mtb.Methods:Induce U937 cells to differentiate into macrophages with phagocytic capacity by phorbol 12-myristate 13-acetate(PMA).The model of macrophages infected with Mtb in vitro was established by exposing the cells to Mtb virulent strains of standard human type H37Rv.These cells were treated with four different treatments:negative control(10%RPMI1640 for 24 hours), vitamin D[100pM 1,25 (OH) 2D3 for 24 hours], autophagy inhibitor plus Vitamin D[10μM 3-methyl adenine(3-MA) plus 100pM 1,25 (OH) 2D3 for 24 hours], positive control (lOnM rapamycin for 18 hours).Infect all these cells with H37Rv at infection ratios(Mtb:target cell) of 10:1 for 4 hours. In the 4th day after infection, Cytotoxicity of the different chemicals at the concentrations used in this experiment was tested by the trypan blue dye exclusion assay.The mRNA expressions of autophagy-related genes(Atg) Atg5, Beclin-1 and the human cathelicidin microbial peptide LL-37,microtubule-associated protein 1 light chain3(LC3) B were determined by semi-quantitative RT-PCR.The protein expressions of LL-37、LC3B-Ⅱ were detected by western blotting.The numbers of colony forming units (cfu) of Mtb in macrophages were counted in the 1 st day and in the 4th day after infection separately.In the 4th day after infection, LC3B-Ⅱ+- and/or Mycobacterium tuberculosis antigen 85A+(Ag85A+)-cells were counted by flow cytometry.Results:None of the chemicals at the concentrations used in this experiment was found to be cytotoxic (viability was>96%).In the 4th day after infection, Semi-quantitative RT-PCR showed that 1,25 (OH) 2D3 enhanced the mRNA expressions of Atg5,Beclin-1,LL-37 and LC3B (P<0.01).Western blotting showed that 1,25 (OH) 2D3 increased the protein expressions of LL-37 (P<0.01) and LC3B-Ⅱ(P<0.05).The colony forming units(cfu) number of Mtb in macrophages treated with 1,25 (OH) 2D3 decreased markedly in the 4th day after infection (P<0.01) but not in the 1st day after infection (P>0.05).In the 4th day after infection, Flow cytometry indicated that the numbers of LC3B-Ⅱ+-cells increased, the numbers of Ag85A+-cells decreased and the numbers of LC3B-Ⅱ+-Ag85A--cells increased(vitamin D 65.1% vs negative control 2.22%) in the presence of 1,25 (OH) 2D3-While in the presence of 1,25 (OH) 2D3 plus autophagy inhibitor 3-MA,the mRNA expressions of Atg5,Beclin-1 and LC3B were suppressed, the mRNA expressions of LL-37 were reduced, the protein expressions of LL-37 and LC3B-Ⅱ were decreased. And 3-MA inhibited the decrease of the cfu number of Mtb in macrophages in the 4th day after infection with inhibition of LC3B-Ⅱ+-Ag85A"-cells increase as well.Conclusion:Vitamin D can induce macrophage autophagy and further contribute to scavenging Mycobacterium tuberculosis.