Study Of Cytokines On The Autophagy In THP-1Derived Macrophages Infected By BCG

Objective:To analyze the possible factors involved in the balance of Th1/Th2andTreg/Th17of the Mycobacterium tuberculosis (MTB) infected patients, we comparedthe levels of different T lymphocyte subsets related cytokines in the serum of subjectsunder the different states of MTB infection. Moreover, we explored the role of TGF-βon the autophagy in macrophages infected by Bacillus Calmette-Guerin (BCG),which may provide preliminary experiment basement for the further study on themolecular mechanisms of TGF-β participating in regulatory of autophagy.Methods:1. Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokine levelsof MTB infected subject sera in the four groups, healthy controls, latent infectionof MTB, tuberculosis(TB) patients and patients of completing course, as well ascorrelation analysis.2. BCG strain was identified using16S rRNA-based phylogeny.3. THP-1derived macrophages were transfected with pcDNA3.1-YFP-LC3plasmidby liposome transfection.4. LC3dots assessed by fluorescence microscopy to monitor autophagy.5. RT-qPCR were used to detect the mRNA level of LC3、Atg5、Atg7、Beclin1.6. Colony-forming unit assay were adopted to calculate survival rate of BCG.Results:1. Compared with the controls, the levels of IFN-γ,IL-4,TGF-β in the serum of TBpatients and patients of completing course group decreased significantly (p＜0.05). Comparatively, the level of IL-17in the latent infection of MTB groupelevated markedly (p＜0.05). While the level of IL-17in TB patients groupsignificantly increased compared with the latent infection (p＜0.05).2. The PCR products of16s rRNA fragment were sequenced and compared with the NCBI-BLAST database by nucleic acid sequences, which confirmed the BCGstain belonging to Mycobacterium tuberculosis complex.3. Obvious fluorescence in the THP-1derived macrophages transfected byYFP-LC3plasmid was observed by fluorescence microscope, suggesting thatLC3plasmid had been transfected into the cells successfully. Compared with thecontrols, LC3dots increasesd by fluorescence microscopy in the treatment groupof Earle’s banlanced salt solution (EBSS)，as well as the mRNA level of LC3,Atg5, Atg7, Beclin1were rised by reverse transcription-polymerase chain reaction(RT-PCR) in macrophages significantly (p＜0.05), which suggested theautophagy cell model induced by EBSS was constructed successfully.4. The mRNA levels of LC3, Atg7, Beclin1were notably increased (p＜0.01) byRT-PCR in macrophages infected by BCG after the treatment of EBSS addingTGF-β, indicating that autophagic flux increased.5. Compared with negative control group, the survival rate of BCG reduced inmacrophages infected by BCG after the treatment of EBSS.Conclusion:1. The protective immunity against MTB in human would be reduced with the lowlevel of IFN-γ， which will lead to the infection with Mycobacteriumtuberculosis.In addition, the lower level of IFN-γ made it more prone to sufferfrom tuberculosis.2. In human immune response against MTB, the immunue imbalances existed on thecytokines in Th1/Th2and Treg/Th17.3. TGF-β may improve the level of autophagy in the macrophages infected by BCGand inhibit the survive of BCG in macrophages through the possible molecularmechanisms such as Beclin1, Atg7, LC3.