Comparison Of The Role Of Autophagy In Anti-tuberculosis Immunity In Macrophages Derived From HiPS And THP-1

Tuberculosis(TB)is still a globally crucial public health issue today.Mycobacterium tuberculosis(MTB)is the causative agent of tuberculosis,and the interaction between MTB and its host macrophages(Macrophages,M?)plays an important role in the pathogenesis and development of tuberculosis.The macrophages used in the current research are mostly tumor cell lines or peripheral blood mononuclear cells(PBMC)-derived macrophages,however,there are problems such as loss of function due to changes in genetic structure or difficulty in cell acquisition.The emergence of induced pluripotent stem cells(iPS)and directed differentiation technology provides a pathway for obtaining macrophages in vitro and to explore macrophage anti-tuberculosis immunity mechanism.Studies have confirmed that autophagy plays a significant role in the interaction of MTB with M?,but the specific mechanism has not been determined past years.To investigate autophagy in anti-tuberculosis immunity similarities and differences between human induced pluripotent stem cells(hiPS)-derived macrophages(hiPS-M?)and THP-1-derived macrophages(THP-1-M?),in this work,we first induced the differentiation of hiPS cells into macrophages(hiPS-M?)in vitro by using Embryonic body(EB)differentiation induction method,then the obtained macrophage-like cells were identified for morphological and biological functions.The results confirmed that hiPS-M? had the characteristics unique to macrophages,including Giemsa staining for purple,phagocytic ink capacity,this indicated that biologically functioning macrophages could be obtained by the EB induction method.On this basis,extracting the total RNA of hiPS-M? and THP-1-M? cells infected with Bacillus calmette-Guerin(BCG),the attenuated strain of Mycobacterium tuberculosis,and analysis of differences in gene expression by high-throughput sequencing.As the result showed that a conspicuous difference occurred in the expression of 832 genes after hiPS-M? infected with BCG compared with hiPS-M?(P<0.05),including 572 genes observably upregulated and 260 genes observably downregulated;compared with THP-1-M?,there were significant differences in the expression of 397 genes after THP-1-M? infected with BCG(P<0.05),including 211 genes that were significantly up-regulated and 186 genes were significantly down-regulated.The differentially expressed gene KEGG enrichment analysis found that after BCG infected hiPS-M? and THP-1-M?,there existed different degrees of enrichment in tuberculosis,Toll-like receptors,TNF,PI3K-AKT,phagosomes,nodular receptors,NF-kappaB,mTOR,MAPK,IL-17,cytokine-cytokine receptor interaction,chemokines and both the autophagy and apoptotic pathways.The submicroscopic structure of hiPS-M? and THP-1-M? cells after infected with BCG was observed by Scanning Electron Microscope(SEM),Transmission Electron Microscope(TEM)and laser confocal microscopy,it was found that after hiPS-M? and THP-1-M? infected with BCG,the cell surface was shrunk and lysed.At the same time,the number of intracellular autophagosomes increased.Infecting cells with an adenovirus expressing the mCherry-GFP-LC3B fusion protein,and autophagosomes increased after hiPS-M? and THP-1-Mcp infected with BCG under laser confocal microscopy.Finally,it was confirmed by qPCR and Western Blot that compared with the autophagy inhibitor 6-amino-3-methylindole(3-MA),hiPS-M? and THP-1-Mcp infected with BCG significantly increased the expression of autophagy-related gene LC3B(P<0.01),while inhibiting the expression of autophagy-related genes GABARAPL2 and P62(P<0.01).It was also confirmed that the expression of autophagy-related genes was regulated by PI3K/Akt/mTOR signaling pathway after infected with BCG,which promoted the occurrence of autophagy.However,there were some differences in the expression of autophagy-related genes induced after infected with BCG in macrophages from different sources.This showed a significant up-regulation of DDIT4 and EXOC8 in hiPS-M?(P<0.01),while RAB7B and CTSD in THP-1-M? were significantly down-regulated(P<0.01),it was indicated that there might exist differences in the regulation mechanism of BCG-induced autophagy by macrophages from different sources.In conclusion,this work successfully obtained hiPS-derived macrophages by EB differentiation method.Through transcriptome sequencing analysis,qPCR and WB verification,and cell submicroscopic structure observation,it was found that after hiPS-M? and THP-1-Mcp infected with BCG,it promoted autophagy by regulating autophagy-related gene expression.However,there are some differences in the mechanism by which BCG infected macrophages from different sources to induce autophagy.This results provided a new idea for further study of the role of autophagy in the anti-tuberculosis immune mechanism of macrophages.