The Investigation Into ASAP1 Influenced Macrophages About The Immunity Of Mycobacterium Tuberculosis

Tuberculosis is one of the most commonest infectious diseases threatening human health.Although huge efforts have been made to prevent and control TB in China,and it is still a serious problem in public health.A large-scale GWAS studies has shown that the single nucleotide polymorphism(SNP)of ASAP1 associates with the susceptibility of TB in humans,but there are no experimental reports on the mechanism underlying how SNP of ASAP1 affects humans susceptibility to TB.Clinical investigation shows that active TB patients with particular SNP of ASAP1 have lower ASAP1 expression than healthy volunteers,accordingly,on which we generated a hypothesis: SNP of ASAP1 affects its protein expression,and ASAP1 expression affects immunophysiological functions.This study used THP-1 differentiated macrophages as a cellular model to investigate into the mechanisms on which SNP of ASAP1 affects humans susceptibility to TB and evaluate our hypothesis aforementioned.We carried out serials experiments and tested immunophisological functions of macrophages with different ASAP1 expression levels after mycobacterial infection with Mtb,including test of phagocytosis and killing of bacteria,cytokine expression,autophagy of macrophages and signal pathway analysis.The completion of my master degree thesis gives following major results: 1.ASAP1 involvement in immune response of macrophages to Mtb H37RaTHP-1 cells,a human peripheral blood mononuclear cell line,were differentiated into macrophages by PMA treatment,and the suspended cells became adhesive and macrophage like monolayer cells,their ASAP1 expression significantly increased This suggested that ASAP1 involved in monocyte differentiation.Infection of Mtb H37 Ra up-regulated ASAP1 in macrophages,indicating that ASAP1 participated in immune response of macrophages to Mtb H37 Ra.2.The establishment of ASAP1 overexpression and knockdown macrophagesLentiviral system was employed for generation of ASAP1 overexpression in THP1 cell line.Transfer vector containing ASAP1 cDNA was contracted,pseudolentivion were produced by co-transfection with transfer vector,packaging vector and enveloping vector into 293 T cell lines,and pseudolentivrion was used from transformation into THP1 cell line into ASAP1 stable expression cell line,and then ASAP1 overexpression macrophages were generated by differentiation of lentivial transformed THP1 using PMA treatment.ASAP1 specific siRNA was designed and used to transfect to THP-1 cells,differentiated with PMA simultaneously.Quatitation of ASAP1 expression uisng Western blotting showed the ASAP1 knockdown in macrophages was established.3.ASAP1 influenced phagocytosis of macrophages and killing of Mtb H37RaThree lines of macrophages expression different levels of ASAP1 were infected Mtb H37 Ra for 24 h,then colony forming number of Mtb H37 Ra was counted from lysate of bacterial infected macrophages.Results showed that ASAP1 overexpression macrophages loaded more bacteria,and ASAP1 knockdown macrophages loaded less bacteria in comparison with wild type macrophages which were directly differentiated from THP1.In ASAP1 lower and high expression macrophages,the expression of pro-inflammatory cytokines were decreased and anti-inflammatory cytokines were increased.Prolonging the time of infection,the percentage of killing bacteria was reduced in high ASAP1 expression macrophages.This result suggested that high ASAP1 expression availed bacterial phagocytosis of macrophages,but excessive or extra lover expression of ASAP1 alternated immunophsilogical functions of macrophages.4.ASAP1 influenced the autophagy of macrophages after Mtb infectionAfter Mtb H37 Ra infection of macrophages expressing different levels of ASAP1,Western blotting analysis of LC3 and P62 expression showed that autophagy was significantly inhibited in ASAP1 overexpression macrophages,however autophagy of ASAP1 knockdown macrophages did not have changes.By analysis of AKT and phosphorylated AKT(p-AKT),mTOR and phosphorylation mTOR(p-mTOR)using Western blotting,results indicated that affect of ASAP1 expression on autophagy of macrophages after Mtb infection were manipulated through alternating activation of AKT-mTOR signaling pathway.The studies above have shown that abnormal ASAP1 expression of macrophages significantly affect the cell immunophisological functions against the infection of Mtb.Both excessive and extra low expression of ASAP1 negatively regulate immune responses of macrophages to Mtb infection,thus increasing the susceptibility of humans to TB,and giving experimental evidence and the scientific foundation for our hypothesis: The particular SNP of ASAP1 which associates humans susceptibility to TB correlates lower level of ASAp1 expression,and abnormal expression of ASAP1 negatively affect immunophsiological functions,thus increasing humans susceptibility to TB.