Aberrated Shelterin Complex Subunits Expression In T Cell Aging And Inflammation In Behcet's Disease

BackgroundBehcet's Disease(BD)is a chronic recurrent vasculitis with various organs affected and unknown etiology.The imbalanced T cell repertoire plays a critical role in the pathogenesis of BD.Telomeres are key regulators of cell senescence as the length of telomeres and the activity of telomerase are involved in regulating cell aging and inflammation.Shelterin protein complex is important for maintaining telomere stability and pretecting telomere DNA.AimsThis study aims to decribe the expression of shelterin subunits in BD CD4+T cells,and to discuss its influence on telomere,telomerase,cell functions and DNA damage response and repair.Methods1.Peripheral blood samples of 40 treatment-naive BD patients referred to Peking Union Medical College Hospital from November 2017 to November 2019 as well as 40 age-and gender-matched healthy controls(HC)were collected.The clinical data and laboratory examination data of the BD patients were documented as well.2.The frequencies and absolute numbers of BD and HC peripheral naive and memory CD4+T cells were determined by flow cytometry.The frequencies of Annexin V positive cells and CD57 positive cells were measured by flow cytometry as well.3.The TNFa level and IFNy level in the cell cultural supernatant of BD and HC naive CD4+T cells upon TCR stimulation were measured by ELISA.4.The expressions of shelterin subunits of BD and HC naive CD4+T cells were deternmined by RT-PCR and their corelation with ESR,hsCRP,BDCAF2006 and disease course was analyzed as well.5.The length of telomeres of BD and HC naive CD4+T cells was measured by RT-PCR.The activity of BD and HC naive CD4+T cells was checked by PCR-ELISA method.6.The expression of ATM,pATM,Mrell,Rad50,Nbsl,CHK2,pCHK2,H2AX,yH2AX,p53,and pp53 in BD and HC naive CD4+T cells were analyzed by western blot.7.The expression of p21 and GADD45A in BD and HC naive CD4+T cells was determined by RT-PCR.8.TRF2 siRNA was transfected into HC naive CD4+ T cells.The cell apoptosis,expression of CD57 and p53 were analyzed as above.Results1.Both of the frequency and absolute number of BD naive CD4+T cells were lower than that of HC naive CD4+T cells,while both of the frequency and absolute number of BD memory CD4+T cells were higher than that of HC memory CD4+T cells(p<0.05).The frequencies and absolute numbers of both naive and memory CD4+T cells remained unchanged after these BD patients received treatment.2.The frequency of Annexin V+cells in BD naive CD4+T cells were increased compared with that in HC(p<0.05),indicating BD naive CD4+T cells were more prone to be apoptotic than that of HC.The expression of senescence marker CD57 was increased in BD naive CD4+T cells as well(p<0.05).3.Compared with that of HC,the level of proinflammatory cytokine TNFa in the supernatant of BD stimulated naive CD4+T cells was notably increased(p<0.05).The level of IFNy was a little higher in the supernatant of BD stimulated naive CD4+T cells,but had not reached statistic significance.4.In comparison with HC,BD naive CD4+T cells had a lower level of TRF2,TIN2 and RAP1(p<0.05).The expressions of TRF1,TPP1 and POT1 between BD and HC naive CD4+T cells were comparable.Moreover,None of the expressions of TRF2,TIN2 or RAP1 was correlated with ESR,hsCRP,BDACF2006 or disease course.5.The activity of telomerase in BD naive CD4+T cells was significantly lower than that in HC resting naive CD4+T cells(p<0.05).Upon stimulation,telomerase in BD naive CD4+T cells was not able to be upregulated while that in HC could(p<0.05).The length of telomres in BD naive CD4+T cells tend to be shorter than that in HC naive CD4+T cells but without reaching a statistic significance.6.The expressions of ATM,phosphor-ATM,Mre11 and Rad50 in BD naive CD4+ T cells were lower than those in HC.The expressions of Nbs1,CHK2,H2AX and p21 were comparable between BD and HC naive CD4+T cells,while those of pCHK2,yH2AX,p53,phosphor-p53 and GADD45A were increased in BD naive CD4+T cells,indicating an aberrant DNA damage repair response in BD naive CD4+T cells.7.After interfered by TRF2 siRNA,the frequencies of Annexin V+cells in naive CD4+T cells were notably increased.The levels of TNFa and IFNy were also elevated in the supernatant of TRF2-knocked down cells.The expression of p53 was upregulated in TRF2 deficient cells as well.ConclusionsThe expression of shelterin protein complex and the telomerase activity were impaired in BD naive CD4+T cells,which aberrantly activated CHK2,H2AX,p53 and GADD45A,promoting T cell senescence and apotosis,leading to inflammatory damage.The insufficiency of ATM and MRN complex also contributed to the accumulation of DNA damage in BD T cells,accelerating cell apoptosis.Knockdown of TRF2 by siRNA further confirmed the role of shelterin aberration in BD pathogenesis.