Study On The Influence Of Aire On The Expression Of Perforin In DC2.4

AIRE is widely expressed in spleen,thymus and lymph nodes,and plays an important role in central and peripheral tolerance.AIRE can affect the apoptosis and maturation of thymic epithelial cells and central tolerance by affecting the negative selection of T cells.AIRE is also expressed in tissues and organs outside the thymus,such as lymph node stromal cells and dendritic cells.It has been shown that transcripts encoding AIRE have been found in both human and mouse dendritic cell subsets in vitro.At present,the role of Aire in peripheral is not clear.It is generally believed that its role in peripheral is only a supplement in the center,and its specific mechanism needs further study.Some studies have found that AIRE expressed on dendritic cells can affect peripheral tolerance by affecting DC maturity,antigen presenting ability and tissue-specific antigen transcription.In addition,our previous study also found that DC cells expressing Aire can induce T cell apoptosis,but the mechanism is not clear.Recently,a group of perforin positive dendritic cell subsets was named perf DC because of its perforin expression characteristics.Perf DC plays an important role in many autoimmune diseases.These cells can clear CD4+T and CD8+T cells by releasing perforin and granzyme A.At the same time,they can participate in immune tolerance.It is found that the mechanism of perforin release depends on TLR7/8activation and TREM-1 binding.Ran orgad,bar nathansohn Levi et al.found that the mechanism of perf DC scavenging CD4+T cells is MHC independent way,which is realized by NO.When perf DC was treated with NO inhibitor,the scavenging effect was inhibited,which confirmed the previous conclusion.The mechanism of perf DC scavenging CD8+T cells is MHC dependent.It has the following characteristics:(1)it depends on TLR7/8 activation and TREM-1 binding;(2)perf DC releases granzyme A and perforin to act on CD8+T cells,and this effect depends on the contact between cells.Therefore,we speculate whether AIRE can regulate the expression of perforin in DC and induce peripheral tolerance.Therefore,we first used DC2.4 cells overexpressing AIRE to observe the effect of AIRE on the expression of perforin by TLR7/8 ligand stimulation,and then explored the possible mechanism of its regulation by detecting the changes of downstream molecules of TLR7/8 signaling pathway.Finally,we studied the effect of AIRE on inducing CD8+T cells by regulating perforin expression and co-culturing with CD8+T cells to explore the peripheral tolerance of AIRE.The possible mechanism of action may provide the basis for understanding the pathogenesis of some autoimmune diseases caused by the lack of AIRE,and provide new ideas for the treatment of autoimmune diseases with AIRE as the target.The specific research contents are as follows:I.To observe the effect of Aire on the expression of perforin in DC2.4 cells1.Determination of stimulation conditions of R848(TLR7/8 agonist)In order to study the best condition of inducing DC 2.4 cells to produce perforin using stimulator R8484,we first selected TLR7/8 activator R848,and explored the effect of R848 with different concentration and stimulation time on perforin expression of DC 2.4 cells.RT-q PCR was used to detect the expression of perforin in DC2.4 cells.The results showed that when the concentration of R848 was 1mg/ml and the stimulation time was 12 h,the expression of perforin in DC2.4 was the highest,while when the concentration of R848 was higher than 3mg/ml,the expression of perforin in DC2.4 cells did not increase but decreased,which may be due to the toxic reaction caused by the high concentration of stimulant.So,we choose 1mg/ml R848 and 12 hours as the best stimulation condition.2.Observe the change of perforin expression in DC2.4 cells overexpressing AIREIn order to further study the effect of AIRE on the expression of perforin in DC2.4 cells,R848 was used to stimulate A1-1(DC2.4 cells transfected with AIRE)and C1-1-1(DC2.4 cells transfected with GFP)respectively,and RT-q PCR and FACS were used to detect the expression of perforin to observe the effect of AIRE on the expression of perforin in DC2.4 cells.The results showed that the expression of perforin increased in DC2.4 cells overexpressing AIRE compare to that of control cells.It is suggested that AIRE can promote the expression of perforin in DC2.4cells.II.Mechanism of the effect of AIRE on the expression of perforin in DC2.4 cellsIn order to further explore the possible mechanism of AIRE’s influence on perforin expression in DC2.4 cells,whether it is through the influence on the related molecules involved in TLR7/8 pathway.The expression of My D88,IRAK4,TRAF6 and IRF5 in TLR7/8 was detected by RT-q PCR and Western blot.The result of RT-q PCR showed that AIRE could promote the expression of My D88,IRAK4,TRAF6,TRAF1 and IRF5 in TLR7/8 pathway,while Western blot showed that AIRE could promote the expression of My D88,IRAK4,TRAF6 and IRF5 in TLR7/8 pathway.The results showed that the mechanism of AIRE regulating perforin expression might be through the regulation of related molecules in TLR7/8signaling pathway.III.To study the effect of AIRE on apoptosis of CD8+T cells induced by perforin expression in DC2.4 cells1.The effect of DC2.4 cells overexpressing AIRE on CD8+T cell apoptosisIn order to study the effect of DC2.4 cells overexpressing AIRE on CD8+T cell apoptosis,we first selected CD8+T cells by magnetic beads,then co-cultured CD8+T cells with A1-1 and c1-1-1 cells respectively,and then detected CD8+T cell apoptosis by FACS using cell apoptosis kit.The results showed that DC2.4 cells overexpressing AIRE had the most apoptosis on CD8+T cells compared with the control group,and the number of CD8+T cells was further increased after R848 stimulation,which indicated that the activation of AIRE and TLR7/8 pathway could promote CD8+T cell apoptosis.2.AIRE induces CD8+T cell apoptosis by regulating perforinThe above study shows that AIRE can promote the expression of perforin,and DC2.4 cells overexpressing AIRE can significantly enhance the apoptosis of CD8+T cells.Therefore,in order to determine whether AIRE can regulate the expression of perforin and then induce the apoptosis of CD8+T cells.We further observed the effect of DC2.4 cells overexpressing AIRE on CD8+T cell apoptosis by silencing perforin and co-culturing with CD8+T cells.(1)Establishment and identification of perforin silenced DC2.4 cellsIn order to establish perforin silenced DC2.4 cells,DC2.4 cells were transfected by perforin si RNA provided by the manufacturer.First,the transfection efficiency was observed by transfection of fluorescent NC.It was found that when 1.5 m L micro RNA and 2.5mL transfection reagent were mixed,the transfection efficiency was the highest.The expression of perforin in DC2.4 cells was detected by RT-q PCR,The result showed that perforin expression was significantly decreased after silence.This indicated that the perforin silenced DC2.4 cells were obtaineduild successfully.(2)The effect of overexpression of AIRE on apoptosis of CD8+T cellsIn order to further clarify that AIRE can induce CD8+T cell apoptosis by regulating the expression of perforin in DC2.4 cells,A1-1 cells and C1-1-1 cells silencing perforin were co-cultured with CD8+T cells and compared with the control group.The results showed that both A1-1 cells and C1-1-1 cells induced CD8+T cell apoptosis significantly decreased after perforin was silenced.At the same time,the ability of A1-1 cells to induce CD8+T cell apoptosis is still slightly stronger than C1-1-1 cell,which indicates that AIRE can induce CD8+T cell apoptosis to a certain extent by regulating perforin expression,but there are some other mechanisms to be studied besides the way of inducing the increase of perforin.In conclusion,this study shows that AIRE can regulate the expression of perforin in DC2.4 cells and promote the activation of TLR7/8 signaling pathway,and finally induce the apoptosis of CD8+T cells by regulating perforin expression,which may play an important role in maintaining peripheral tolerance.This study provides a new explanation for understanding the role and mechanism of peripheral AIRE expression,and also provides a new treatment for autoimmune diseases.