The Role And Mechanism Of AIRE In DC Regulating Th17/treg Balance

AIRE,as a key regulatory factor for central and peripheral immune tolerance,is widely expressed in the thymus,spleen and lymph nodes,so that it can induce and maintain the immune tolerance.AIRE in the central immune system maintains tolerance mainly by adjusting the TSAs expression on the mTECs,and then participates in negative selection.In periphery,there are many other ways for AIRE to participate in tolerance,for instance,affecting APC's presentation ability and maturity status,affecting the tolerance and differentiation of T cells in addition to regulating the TSAs expression by the eTACs,DC and so on.The role of AIRE in periphery is considered to be complementary to the central role,and the function in periphery are more diverse;however,the mechanism of the role of AIRE in periphery still needs deeper study to figure out.DC is the best kind of APC with the ability to present antigen,and its function has been studied more deeply with the clarity of its classification.DC,while presenting antigen,can give T cells different stimuli based on microenvironment,actives T cells and then affects T cell's effect.T helper cells can link innate immunity with adaptive immunity.Receiving antigens from APC,Th cells can transmit the stimulation information to the adaptive immune system,then make corresponding immune responses.DC plays an important role in inducing the differentiation of various Th cell subsets and their response process,such as regulating the balance of Th17/Treg.Th17 cells are mainly involved in resisting exogenous infection,triggering appropriate inflammatory response.Treg cells,as immunosuppressive cells,control the normal level of inflammation in the physiological state to avoid excessive inflammation which may cause disorder.Th17/Treg cells not only coordinate with each other in the process of immune response,but also have a plastic balance state in the process of cell differentiation,and it changes according to the microenvironment.Under normal physiological condition,the differentiation of Th17/Treg are both induced by TGF-?,and there is a balance between the differentiation of the two by FoxP3 suppression of ROR?t.But in the inflammatory microenvironment such as IL-6/IL-21,FoxP3 suppression of ROR?t is inhibited,Th17/Treg balance was broken and towards to Th17.The imbalance of Th17/Treg can lead to the occurrence of a variety of diseases,such as chronic inflammation,autoimmune diseases,tumors and so on.At present,some treatments for regulating Th17/Treg balance have been applied in clinic.In addition,the previous study of the group found that the BMDC with transduced AIRE can affect the balance state of Th17/Treg in the spleen of NOD mice,inhibiting Th17 and promoting Treg differentiation.Thus,we wonder that whether AIRE can affect the activity and function of DC,and then affect the balance state of Th17/Treg,and ultimately maintain immune toleranceBased on the above background,this topic is proposed in the co-culture system in vitro,using AIRE^-/--BMDC and WT-BMDC,to explore whether AIRE can affect Th17/Treg balance through DC,and how to influence Th17/Treg balance through DC.The aim is to provide a new breakthrough point for treatments for Th17/Treg balance.The specific research contents are as follows:1.Observe the effect of AIRE in DC on Th17/Treg differentiationFirst,the cells were obtained from the bone marrow of AIRE^-/-mice and wild mice,and BMDC was induced by GM-CSF and IL-4,then stimulated with LPS.Aand na?ve CD4⁺T cells were obtained from the spleen cells of C57 mice selected by magnetic bead separation,then the BMDC and na?ve CD4⁺T cells were co-cultured for 72h.The differentiated T cells were then collected and detected by RT-qPCR and FACS from RNA levels and cell levels.The results showed that the FoxP3 expression level of AIRE^-/--BMDC group was lower than that of WT-BMDC group,and the expression level of ROR?t was higher.The FACS results showed that under LPS stimulation,the Th17 proportion of AIRE^-/--BMDC group was significantly higher than that of WT group,while the proportion of Treg was also increased,but the increasing degree was lower than that of WT group,and the ratio of Th17/Treg was significantly higher than that of WT group.The results suggest that in the DC and T cell co-culture system in vitro,AIRE can inhibit the differentiation of Th17 while promote Treg,causing the Th17/Treg balance tends to Th17 direction.2.Detect effect of AIRE on Th17/Treg differentiation related molecules in DCIt is known that DC can affect the activation of T cells through a variety of molecules,including cellular surface immunomodulatory molecules such as CTLA-4,PD-L1,CD40;secretory cytokines such as IL-12 and IL-23;and intracellular immunomodulatory enzymes such as IDO and RA.Among them,researchers found that IL-6,IL-1?and IL-23 in the pathological process of disease can promote Th17differentiation,while TGF-?,IL-10,IDO1,Aldh1?2,PD-L1,PD-L2,Integrin?v,Integrin?8 plays an important role in inducing Treg differentiation.Therefore,to make clear whether AIRE can affect the expression of the above molecules in DC to participate in the differentiation of Th17 and Treg,we use RT-qPCR to detect the expression of the above molecules in AIRE^-/--BMDC and WT-BMDC before and after LPS stimulation.The results showed that both in normal state and LPS stimulation,the mRNA expression of IDO1 and Aldh1?2 in AIRE^-/--BMDC decreased and the mRNA expression of IL-23 increased compared with WT-BMDC.To confirm whether the protein level of these three molecules also changed as same as the mRNA level,we tested by ELISA and found that IL-23 showed the same trend of change as mRNA expression,but there was no significant difference in protein levels of IDO and Aldh1?2 between AIRE^-/--BMDC and WT-BMDC groups.It suggested that AIRE can reduce the expression of IL-23 in DC.In order to confirm whether AIRE affects Th17 differentiation and Th17/Treg balance by regulating the expression of IL-23 in DC,we add anti-IL-23 to the co-culture system of BMDC and T cells,neutralize IL-23 secreted by DC,and then detect the differentiation of Th17 and Treg cells by RT-qPCR and FACS.The results showed that after adding anti-IL-23,the mRNA expression of ROR?t decreased and the Th17/Treg ratio obviously decreased;compared with NC group,AIRE^-/--BMDC group after LPS stimulation and anti-IL-23 neutralization,Th17/Treg is lower and is statistically significant.It indicates that AIRE may inhibit the differentiation of na?ve CD4⁺T cells to Th17 by suppressing the secretion of IL-23 by BMDC,so that the Th17/Treg differentiation balance is tilted to Treg.3.Explore on the molecular mechanism of AIRE regulating IL-23 in DCThe above studies have confirmed that in DC,AIRE can regulate the transcription and expression of IL-23,and then promote the balance of Th17/Treg in the direction of Treg.However,it is not clear how AIRE regulates IL-23,by directly affecting its transcription process or indirectly affecting IL-23 expression through other molecules,which still needs to be explored.It has been reported that LPS can be combined with TLR4 on the DC to activate the intracellular NF-?B pathway and promote the production of a variety of cytokines,including IL-23.Therefore,in order to explore whether the AIRE can regulate the production of IL-23 by affecting the related signal molecules in NF-?B pathway in DC,we detect the expression of related molecules in the NF-?B pathway through the Western Blot.The results showed that after LPS stimulation,the expression of p-I?B?and p-p65 in AIRE^-/--BMDC group was higher than that of WT-BMDC group,which indicated that AIRE might directly affect the change of key molecules in NF-?B pathway,and then affect the expression of IL-23.In summary,this study confirmed that AIRE can promote the differentiation of Th17/Treg towards to Treg through DC in vitro.And this function is mediated by AIRE inhibition of IL-23 expression in DC.In addition,AIRE can affect the signaling molecules in NF-?B pathway which mediates the production of IL-23.This study provides a new breakthrough point and possibilities for disease treatment solutions by regulating Th17/Treg balance.