The Effects And Mechanisms Of Aire Expressing In DC On Differentiation And Tolerance Of CD4~+T Cell

Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)also known as Autoimmune Polyendocrinopathy Syndrome type1(APS-1) isresulted from Autoimmune Regulator (Aire) mutation or dysfunction. Patients withAPECED may develop Addison’s disease, hypoparathyroidism, and diabetes et al. Inaddition to these autoimmune diseases, APECED patients are also susceptible toCandida albicans infections due to immunodeficiency, but the underliningmechanism is largely unknown. Aire is highly expressed in medullary thymicepithelial cells mTECs, where it regulate tissue restrictive antigen (TRA) expressionto prevent autoimmunity, it could induce central immune tolerance through deletingauto-reactive T cells or peripheral immune tolerance through inducing regulatory Tcells (Tregs). Aire is also reported to be expressed in monocytes, macrophages anddendritic cells (DCs) in the peripheral lymphoid tissues, although its functions in theperipheral immune system is still unclear, they were reported to be different from itwas in central immune system. The research reported function of Aire seems to bedissimilar with its role in thymus. These differences may explain the highsusceptibility to C albicans infection in APECED patients, and further studies couldimprove the understanding of the functions and mechanisms of Aire in immunesystem.It was reported that Aire could regulate TRAs expression in DCs，and reducedexpression of Aire in peripheral organs could exacerbate APECED relatedautoimmune diseases. Moreover, it was also reported that DCs and macrophagesfrom Aire knockout (KO) mice had irregular antigen presenting capability, and thiscould affect immune response against auto-or foreign-antigens. These suggestedthat Aire in peripheral DC cells may play an important role in the immune toleranceand response.CD4+T lymphocyte (also known as T helper (Th) cells) play a crucial role in regulating the immune response and in maintaining peripheral tolerance. NaiveCD4+T cells may differentiate into distinct subsets, including Th1, Th2, Th9, Th17,Th22and Tregs, after receiving different stimulation signals and cytokine signals.And DCs have the ability to affect CD4+T cell activation and polarization. Therefore,we hypothesized that Aire expressed in DCs may play a key role in the immuneresponse and peripheral tolerance by affecting CD4+T cell subsets.The clearance and induced tolerance of auto-reactive CD4+T cells play animportant role in maintaining immune tolerance and prevention of the autoimmunediseases. In the thymus, T cells develop, mature and form a polyclonal T cellrepertoire capable of identifying large numbers of exogenous pathogens. During thisprocess, T cell clones that recognize self-antigens are inevitably generated. In orderto maintain self-tolerance of a normal body, T-cell clones that reactive withself-antigens need to be depleted.Thymic negative selection mediated by Aire is themain mechanism underlying the elimination of autoreactive T cells in the centralimmune system.However, a portion of the autoreactive T cells may escape negativeselection and enter into the periphery. To prevent the occurrence of autoimmunediseases, further elimination of the autoreactive T cells in the periphery is necessary.Studies shown that peripheral splenic and lymph node stromal cells and dendriticcells (DCs) play an important role in the clearance of autoreactive T cells escapedthymic negative selection and entered the periphery. But the mechanism is unclear.Based on these, we hypothesize that Aire expressing DCs may participate inperipheral tolerance and immune response by affectting differentiation of CD4+T cellsubsets, and inducing auto-reactive CD4+T cell tolerance or eliminate in peripheral.In order to understand the effects and functions of AIRE expressing DCs onCD4+T cells in peripheral immune system, tolerance and differentiation of CD4+Tcells were studied.Part I: The functions and mechanisms of Aire-overexpressing dendritic cells onCD4+T cell subsets.1. Aire-overexpressing DC2.4cells polarize native CD4+T cell to Th1and Th17 subsetsTo examine the effects of Aire cells on the CD4+T cell subsets, we co-culture theAire cells and control cells with CD4+T cells from splenocytes using Transwellinserts48h. the mRNA expression levels of the master regulators of the differentCD4+T cell subsets were first detected by RT-qPCR. Foxp3and the cytokines fromdifferent CD4+T cell subsets were detected by FACS. The results showed that T-betand IFN-γ，which were the markers of Th1cells, and RORγT and IL-17A, whichwere the markers of Th17cells, were up-regulated in CD4+T cells co-cultured withAire cells compared with the control cells. These results suggest that Aire plays apositive role in inducing Th1and Th17development from na ve CD4+T cells.2. Aire-overexpressing DC2.4cells induce Th1and Th17via up-regulating IL-12,IL-6, and TGF-βexpressionTo investigate the mechanism by which Aire cells induce Th1and Th17development, the mRNA and protein expression levels of IL-12, IL-6and TGF-β inAire cells were detected by RT-qPCR and ELISA, respectively. As shown in results,IL-12, IL-6, and TGF-β mRNA protein levels were higher in Aire cells than in thecontrol cells. Additionally, IL-12, IL-6and TGF-β antibody was used to block thesupernatants of the Aire cells prior to the addition of the CD4+T cells in Transwellinserts. No differences in the expression levels of these cytokines observed forIFN-γ-expressing CD4+T cells compared Aire cells with control cells when IL-12was blocked. Additionally, no differences were observed for IL-17A-expressingCD4+T cells after anti-IL-6antibody, anti-TGF-βa ntibody or their combination wasused. These data demonstrated that Aire cells induce Th1and Th17differentiation byenhancing IL-12, IL-6and TGF-β secretion.3. Aire up-regulates the expression of IL-12, IL-6and TGF-β through MAPKsignalingTo explore the signal transduction pathway by which Aire affects IL-12, IL-6,and TGF-βexpression in DCs, the activities of ERK and p38, which belong to thefamily of MAPKs related to the production of IL-12, IL-6, and TGF-β, were detected by FACS. The results showed that the phosphorylation levels of ERK and p38weresignificantly increased in Aire cells, as expected. These data provided an explanationfor Aire up-regulating IL-12, IL-6and TGF-β by enhance ERK and P38signaling inDCs.Part II: The funtions and mechanisms of Aire-overexpressing dendritic cells onCD4+T cell tolerance in peripheral immune system1. The effect of Aire on tolerance related molecules in DC2.4cellsTo investigate whether Aire maintains the immature state of DCs and inducesimmune tolerance by affecting the expression of molecules related to tolerance onDCs, FACS was conducted to examine the expression of cell surface molecules，andexpression of T1D-related TRAs in DCs were detected by RT-qPCR.The resultsshowed that stimulation of the two groups of cells with10μg/ml of LPS for48hresulted in elevated expression of the control surface molecules. However, theexpression of CD40, CD80, CD83, CD86and CD11c was significantly lower in Airecells compared to those in the control group; the mRNA levels of GAD65/67、Ins2、IA-2、IGRP were significantly elevated in Aire cells compared those in to controlcells. This suggest Aire regulating moleculars related tolerance in DCs, maintainingimmune tolerance.2. The effect of Aire-overexpressing DC2.4cells on the tolerance status of CD4+TcellsTo investigate the effect of Aire-overexpressing DC2.4cells on CD4+T celltolerance, splenocytes derived from mice with STZ-induced T1D (STZ-T1D) andwild type (WT) mice were co-cultured with either Aire and control cells for48h,then the apoptosis,fuction of CD4+T cells and molecules related to tolerance mRNAexpression of splenocyte were detected. The results showed that the apoptotic rate ofSTZ-T1D mouse-derived CD4+T cells was higher in the Aire group compared to thatin the control group,and STZ-T1D mouse and WT mouse-derived splenocytes fromthe Aire group had significantly reduced numbers of CD4+IFNγ+T cells compared tothe splenocytes from the control group. In addition, the expression levels of Lag3 and FR4were significantly higher in STZ-T1D mouse-derived and WTmouse-derived splenocytes from the Aire group compared with those from thecontrol group. These results indicated that Aire-transfected DCs induced tolerance ofperipheral CD4+T cells.3. The mechanism by which Aire-overexpressing DC2.4cells down-regulate thefunction of CD4+T cellsTo further investigate whether Aire cells reduce IFN-γ production in CD4+Tcells derived from STZ-T1D mice by affecting TCR signaling pathways.FACS wasperformed to examine the key molecules of the TCR signaling pathways in CD4+Tcells, including Ca2+and p-ERK. The results show that after stimulation with CD3antibody and Insulin, the Ca2+level and p-ERK expression were lower in STZ-T1Dand WT mouse-derived CD4+T cells from the Aire group compared with those fromthe Control group. Moreover, no significant differences were detected in the Ca2+level and p-ERK expression between the Aire and the control group after ConAstimulation,which as positive control. In summary, Aire-transfected DCs reduced theproduction of Ca2+and p-ERK in CD4+T cells, thereby inhibiting TCR signalingpathways and inducing tolerance in STZ-T1D mouse-derived CD4+T cells.4. Aire-overexpressing DCs induce type1diabetes (T1D) in mice via CD4+T cell.We speculated whether Aire-induced CD4+T cells would inhibit or delay theoccurrence of T1D in STZ-induced mice. In order to verify this theory, WTmouse-derived splenocytes were co-cultured with either Aire or control cells andthen intravenously injected into WT mice through the tail vein. Subsequently, themice were induced with STZ to establish a model of T1D. The results showed thatSTZ induction resulted in the development of T1D symptoms in mice. However, theblood sugar level was significantly lower in mice transplanted with splenocytes ofthe Aire group in comparison to the mice transplanted with splenocytes belonging tothe Control group,and the degree of inflammatory infiltration was significantly lowerin the pancreatic tissues of mice transplanted with splenocytes from the Aire groupcompared to mice transplanted with splenocytes from the control group. In summary, Aire cell-induced splenocytes delayed the occurrence of T1D in STZ-induced mice.Conclusions:1. Aire up-regulating IL-12, IL-6and TGF-β by enhance ERK and P38signaling inDCs, which results in inducing Th1and Th17development from naive CD4+T cells.2. Aire-overexpressing DCs induce autoreactive CD4+T cells functionalinactivation and apoptosis. which maintain the peripheral CD4+T cell tolerancethrough this two kinds of mechanism combination.In summary, DCs expressed Aire induce the polarization of Th1and Th17subsets through up-regulating the production of relevant cytokines. On the otherhand, it induce auto-reactive CD4+T cells undergo apoptosis or functionalinactivation by up-regulating tolerance related molecular in DCs. So Aire couldmaintain peripheral tolerance while prevent pathogen infection via these twodistinguish effects on CD4+T cells.