The Effects Of Autoimmune Regulator On The Phenotype And Function Of DC2.4Cells

The autoimmune regulator (Aire) is a transcription factor. Studies have shownthat Aire played an essential role in maintaining immune tolerance. Aire genemutations can lead to APECED (Autoimmune ployendocrinopathy candidiasisectodermal dystrophy, APECED), also called the APSI (Autoimmune ployglandualrsymndrom type I, APS-I), which characterize with multiple organs diseases, A lot ofautoantibodies against self organs are produced in the APECED patients, resulting inmultiple organ autoimmune diseases. Aire is expressed at high levels in thymus, andmainly expressed in thymic medullary epithelial cell (mTEC), lesser in dendritic cells(DC). In periphery, Aire is mainly expressed in lymph nodes and spleen, and CD14+monocytes, dendritic cells and extrathymic Aire-expressing cells (eTACs) are theprimary cells for Aire expression.Study confirmed that central Aire could regulate the expression of TRAs inmTECs to remove the autoreactive T cells and maintain the central immune tolerance.But the function of Aire expression in periphery is not clear. In periphery, Aire ismainly expressed in DC. DC as a kind of heterogeneous cell population can bebroadly classified into two groups: mature and immature DC, which characterize withdifferent phenotypes, such as mature DC expresses CD83specifically, as well as highlevel of MHC class II molecules, CD80and CD86et al. In contrast, immature DC canexpress the relative low levels of MHC class II molecules and costimulatorymolecules. But some ligands of pattern recognition receptors, such asPolyinosinic:polycytidylic acid[Poly(I:C)] as Toll-like receptor3(TLR3) ligand、Curdlan as C type lectins Dectin-1ligand and lipopolysaccharide (LPS) as Toll-likereceptor4(TLR4) ligand treatment can promote DC maturation. The functions of two types of DC are also different, such as the immature DC has strong phagocytosis andmature DC no longer has. Immature DC can induce immune tolerance, but there arereports that some mature DC can also participate in the induction of immune tolerance.In addition, peripheral DC can also express various TRAs to induce removal ofself-reactive T cells to maintain immune tolerance. Our previous research confirmedthat Aire expressing in macrophages induced Treg generation by promoting thesecretion of TGF-, thus play a role in maintaining peripheral immune tolerance. Butthe function and significance of Aire expressing in DC are not clear.Based on the above background, we suppose that Aire may affect the phenotypeand maturation of DC, and or regulate the expression of TRAs to participate inmaintaining peripheral immune tolerance. Therefore, this study was performed fromthe following aspects:1. Identification of DC2.4cell line stably expressing AireIn order to determine whether Aire were expressed stably in DC2.4cell lines transfected with pEGFPC1/Aire (A1-1) or pEGFPC1(C1-1-1) plasmids, which previously constructed and kept in liquid nitrogen by our lab. The expression of Aire and GFP in A1-1and C1-1-1cells were examined by flow cytome-try, immunofluorescence and RT-PCR, respectively. These results showed thatAire and GFP were respectively expressed in A1-1and C1-1-1cells at high level. which can be used in subsequent experiments.2. Effect of Aire on the phenotype of DC2.4cellsIn order to study whether Aire can effect on the phenotype of DC2.4cells, DC2.4cells, A1-1and C1-1-1cells were treated with LPS, Poly(I:C), Curdlan or Poly(I:C)combination with Curdlan, the expression of CD40, CD80, CD83, CD86, CD11c andMHC class II molecule on three kinds of cells were examined by flow cytometry. Theresults showed that the expression of CD40, CD80, CD83, CD86, CD11c, MHC-II inA1-1cells reduced comparison with C1-1-1cells after LPS, Poly(I:C) or Curdlanstimulation. But their expressions were similar in A1-1and C1-1-1cells after Poly(I:C) combination with Curdlan stimulation. These results showed that Aire could keepDC2.4cells at immature stage after LPS, Poly(I:C), Curdlan stimulation.3. Effect of Aire on the proliferation of DC2.4cellsIn order to define whether Aire can affect DC2.4cells’ proliferation, theproliferation of DC2.4, C1-1-1and A1-1cells were detected using the Cell CountingKit-8(CCK-8Kit). The results showed that the proliferation of three groups cells wasno significant difference. It indicated that Aire did not affect the proliferation abilityof DC2.4cells.4. Effect of Aire on phagocytosis of DC2.4cellsIn order to define whether Aire affect the phagocytosis DC2.4cells, the intensityand percentage of phagotrophic fluorescent microspheres in DC2.4, C1-1-1and A1-1cells were tested by flow cytometry. The results showed that the enhancement ofphagocytosis ability in A1-1cells compared with C1-1-1cells. And after LPSstimulation, the phagocytosis ability of A1-1cells was still higher than its in C1-1-1cells. These above results indicated that Aire can enhance the phagocytosis of DC2.4cells, which help to keep their immaturation.5. Effect of Aire on TRAs expression of DC2.4cellsIn order to further understand whether Aire can regulate the expression of TRAsin peripheral DC to participate in peripheral immune tolerance, the expressions ofmany kinds of TRAs on the DC2.4, C1-1-1and A1-1cells were detected by RT-PCRand RT-qPCR. The results showed that GAD65/67(Glutamate decarboxylase67/65)、IA-2(Insulinoma associated protein2)、IGRP (Islet-specific glucose-6phosphataserelated protein) as type I diabetes-related autoantigens; Nalp5(Pyrin domaincontaining5) as hypothyroidism autoantigen; Rbp3(Retinol-binding protein3) asuveitis autoantigen; Spt2(Salivary protein2) as Sj gren’ autoantigen; GR NMDA2C(Glutamate receptor NMD2C) as systemmic lupus erythematosis (SLE) autoantigen;Mup1(major urinary protein-1) as Graves disease’s autoantigen; Mog(Mylin-oligodendrocyte glycoprotein) and Plp1(Proteolipid protein (myelin)1) as EAE and multiple sclerosis autoantigen were up-regulated in A1-1cells comparedwith the C1-1-1cells. The above results showed that Aire may regulate the expressionof TRAs in DC2.4cells to maintain peripheral immune tolerance.From the studies of above, we made the following conclusions:1. Aire can regulate the expression of surface molecules on DC2.4cells.2. There is no effect of Aire on proliferation ability of DC2.4cells.3. Aire can enhance the phagocytosis ability of DC2.4cells.4. Aire can enhance part of TRAs expressions in DC2.4cells.This study reveals that the effects of Aire on phenotype and phagocytosis abilityof DC which are possibly benefit to keep its immature state. As well as by regulatingthe expression of TRAs on peripheral DC, Aire may participate in maintainingperipheral immune tolerance. This study will be better to reveal the functions andsignificances of Aire expressing in peripheral immune system, and further to providenew means by using the Aire gene as targets, to induce (transplantation rejection orhypersensitivity) or break down (chronic infection disease or tumor) the peripheralimmune tolerance for diseases’ precaution or treatment.