The Role Of A20 In The Pathogenesis Of Behcet's Disease And Vogt-koyanagi-harada Disease

Uveitis is an intraocular inflammation that often leads to visual handicap.The most frequent uveitis entities leading to blindness in China are Behcet's disease(BD)and Vogt Koyanagi Harada(VKH)disease.BD is a chronic systemic inflammatory disease that involves recurrent uveitis,genital ulcers,oral aphthae,and skin lesions.VKH disease is a systemic autoimmune disease that affects the eyes,skin,and central nervous system.The characteristics of the disease include bilateral granulomatous panuveitis,poliosis,vitiligo,alopecia,central nervous system symptoms,and hearing loss.Uveitis is often treated with corticosteroids and immunosuppressive drugs,which have serious side effects and elucidating the mechanisms involved in the inflammatory response may hopefully lead to the development of more specific drugs.Genetic studies from our group recently identified the association of certain gene variants of TNFAIP3 with both VKH and BD,although the exact mechanisms were not clear.The study reported here was therefore devised to further examine the role of this protein in the development of both BD and VKH.TNFAIP3,also known as A20 was discovered as a protein that was readily induced following the stimulation of endothelial cells with tumor necrosis factor,although later studies showed that it can also be induced by many other stimuli.A20 is expressed in several immune organs,including thymus,spleen and gut associated immune tissue.A20 is now known as a protein that can block NF-?B activation thereby markedly dampening the inflammatory response.A20 mediates this effect via a deubiquination of proteins involved in the NF-?B signaling cascades such as RIP1.Further studies have found that A20 also plays an important role in restricting TLR induced ubiquitination of TNF receptor–associated factor(TRAF)6.In recent years,A20 has shown to be involved in the regulation of DC activation,maturation and function,and intense research has addressed its role in immune mediated diseases.Studies have shown that mice lacking A20 are able to spontaneously activate DCs and T cells in vivo.A20fl/flCd11c-Cre mice can spontaneously develop sero-negative ankylosing arthritis,lymphocyte-dependent colitis and enthesitis.The lack of A20 in DCs increases the ability of the cell to resist apoptosis,which may lead to an uncontrolled inflammatory response.Since the role of A20,except for the genetic studies mentioned above,has not been studied during intraocular inflammation,we decided to investigate the levels of this protein in the blood of patients with uveitis,whereby we chose BD and VKH because these are two of the most frequent uveitis entities observed in our clinic.To elucidate the possible mechanism of action of A20,we focused on the role of this protein in the functioning of DCs.PART ? THE EXPRESSION OF A20 MRNA IN PATIENTS WITH BD AND VKH DISEASEPurposeA20 has been found to play a significant regulatory role in the innateimmune response.This study was set up to investigate the mRNA expression of A20 in patients with BD and VKH disease.MethodsVKH disease patients and BD patients and age-matched and sex-matched healthy individuals were included in the present study.The diagnosis of BD and VKH disease followed the criteria of the International Study Group and international committee on nomenclature.PBMCs in VKH patients(including patients with active and inactive disease),BD patients(including patients with active and inactive disease)and healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation.Total RNA of the PBMCs was extracted using Trizol reagent.A Reverse Transcription Kit was used to reverse RNA into cDNA according to the manufacturer instructions.Real-time quantitative PCR was performed to detect the expression of A20 in PBMCs.Results1.Quantitative real time-PCR showed that the mRNA expression of A20 in PBMCs was significantly lower in patients with active BD as compared to inactive BD patients and normal subjects.The relative mRNA level of A20 in PBMCs from many active BD patients was very low,but still detectable.2.No difference was observed between inactive BD patients and normal subjects.3.No difference was observed between active VKH patients,inactive VKH patients or control individuals.Conclusionswe found that the mRNA expression of A20 in PBMCs was significantly lower in patients with active BD as compared to inactive BD patients and normal subjects.The relative mRNA level of A20 in PBMCs from many active BD patients was very low,but still detectable.The reduction of A20 is closely related to the occurrence and development of BD.No difference was observed between active VKH patients,inactive VKH patients or control individuals.The results showed that the decrease of A20 was not associated with the occurrence of VKH disease.PART ? THE EXPRESSION OF A20 MRNA IN DCS FROM ACTIVE BD PATIENTSPURPOSEThis study was set up to investigate the expression of A20 mRNA in DCs from active BD patients.MethodsBD patients and age-matched and sex-matched healthy individuals were included in the present study.The diagnosis of BD followed the criteria of the International Study Group and international committee on nomenclature.PBMCs in BD patients(including patients with active and inactive disease)and healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation.CD14~+monocytes were isolated using magnetic cell sorting(MACS),following the manufacturer's instructions.Immature DCs(iDCs)were cultured in Roswell Park Memorial Institute1640(RPMI 1640)medium supplemented with recombinant human granulocyte-macrophage colony-stimulating hormone(GM-CSF)and recombinant human IL-4 for 6 days.Total RNA of the DCs was extracted using Trizol reagent.A Reverse Transcription Kit was used to reverse RNA into cDNA according to the manufacturer instructions.Real-time quantitative PCR was performed to detect the expression of A20 in DCs.Results1.The expression of A20 in DCs from active BD patients was significantly lower than that seen in patients with inactive BD.2.The expression of A20 in DCs from active BD patients was significantly lower than that seen in normal subjects.3.No difference was observed between inactive BD patients and normal subjects.Conclusionswe found that the expression of A20 in DCs from active BD patients was significantly lower than that seen in patients with inactive BD or normal subjects.We have now clearly known that there is a close connection between A20 and DCs.PART ? THE EFFECT OF A20 ON THE FUNCTION OF DCSPurposeIn the present study,we investigated effect of A20 on maturation of DCs and cytokine expression.MethodsBD patients and age-matched and sex-matched healthy individuals were included in the present study.The diagnosis of BD followed the criteria of the International Study Group and international committee on nomenclature.PBMCs in BD patients(including patients with active and inactive disease)and healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation.CD14~+monocytes were isolated using magnetic cell sorting(MACS),following the manufacturer's instructions.Immature DCs(iDCs)were cultured in Roswell Park Memorial Institute1640(RPMI 1640)medium supplemented with recombinant human granulocyte-macrophage colony-stimulating hormone(GM-CSF)and recombinant human IL-4.On day 3 of incubation,iDCs were transduced with A20-shRNA at 50,100,300,500,or 1000 multiplicities of infections(MOIs)for 72h at 37°C.The transduction efficiency of DCs was evaluated by observing green fluorescent protein(GFP)-positive cells under a fluorescence microscope and Flow Cytometry.The most suitable MOI value was selected according to the result of transduction efficiency.Human CD83,CD86,CD40,CD80 and HLA-DR antibodies were used to detect the influence of A20 on DCs surface marker expression using a Flow Cytometer.ELISA development kits(R&D Systems,Minneapolis,MN)were used to measure supernatant levels of IL-1?,IL-6,IL-10,IL-17 and IL-27 based on the manufacturer's protocols.Results1.The expression of GFP was observed under the fluorescence microscope.Most of the DCs showed a green signal under the fluorescence microscope at an MOI of 500.2.The transduction efficiency of adenovirus was detected by flow cytometry.We found that the transduction efficiency of adenovirus was approximately 80%in DCs in vitro at an MOI of 500.3.The data indicated that the transduction of A20-shRNA had no effect on the expression of the cell surface markers CD40,CD80,CD83,CD86 and HLA-DR.4.The secretion of IL-1?and IL-6 was increased,whereas the expression of IL-10 and IL-27 was decreased following silencing of A20.Collectively,these data suggest that A20 negatively regulates inflammatory cytokine expression in DCs.ConclusionsAdenovirus can effectively transfect DCs,and the transfection efficiency is high.Compared with the control,the expression level of A20decreased significantly at 48 hours after transduction with A20-sh RNA.Transduction of A20-shRNA had no effect on the expression of the cell surface markers CD40,CD80,CD83,CD86 and HLA-DR.The secretion of IL-1?and IL-6 was increased,whereas the expression of IL-10 and IL-27was decreased following silencing of A20.Collectively,these data suggest that A20 negatively regulates inflammatory cytokine expression in DCs.It is suggested that A20 may play a role in the pathogenesis of Behcet disease by affecting the inflammatory cytokines secreted by DCs.Part ? The effect of A20 on the function of CD4~+T cellPurposeTo investigate whether silencing of the A20 gene in DCs affects the percentage of IL-17 expressing CD4~+T cells.MethodsBD patients and age-matched and sex-matched healthy individuals were included in the present study.The diagnosis of BD followed the criteria of the International Study Group and international committee on nomenclature.PBMCs in BD patients(including patients with active and inactive disease)and healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation.CD14~+monocytes were isolated using magnetic cell sorting(MACS),following the manufacturer's instructions.Immature DCs(iDCs)were cultured in Roswell Park Memorial Institute1640(RPMI 1640)medium supplemented with recombinant human granulocyte-macrophage colony-stimulating hormone(GM-CSF)and recombinant human IL-4.On day 3 of incubation,iDCs were transduced with A20-shRNA at 500 multiplicities of infections(MOIs)for 72h at 37°C.LPS(100ng/ml)was added for 24h to stimulate the maturation of DCs.DCs and purified CD4~+T cells were mixed at a ratio of 1:4 and cultured for7 days.After fixation,Anti-human IL-17A was added to the cells to analyze the proliferation of IL-17 using a FACScan Flow Cytometer.ResultsIn the T cell/DC coculture system,A20-shRNA transducted DCs resulted in an increased level of IL-17 expressing CD4~+T cells.Conclusionswe cocultured transducted DCs with CD4~+T cells,and showed that silencing A20 gene expression in DCs significantly increased the population of IL-17 positive CD4~+T cells.These findings are in agreement with our earlier studies showing an important role of Th17 cells in the pathogenesis of BD.We thus hypothesize that a decreased A20 expression during an active episode of disease would be associated with an increase in Th17 cells.Part?The effect of A20 on MAPK signaling pathwayPurposeIt is not exactly known how A20 affects DC signaling cascades.In this study,we investigated the effect of A20 on MAPK signaling pathway.MethodsBD patients and age-matched and sex-matched healthy individuals were included in the present study.PBMCs in BD patients(including patients with active and inactive disease)and healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation.CD14~+monocytes were isolated using magnetic cell sorting(MACS),following the manufacturer's instructions.On day 3 of incubation,iDCs were transduced with A20-shRNA at 500 multiplicities of infections(MOIs)for 72h at 37°C.DCs were transducted with adenovirus for 48 hours,whereafter cells were stimulated with LPS(100ng/ml)for 30min.Phosphorylation levels of JNK,ERK1/2 and p38 were then detected by flow cytometry.ResultsSilencing of A20 lead to an increased phosphorylation of p38 and JNK,whereas ERK1/2 phosphorylation was not affected.ConclusionsSilencing of A20 caused an increased JNK and p38 activation,but did not affect ERK1/2.This is similar to the results of other studies.The present results showed that the effect of A20 on the function of DCs was involved in the pathogenesis of BD through JNK and p38 dependent pathway.