Endoplasmic Reticulum Stress-related Gene Expression In Autoimmune Diseases

Systemic lupus erythematosus(SLE), Rheumatoid athritis(RA) and Ankylosingspondylitis(AS) are classified as complicated systemic autoimmune diseases and theirexact pathogenesis remains uncertain. Recently it was reported that in the differentiationfrom B cell to plasma cell, the development and survival of plasma cell relied on theintact unfolded protein response(UPR) and autoimmune diseases are characterised bythe overactivation of B cell and the inappropriate production of auto-antibodies.Endoplasmic reticulum(ER) stress could induce cell apoptosis and the formation ofapoptotic bodies, while the source of auto-antigens is nucleosome, a component ofapoptotic bodies. Therefore, ER stress might be involved in the regulation ofautoimmune diseases. In order to testify the hypothesis, UPR-related gene expressionlevels were investigated in patients and animal models; RNA interference method wasused to determine the critical factor in the UPR pathway which related to autoimmunediseases. The susceptibility to ER stress was compared between T and B lymphocytes tomake sure the main resource of autoantibodies. To screen out the genes beneficial to thediagnosis in SLE and RA, the gene expression levels of UPR-related genes weredetected from SLE and RA patients. These studies help us to understand the role ofnon-immunologic mechanisms involved in the regulatory of autoimmune diseases, andoffer new insights for the pathogenic study and targets for clinical therapeutics of autoimmune disease pathogenesis.OBJECTIVE:The purpose of this research is to evaluate the role of ER stress in the autoimmunediseases by detecting UPR-related genes expression in peripheral blood white cells fromSLE, RA and AS patients, and to determine the key molecules of the UPR pathwaywhich participate in the disease modulation, and to compare the susceptibility to ERstress between T and B lymphocytes to identify the resource of autoantibodies in SLE tomake a better understanding of the pathogenesis of autoimmune diseases.METHODS:In order to cofirm the relationship between autoimmune diseases and ER stress, realtime qPCR method was applied to detect ER stress-associated genes in SLE, RA and ASpatients. The siRNA delivery technology was performed to ensure the key factors ofUPR pathway involved in autoimmune diseases. ConA and LPS were used to activateT/B lymphocytes and tunicamycin was used to induce ER stress in mice and comparethe susceptibility to ER stress between T and B lymphocytes by using RT-PCRtechnique.RESULTS:1. Clinical samples collectionAfter informed consent,65SLE patients,63RA patients and26AS patients whofulfilled the American Rheumatism Association criteria for the diagnosis of SLE, RA,AS and69normal unrelated blood donors were enrolled in this study. Peripheral bloodwhite cells were isolated from fresh whole blood in anticoagulation tubes for later use incultivation and RNA exaction.2. Establishment of quantitative real time PCR method using SYBR Greenâ… DNA fragments of ER stress-associated genes were amplified and inserted into pMD19-T vector, respectively. All the constructs were determined by enzymatic digestionand confirmed by sequencing, followed by using as the standards for qPCR. The qPCRmethod we established with a detectable limitation at104copies per reaction. There was a significantly linear correlation (r2=0.9919) between the threshold Ct and the templateDNA concentration ranged from106to1013copies.3. Gene expression of UPR pathway members in SLE, RA and AS peripheral bloodwhite cellsUsing real time PCR method, ATF6, XBP1and MANF expression were detected to beupregulated in SLE patients, but CHOP, PERK and IRE1were downregulated. Besides,MANF expression was dramastically upregulated in RA patients, but ATF6and XBP1were downregulated. In AS patients, all the UPR-related genes were downregulatedexcept MANF, which was sharply increased. This indicated that MANF might beinvolved in the regulation of autoimmune diseases, ATF6and XBP1could be used todiscriminate RA from SLE.4. ATF6knockdown in EL4cellsRNAi method was used to knockdown the ATF6transcription in EL4cells.24hoursafter transfection, the total RNA was isolated with Trizol reagent to reverse transcribeinto cDNA.The RT-PCR results showed that one of three siRNAs was effective in theinhibition of the mRNA level. Therefore, the effective siRNA for ATF6was chosen forthe subsequent experiments. After the knockdown of ATF6with specific siRNA, therewere significant changes when the cells were subjected by ER stress stimulate.5. Activated T, B cell could induce ER stressActivated T, B lymphocytes with ConA and LPS respectively, were treated withtunicamycin to induce ER stress. BIP, ATF6, IRE1and PERK expression levels in Tcells were lower than B cells, but CHOP and XBP1expression levels were opposite.This result showed the response to ER stress in B cells was higher than T cells, andthere might be ER stress functional disorder in T cells.CONCLUSIONS:In this study, a quantitative real-time PCR method with SYBR Green â… was establishedand used to detect the mRNA expression levels of ER stress-associated genes in a large number of human peripheral blood samples. The response to ER stress in B cells washigher than T cells and ER stress might participate in the regulation on SLE and RA.