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Document Type
Graduate Research
Creation Date
Spring 4-8-2015
Department
Biology
Abstract
SLE is gender biased autoimmune disease (9:1 female to male). The immune system is a complex biological system that fights with foreign antigens including bacteria and viruses. The immune system works by producing antibodies against antigens. In SLE, the immune system produces antibodies against its own tissues. These antibodies are known as auto-antibodies. The female sex steroid, estradiol, alters T cell signaling pathways in SLE T cells and contributes to SLE onset and progression. Estradiol inappropriately regulates genes involved in T cell signaling pathways only in SLE T cells and not in control T cells. This suggest that the changes in the gene expression patterns in SLE T cells could lead to impaired peripheral tolerance in women and SLE onset. Peripheral tolerance is immunological tolerance developed after T and B cells mature and enter the periphery. Dead end homolog (Dnd1), an RNA-binding protein, inhibits miRNA by binding to target mRNAs. DND1 targets, such as p27 –Kip1, contribute to the maintenance of peripheral tolerance. Microarray analysis suggested estradiol suppresses DND1 expression in SLE T cells but no in normal T cells. The present study measured DND1 protein in freshly isolated normal T cells and tested the effect of estradiol (10-8M) on DND1 expression. T cells were purified from normal blood samples (n = 16) by Histopaque gradient and negative selection. Proteins were extracted and DND1 immunoprecipitated. The immunoprecipitates were size fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were reacted with DND1 antibody. The protein amount was determined in triplicate using chemiluminesence. T cells from women <35 years contained significantly higher amounts (p = 0.37, Pearson’s rho test, resulting R value was used to calculate significance p-value) of DND1 compared with T cells from donors >35 years. DND1 was 63% higher in younger age group than older age donors. DND1 increased in response to estradiol with maximum expression (3.5-fold) at 3 h post estradiol stimulation. p27 expression was measured by the real-time (Step-one, Applied Bio systems) polymerase chain reaction (PCR). p27 expression was suppressed (p = 27, two tail T test was used to calculate the significance P value) in SLE T cells (n=12) compared with normal T cells (n=7). Together, the results confirm the microarray data indicating that estradiol does not suppress DND1 nor p27 mRNA expression in normal T cells. p27 has an established role in the development/maintenance of tolerance via complex regulation of CD4+ T cell proliferation and effector function. Future experiments will test the hypothesis that the inappropriate suppression of Dnd1 allows miRNA inhibition of p27 (Kip 1) and thereby alters CD4+ T cell effector subtypes in SLE. Changes in CD4+ T cells subtypes lead to a reduction in suppressor function and impaired peripheral tolerance. Also look at the aspect in detail, weather the estradiol effect is age dependent.
