Ageing is associated with a progressive decline in immune function that contributes to age-related diseases. Growing evidence suggests that increased reactive oxygen species (ROS) production occurs with age. This can alter a variety of immune functions including T cell function and proliferation. Key antioxidants are involved in removing ROS and maintaining the cellular redox state of the cell such as glutathione (GSH), peroxiredoxin (Prx) and thioredoxin (Trx) systems. The main objective of this study was to look at the effect of oxidative stress to these key systems and to identify the changes to binding partners and Trx1 distribution. Activation of T cells in PBMCs using anti-CD3/CD28 antibodies was undertaken to look at Trx1 distribution and secretion during activation. T cells were treated with non-cytotoxic concentrations of buthionine sulfoximine (BSO; 25 µM, 24 h) and auranofin (ARF; 25 µM, 2 h) followed by isolation of cytosolic and membrane protein fractions and total GSH measure. Protein analysis was carried out using reducing and non-reducing SDS-PAGE and western blot to identify Trx1 monomers and mixed disulphides. Also cells treated with ARF show a trend towards increased GSH levels as compared to BSO-treated cells. A major 12KDa Trx1 band was detected in lysates when treated T cells were analysed under reducing SDS-PAGE irrespective of treatment. In contrast, a number of higher molecular weight Trx1 bands were present in treated and untreated cells with varying intensity after separating on a non-reducing gel suggesting possible binding partners. More intense Trx1 bands were evident in western blots of membrane proteins after ARF treatment and an increase in surface Trx1 was observed using flow cytometry in ARF-treated compared to untreated T cells was observed; the MdX surface Trx1 expression was 551±194 for ARF-treated T cells, but was 128±4 and 142±55 for untreated and BSO-treated T cells respectively. These preliminary data may suggest that the T cell response to oxidative stress conditions shows an increase in total GSH levels in the absence of reduced Trx1 in order to maintain the redox state. Also, an increased expression of Trx1 on the surface of T cells after ARF treatment suggests that Trx1 distributes to the membrane when in oxidised form.
|Publication status||Published - 1 Jul 2016|