Investigating the role of T cell and B cell DNA release

Neutrophils, T cells and B cells are all critical cells of the immune system which participate in the vicious cycle of inflammation in ANCA associated vasculitis a rare autoimmune kidney disease with sup-optimal treatment options.. A common feature between all 3 of these cells is that they use reactive oxygen species (ROS) produced by the power house of the cell known as mitochondria to produce their anti-bacterial fighting capabilities. Overactivated cells produce excess ROS which initiates a unique form of cell death that results in large webs of DNA being released covered in injurious molecules that cause direct damage to the blood vessels. In neutrophils this is known as NETosis and is associated with the worst outcomes in patients with AAV. T cells and B cells have also been reported to release webs of DNA under inflammatory conditions in other forms of autoimmune diseases. It is not yet known if this same phenomenon occurs in AAV. Inhibiting ROS in all 3 cell types prevents the formation of these DNA webs. The unique feature of SkQ1 is that at low concentrations it only removes excess ROS to prevent extrusion of DNA without depleting the required ROS for the cell to perform its infection fighting role. The primary aim of this study is to study the release of pro inflammatory DNA from B and T cells and determine what effect this has on the immune system and perpetuating inflammation in autoimmune kidney diseases. This project will involve the use of high-end super resolution microscopy to tease out the role of different pathways that case the release of DNA webs from T and B cells. Primarily animal models of vasculitis will be used and human blood cells. Other techniques instrumental to this project will be the use of FACS, image analysis using (IMARIS and Image J), supervised computer learning for image analysis, and basic laboratory techniques such as immunohistochemistry, ELISA, western blots, cell culture and basic histology