The objectives of the proposed integrated programme of work are: Aim 1 will investigate the mechanisms responsible for endothelial cell killing by Ly6Clow monocytes. We will analyse in vivo using intravital microscopy and electron microscopy the role of membrane dependent necroptosis, reactive oxygen species, NETs formation, IFNAR, and platelets, in Ly6Clow monocyte retention inside capillaries and TLR7 dependent necrosis of endothelial cells. Aim 2 will test the hypothesis that NR4A1 controls the differentiation and homeostasis of murine and human Ly6Clow/CD14 dim monocytes. We will examine the role of NR4A1 in a conditional hematopoietic Nr4a1 deficient model, and perform RNA-seq and ChIP-seq analysis to analyse and compare the target genes of Nr4a1 in mice and human monocytes. Aim 3 will test the hypothesis that Ly6Clow monocytes control endothelial damage in glomerulonephritis due to immune complex deposition and in a TLR7/8 sensitive tumor model. We will examine the role of Ly6Clow monocytes and TLR signalling in glomerulonephritis following injection of an antibody against the glomerular basement membrane (GBM) which generates a glomerular inflammatory response via immune complex formation in the glomerulus and in the vascularisation of B16F10 tumors. Aim 1 and 2 will be started in parallel at the start of the project. They address at the cellular and molecular levels the mechanisms that control the differentiation and functions of ly6C-low monocytes and their human CD14dim counterparts. The experimental systems, technological approaches and mouse lines we will be using are in place in the laboratory, and we have assembled a team of outstanding collaborators with whom we have established collaborations. Aim 3 will be started in year 3, to best incorporate results from the previous aims. This aim will investigate the roles of ly6C-low monocytes in pathological conditions such as glomerulonephritis and tumor growth, and the mechanisms involved