Live imaging of monocyte subsets in immune complex-mediated glomerulonephritis reveals distinct phenotypes and effector functions

Abstract

Crescentic glomerulonephritis (GN) caused by deposition of immune complexes within the glomerular capillary wall is a common cause of rapidly progressive glomerulonephritis and an important cause of end-stage renal failure. Current treatment with generalised immunosuppression is suboptimal in terms of efficacy, and is associated with significant side effects and long-term toxicity. Monocytes and macrophages are important in mediating crescentic GN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. I developed a live glomerular imaging model using confocal microscopy in order to monitor intravascular monocyte subset behaviour during nephrotoxic nephritis (NTN) in a novel WKYhCD68- GFP monocyte/macrophage reporter rat strain. Additionally, I used flow cytometry and gene expression analysis to analyse ex vivo the glomerular leukocyte infiltrate during NTN. Using live glomerular imaging, I showed that non-classical monocytes surveyed the glomerular endothelium via the b2 integrin LFA-1 in the steady state. During NTN, nonclassical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage and the onset of proteinuria. Importantly, monocytes recruited to the glomerular vasculature during NTN did not undergo transendothelial migration. This finding suggests that inflammation in crescentic GN caused by immune complex formation within the glomerulus is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelial cells and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Lewis rats, that are inherently resistant to experimental GN, demonstrated reduced recruitment of classical monocytes during NTN, suggesting a role for CD16 in mediating glomerular damage. In conclusion, my data suggest that monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental crescentic GN caused by immune complex deposition within the glomerulus. LFA-1 dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria.