Live Imaging of Monocyte Subsets in Immune Complex-Mediated Glomerulonephritis Reveals Distinct Phenotypes and Effector Functions
Authors: Turner-Stokes Tabitha , Garcia Diaz Ana , Pinheiro Damilola , Prendecki Maria , McAdoo Stephen P. , Roufosse Candice , Cook H. Terence , Pusey Charles D. , Woollard Kevin J. 
Published: August 2020

Significance Statement
Monocytes and macrophages are important in mediating crescentic GN (CrGN), but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. Using nephrotoxic nephritis in the rat as a clinically relevant experimental model of CrGN, we show that this is a predominantly intravascular disease and that glomerular inflammation and damage is driven by dynamic interactions between intravascular blood monocytes and the endothelium. Monocyte subsets had distinct phenotypes and effector functions: non-classical monocytes were recruited to the glomerulus first, and may orchestrate the inflammatory response. Later recruitment of classical monocytes was associated with glomerular damage and proteinuria. Targeting specific monocyte subpopulations may generate less toxic and more effective therapies for patients with GN.

Background
Immune complexes within glomerular capillary walls cause crescentic GN (CrGN). Monocytes and macrophages are important in mediating CrGN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation.

Methods
Live glomerular imaging using confocal microscopy monitored intravascular monocyte subset behavior during nephrotoxic nephritis (NTN) in a novel WKY-hCD68-GFP monocyte/macrophage reporter rat strain. Flow cytometry and qPCR further analyzed ex vivo the glomerular leukocyte infiltrate during NTN.

Results
Non-classical monocytes surveyed the glomerular endothelium via lymphocyte function-associated antigen 1 (LFA-1) in the steady state. During NTN, non-classical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage. Monocytes recruited to the glomerular vasculature did not undergo transendothelial migration. This finding suggests that inflammation in immune complex-mediated CrGN is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelium and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Reduced classical monocyte recruitment in Lewis rats during NTN confirmed a role for CD16 in mediating glomerular damage.

Conclusions
Monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental CrGN resulting from immune complexes formed within the glomerular capillary wall. 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.