Monocytes transition to monocyte‐macrophages within the inflamed vasculature via CCR2 on monocytes and endothelial TNFR2

Abstract

ObjectiveTo understand the cellular pathways that drive the maturation of classical monocytes into macrophages in inflammationHypothesisMonocytes play essential roles in homeostasis, infection and sterile inflammation and can acquire macrophage phenotypes by transitioning through intermediate states. The mechanisms leading to the accumulation of these monocyte intermediates within tissues is not well understood; in the intestine, it has been attributed to a disruption in the differentiation along the monocyte–macrophage continuum under steady state and following inflammation. We hypothesize that monocytes double positive for chemokine receptors, CX3CR1 and CCR2 are preferentially recruited to the glomerulus and transition to immature, inflammatory macrophages while still within the vasculature in a CCR2 dependent manner.MethodsMonocyte recruitment and maturation was evaluated using mouse models of acute/ chronic glomerulonephritis, intravital microscopy of the kidney, cell sorting, multi‐parameter flow cytometry and adoptive transfer approaches. Mechanistic insights into this conversion were provided using in vivo intravascular staining, in vitro monocyte‐endothelial cell co‐cultures and single cell transcriptional profiling. Evidence of the conversion was also evaluated by flow cytometric analysis of blood and urine samples from SLE patients with active nephritis.ResultsWe show that renal Ly6C+ monocytes acquire macrophage markers (MHCII, CD64, F4/80) upon renal inflammation and are distinguished by the expression of both CCR2 and CX3CR1. These cells, which we refer to as immature macrophages, may be derived from a CCR2+CX3CR1+ double‐positive monocytes in blood that are preferentially recruited and retained within glomerular capillaries, and acquire pro‐inflammatory characteristics. Mechanistically, the monocyte acquisition of macrophage markers occurs within the vasculature and relies on CCR2 on hematopoietic lineages and TNFR2 on parenchymal cells, findings that are recapitulated in vitro with monocytes co‐cultured with TNF‐TNFR2 activated endothelial cells, which generate CCR2 ligands. Single‐cell RNA‐sequencing defines a monocyte differentiation path associated with the acquisition of immune effector functions, and monocyte CCR2‐dependent generation of CCR2 ligands. Immature macrophages are detected in the urine of patients with lupus nephritis and their frequency correlates with clinical disease.ConclusionsCCR2 dependent functional specialization of monocytes into immature macrophages occurs within the TNF‐TNFR2 activated vasculature and establishes a chemokine‐based autocrine, feed‐forward loop that may amplify inflammation and renal injury.