Single cell RNA-seq in regenerative and fibrotic biomaterial environments defines new macrophage subsets

Macrophages play diverse roles in the immune response to infection, cancer, and wound healing where they respond to local environmental signals, yet identification and phenotypic characterization of functional subsets in vivo remains limited. We performed single cell RNA sequencing analysis on differentiated macrophages sorted from a biologic matrix-induced regenerative environment versus a synthetic biomaterial foreign body response (FBR), characterized by T H 2/interleukin (IL)-4 and T H 17/IL-17, respectively. In the regenerative environment, unbiased clustering and pseudo time analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle. In the FBR environment, we identified a CD9 hi+ IL-36γ + macrophage subset that expressed T H 17-associated molecules characteristic of certain auto-immune responses that were virtually absent in mice lacking the IL-17 receptor. Surface marker combinations including CD9 and CD301b defined macrophage fibrotic and regenerative subsets enabling functional assessment and identification in human tissue. Application of the terminal macrophage subsets to train the SingleCellNet algorithm and comparison to human and mouse macrophages in tumor, lung, and liver suggest broad relevance of macrophage classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide new targets for potential therapeutic modulation of certain pathologic states and tissue repair.