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

Abstract

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 TH2/interleukin (IL)-4 and TH17/IL-17, respectively. In the regenerative environment, unbiased clustering and pseudotime 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-36 + macrophage subset that expressed TH17-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.

Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages

“…For example, polarization with IL4 and IL13, which we used in this study, is considered to promote an M2a phenotype, while polarization with IL10 is considered to promote an M2c phenotype characterized by distinct surface markers like CD163 and increased secretion of numerous matrix-degrading enzymes like matrix metalloprotease (MMP)-7 and -8 [69], suggesting distinct effects on ECM remodeling. Furthermore, transcriptomic studies of macrophages isolated from the in vivo environment suggest that they are difficult to characterize as precisely as macrophage phenotypes studied in vitro [9,19,70]. Therefore, it appears likely that the choice of particular M2 markers used to characterize macrophages strongly affects interpretation, and that large panels of markers should be used to facilitate comparison between studies.…”

“…While the field continues to evolve, the current state of knowledge is that M1 macrophages initiate healing while M2 macrophages promote healing resolution (for review, see [7,8]). In addition, recent studies employing single cell transcriptomic profiling have shown that macrophages often exist as hybrid phenotypes in vivo, simultaneously expressing markers of both archetypal M1 and M2 phenotypes [9]. Because macrophages switch from M1 to M2 during the course of normal wound healing, the generation of a hybrid M1/M2 phenotype in between these phases is likely.…”

“…On the other hand, the potent M2-promoting cytokines IL4 and IL13 as well as M2-secreted factors like transforming growth factor-b (TGFb), platelet-derived growth factor-BB (PDGFBB), CCL17, and CCL22 are also hallmarks of fibrosis [14][15][16]. Other studies that analyzed macrophages participating in fibrous encapsulation of biomaterials have reported co-expression of both M1 and M2 markers [9,[17][18][19]. Also, while the M2-promoting cytokine IL4 stimulates the fusion of macrophages into multinucleated foreign body giant cells [20], several recent reports have shown that controlled release of IL4 from biomaterials promoted a significant reduction in fibrous capsule formation around implanted biomaterials [21,22].…”

Classical Dichotomy of Macrophages and Alternative Activation Models Proposed with Technological Progress

“…This emphasizes the need for researchers to develop new tools to isolate and explore this population [ 69 ]. Sommerfeld et al [ 70 ] used scRNA-seq to describe the relationship between macrophages isolated from mouse tissue repair models and tissue environmental fibrosis after the use of model biomaterials. They used an unbiased clustering algorithm to reveal the diversity of macrophages, calculated and analyzed the phenotypic characteristics of macrophage clusters, defined phenotypic and functional macrophage populations, identified macrophage surface markers by flow cytometry and immunofluorescence techniques, and identified new CD9hi+IL-36 γ + macrophage populations.…”

The role of macrophages in osteoarthritis and cartilage repair

“…Here, we review recent key findings of the origin of tissue macrophages and how these cells modulate anabolic and catabolic responses in OA, with a particular focus on the constituents of the joint organ system. Furthermore, recent advances in next-generation sequencing technologies and transcriptomic analysis have greatly facilitated our understanding of phenotypic identity and heterogeneity of the macrophage populations 4,5 . Thus, we also summarize the potential contributions of previously unrecognized macrophage subpopulations to OA pathogenesis.…”