Research Article

Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation

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Science  25 Jan 2019:
Vol. 363, Issue 6425, eaat7554
DOI: 10.1126/science.aat7554

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A myeloid cell atlas of neuroinflammation

Myeloid cells, such as dendritic cells and macrophages, in the central nervous system (CNS) play critical roles in the initiation and exacerbation of multiple sclerosis (MS). Jordão et al. combined high-throughput single-cell RNA sequencing and intravital microscopy to compile a transcriptional atlas of myeloid subsets in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Microglia and other CNS-associated macrophages expanded and transformed into various context-dependent subtypes during EAE. Furthermore, dendritic cells and monocyte-derived cells, but not resident macrophages, played a critical role by presenting antigen to pathogenic T cells. This exhaustive characterization may inform future therapeutic targeting strategies in MS.

Science, this issue p. eaat7554

Structured Abstract


Under homeostasis, the central nervous system (CNS) hosts microglia (MG) and CNS-associated macrophages (CAMs). During experimental autoimmune encephalomyelitis (EAE), myeloid complexity drastically increases, with dendritic cells (DCs) and monocytes seeding the CNS. However, which disease-specific populations can be found during neuroinflammation remains largely unknown.


An important step for the initiation of EAE and multiple sclerosis (MS) is the infiltration of the CNS by encephalitogenic T cells, which potentially become reactivated by encountering their self-cognate antigens presented at the brain interfaces. Myeloid cells have been shown to play a critical role in antigen presentation. Consequently, their transcriptomic profile and dynamics during neuroinflammation are crucial for understanding neuroinflammatory pathology.


High-throughput single-cell sequencing (scRNA-seq) of CD45+ cells isolated from several CNS compartments (including leptomeninges, perivascular space and parenchyma, and choroid plexus) allowed us to assemble a transcriptional atlas comprising 3461 immune cells, identified as homeostatic (“h”) or disease-associated (“da”) myeloid subsets. Profiling of all CAMs unraveled a core signature that consists of Mrc1, Pf4, Ms4a7, Stab1, and Cbr2. During disease, only Ms4a7 remained stably expressed, and a strong increase of antigen-presentation molecules (such as Cd74) was observed. Microglia expressed genes that included P2ry12, Tmem119, Sparc, and Olfml3. Although most of the core genes were down-regulated during disease, Sparc and Olfml3 expression remained unaltered and were accompanied by an up-regulation of Ly86. Several monocyte populations were observed during EAE, including monocyte-derived cells expressing Mertk and Mrc1 or expressing Zbtb46 and Cd209a. Although DCs were scarce in the homeostatic CNS, their density highly increased during disease, and diverse disease-associated DCs could be identified.

We next established the spatiotemporal relationship between infiltrating monocytes and resident macrophages using the Cx3cr1CreERT2 system. Local proliferation of resident macrophages occurred alongside continuous monocytic infiltration up to the peak of disease. Monocytes were transiently integrated into the CNS, and resident macrophages underwent apoptosis during the chronic phase. An evaluation of microglial expansion by using Cx3cr1CreER:R26Confetti mice revealed their clonal expansion during neuroinflammation.

We then investigated the capacity of resident and hematopoietic stem cell–derived myeloid cells for antigen presentation. Time-lapse imaging of Cx3cr1CreERT2:R26tdTomato:Cd2GFP and Ccr2RFP: Cd2GFP mice showed prolonged T cell interactions with circulating myeloid cells rather than tissue-resident macrophages during neuroinflammation. Accordingly, MOG35-55 immunization of Cx3cr1CreERT2:H2-Ab1flox mice showed no overt changes in disease development, indicating that resident macrophages are redundant for antigen presentation. By contrast, Cd11cCre:H2-Ab1flox mice were highly resistant to EAE, pointing to the potential role of DCs and monocyte-derived cells in EAE onset.


In this study, we unraveled the complexity of the CNS myeloid landscape and the dynamics of several myeloid populations during neuroinflammation. Although CNS-resident macrophages quickly generated context-dependent subsets during disease, their role as APCs was irrelevant for the initiation of pathology. DCs and monocyte-derived cells, highly diverse during EAE, remain the major players in antigen presentation. The comprehensive characterization presented here will provide a strong basis for their future targeting.

Myeloid cell diversity during neuroinflammation.

The homeostatic CNS includes microglia and different CAMs. During disease, microglia clonally expand, and the transcriptomic profile of microglia and CAMs drastically change. Diverse DC and monocyte subsets simultaneously populate the CNS. The role of resident macrophages for antigen presentation is redundant, whereas DCs and/or monocyte-derived populations show high antigen-presentation capacity, pointing to their crucial role in experimental autoimmune encephalomyelitis.


The innate immune cell compartment is highly diverse in the healthy central nervous system (CNS), including parenchymal and non-parenchymal macrophages. However, this complexity is increased in inflammatory settings by the recruitment of circulating myeloid cells. It is unclear which disease-specific myeloid subsets exist and what their transcriptional profiles and dynamics during CNS pathology are. Combining deep single-cell transcriptome analysis, fate mapping, in vivo imaging, clonal analysis, and transgenic mouse lines, we comprehensively characterized unappreciated myeloid subsets in several CNS compartments during neuroinflammation. During inflammation, CNS macrophage subsets undergo self-renewal, and random proliferation shifts toward clonal expansion. Last, functional studies demonstrated that endogenous CNS tissue macrophages are redundant for antigen presentation. Our results highlight myeloid cell diversity and provide insights into the brain’s innate immune system.

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