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 Cx3cr1^CreERT2 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 Cx3cr1^CreER:R26^Confetti 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 Cx3cr1^CreERT2:R26^tdTomato:Cd2^GFP and Ccr2^RFP: Cd2^GFP mice showed prolonged T cell interactions with circulating myeloid cells rather than tissue-resident macrophages during neuroinflammation. Accordingly, MOG_35-55 immunization of Cx3cr1^CreERT2:H2-Ab1^flox mice showed no overt changes in disease development, indicating that resident macrophages are redundant for antigen presentation. By contrast, Cd11c^Cre:H2-Ab1^flox 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 …