Molecular and spatial profiling of multiple sclerosis lesions identifies dysfunctional lipid efflux as a driver of chronic active inflammation

Compartmentalized inflammation is considered a critical factor in driving the progression of multiple sclerosis (MS). Yet, the mechanisms sustaining its persistence remain poorly understood. A hallmark of this persistent and slowly evolving inflammatory process are chronic active MS lesions. In this study, we created a high-resolution, single-cell molecular and spatial atlas of chronic inflammation in MS. To accomplish this, we combined single-nucleus RNA sequencing (snRNA-seq) with single-cell spatial transcriptomics using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to examine MS lesions, specifically focusing on those exhibiting chronic active immune pathology characterized by lymphocyte presence. Our integrative profiling uncovered the molecular landscape of glial and immune cells, their disease-associated states, and the surrounding microenvironments. Within the lesion rim, we identified CD8+ T cell niches with lipid-associated microglia. To demonstrate the utility of this spatially resolved atlas of chronic active MS lesions as a resource for future discovery, we investigated the role of lipid-associated microglia in experimental autoimmune encephalomyelitis (EAE). Our findings showed that inhibiting cholesterol efflux increased the formation of lipid-storing phagocytes, "foamy microglia," which actively drive inflammatory processes in EAE and represent tractable therapeutic targets for pharmacological modulators of sterol metabolism. These results provide a framework for system-level insights into cell-type diversity and represent a valuable resource for advancing the study of neuroinflammation in MS. Overall design: 8-week-old male Cx3cr1CreERT2/+; Abca1flox/flox; Abcg1flox/flox experimental animals (KO) and Cx3cr1CreERT2/+ controls (Ctrl) were used for EAE experiments. Myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in mice purchased from Charles River Laboratories by subcutaneous immunization with 200 mg of MOG peptide 35–55 (MOG35–55) in complete Freund's adjuvant (Mycobacterium tuberculosis at 3.75 mg ml-1; BD) and intraperitoneal injection twice with 500 ng of pertussis toxin (Sigma). Animals were examined daily and scored for clinical signs of disease. If disease did not start within 15 d of induction or the clinical score rose above 4, animals were excluded from the analysis. Clinical scores were defined as: 0, normal; 0.5, loss of tail tip tone; 1, loss of tail tone; 1.5, ataxia and mild walking deficits (slip off the grid); 2, mild hind limb weakness, severe gait ataxia and twist of the tail causing rotation of the whole body; 2.5, moderate hind limb weakness and inability to grip the grid with the hind paw but ability to stay on an upright tilted grid; 3, mild paraparesis and falls from an upright tilted grid; 3.5, paraparesis of the hind limbs (legs strongly affected but move clearly); 4, paralysis of the hind limbs and weakness in the forelimbs; 4.5, forelimbs paralyzed; 5, moribund or dead. Tamoxifen administration commenced 4 weeks before immunization. Mice were deeply anesthetized and perfused with cold PBS. The spinal cord was removed from vertebrae and cervical and thoracal parts (C1-Th10) of the spinal cord were used for scRNA-seq experiments. Cells were isolated with a previously established isolation protocol using gentleMACS with the Papain Neural Tissue Dissociation Kit (Miltenyi Biotec) and actinomycin D (catalogue no. A1410, Sigma-Aldrich) at a final concentration of 45 mM. After dissociation, myelin debris was removed from the cell suspension using Myelin Removal Beads II (Miltenyi Biotec). Cells were resuspended in 0.04% BSA + PBS and counted using an automated cell counter (TC20, Bio-Rad).