The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22‐C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22‐C24 sphingolipid synthesis in myelin biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2^ΔO/ΔO). At 6 weeks of age, normal‐appearing myelin had formed in CerS2^ΔO/ΔO mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22‐C24 sphingolipids in myelin of CerS2^ΔO/ΔO mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2^ΔO/ΔO mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6‐week‐old CerS2^ΔO/ΔO mice. By 16 weeks, CerS2^ΔO/ΔO mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2^ΔO/ΔO mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases.

As the resident parenchymal myeloid population in the central nervous system (CNS), microglia are strategically positioned to respond to neurotropic virus invasion and have been implicated in promoting both disease resolution and progression in the acute and post-infectious phase of virus encephalitis. In a mouse model of West Nile virus encephalitis (WNE), infection of the CNS results in recruitment of large numbers of peripheral immune cells into the brain, the majority being nitric oxide (NO)-producing Ly6C^hi inflammatory monocyte-derived cells (MCs). In this model, these cells enhance immunopathology and mortality. However, the contribution of microglia to this response is currently undefined. Here we used a combination of experimental tools, including single-cell RNA sequencing (scRNA-seq), microglia and MC depletion reagents, high-dimensional spectral cytometry and computational algorithms to dissect the differential contribution of microglia and MCs to the anti-viral immune response in severe neuroinflammation seen in WNE. Intriguingly, analysis of scRNA-seq data revealed 6 unique microglia and 3 unique MC clusters that were predominantly timepoint-specific, demonstrating substantial transcriptional adaptation with disease progression over the course of WNE. While microglia and MC adopted unique gene expression profiles, gene ontology enrichment analysis, coupled with microglia and MC depletion studies, demonstrated a role for both of these cells in the trafficking of peripheral immune cells into the CNS, T cell responses and viral clearance. Over the course of infection, microglia transitioned from a homeostatic to an anti-viral and then into an immune cell-recruiting phenotype. Conversely, MC adopted antigen-presenting, immune cell-recruiting and NO-producing phenotypes, which all had anti-viral function. Overall, this study defines for the first time the single-cell transcriptomic responses of microglia and MCs over the course of WNE, demonstrating both protective and pathological roles of these cells that could potentially be targeted for differential therapeutic intervention to dampen immune-mediated pathology, while maintaining viral clearance functions.

Inflammation of the brain parenchyma is characteristic of neurodegenerative, autoimmune, and neuroinflammatory diseases. During this process, microglia, which populate the embryonic brain and become a permanent sentinel myeloid population, are inexorably joined by peripherally derived monocytes, recruited by the central nervous system. These cells can quickly adopt a morphology and immunophenotype similar to microglia. Both microglia and monocytes have been implicated in inducing, enhancing, and/or maintaining immune-mediated pathology and thus disease progression in a number of neuropathologies. For many years, experimental and analytical systems have failed to differentiate resident microglia from peripherally derived myeloid cells accurately. This has impeded our understanding of their precise functions in, and contributions to, these diseases, and hampered the development of novel treatments that could target specific cell subsets. Over the past decade, microglia have been investigated more intensively in the context of neuroimmunological research, fostering the development of more precise experimental systems. In light of our rapidly growing understanding of these cells, we discuss the differential origins of microglia and peripherally derived myeloid cells in the inflamed brain, with an analysis of the problems resolving these cell types phenotypically and morphologically, and highlight recent developments enabling more precise identification.

Microglia and bone marrow‐derived monocytes are key elements of central nervous system (CNS) inflammation, both capable of enhancing and dampening immune‐mediated pathology. However, the study‐specific focus on individual cell types, disease models or experimental approaches has limited our ability to infer common and disease‐specific responses. This meta‐analysis integrates bulk and single‐cell transcriptomic datasets of microglia and monocytes from disease models of autoimmunity, neurodegeneration, sterile injury, and infection to build a comprehensive resource connecting myeloid responses across CNS disease. We demonstrate that the bulk microglial and monocyte program is highly contingent on the disease environment, challenging the notion of a universal microglial disease signature. Integration of six single‐cell RNA‐sequencing datasets revealed that these disease‐specific signatures are likely driven by differing proportions of unique myeloid subpopulations that were individually expanded in different disease settings. These subsets were functionally‐defined as neurodegeneration‐associated, inflammatory, interferon‐responsive, phagocytic, antigen‐presenting, and lipopolysaccharide‐responsive cellular states, revealing a core set of myeloid responses at the single‐cell level that are conserved across CNS pathology. Showcasing the predictive and practical value of this resource, we performed differential expression analysis on microglia and monocytes across disease and identified Cd81 as a new neuroinflammatory‐stable gene that accurately identified microglia and distinguished them from monocyte‐derived cells across all experimental models at both the bulk and single‐cell level. Together, this resource dissects the influence of disease environment on shared immune response programmes to build a unified perspective of myeloid behavior across CNS pathology.

Microglia are the innate myeloid cells of the central nervous system (CNS) parenchyma, functionally implicated in almost every defined neuroinflammatory and neurodegenerative disorder. Current understanding of disease pathogenesis for many neuropathologies is limited and/or lacks reliable diagnostic markers, vaccines, and treatments. With the increasing aging of society and rise in neurogenerative diseases, improving our understanding of their pathogenesis is essential. Analysis of microglia from murine disease models provides an investigative tool to unravel disease processes. In many neuropathologies, bone‐marrow‐derived monocytes are recruited to the CNS, adopting a phenotype similar to that of microglia. This significantly confounds the accurate identification of cell‐type‐specific functions and downstream therapeutic targeting. The increased capacity to analyze more phenotypic markers using spectral‐cytometry‐based technologies allows improved separation of microglia from monocyte‐derived cells. Full‐spectrum profiling enables enhanced marker resolution, time‐efficient analysis of >40 fluorescence parameters, and extraction of cellular autofluorescence parameters. Coupling this system with additional cytometric technologies, including cell sorting and high‐parameter imaging, can improve the understanding of microglial phenotypes in disease. To this end, we provide detailed, step‐by‐step protocols for the analysis of murine brain tissue by high‐parameter ex vivo cytometric analysis using the Aurora spectral cytometer (Cytek), including best practices for unmixing and autofluorescence extraction, cell sorting for single‐cell RNA analysis, and imaging mass cytometry. Together, this provides a toolkit for researchers to comprehensively investigate microglial disease processes at protein, RNA, and spatial levels for the identification of therapeutic targets in neuropathology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Processing the mouse brain into a single‐cell suspension for microglia isolationBasic Protocol 2: Staining single‐cell mouse brain suspensions for microglial phenotyping by spectral cytometryBasic Protocol 3: Flow cytometric sorting of mouse microglia for ex vivo analysisBasic Protocol 4: Processing the mouse brain for imaging mass cytometry for spatial microglia analysis

CD8⁺ T cells are critical to the adaptive immune response against viral pathogens. However, overwhelming antigen exposure can result in their exhaustion, characterised by reduced effector function, failure to clear virus, and the upregulation of inhibitory receptors, including programmed cell death 1 (PD-1). However, exhausted T cell responses can be “re-invigorated” by inhibiting PD-1 or the primary ligand of PD-1: PD-L1. Further, the absence of the type I interferon receptor IFNAR1 also results in T cell exhaustion and virus persistence in lymphocytic choriomeningitis virus Armstrong (LCMV-Arm)-infected mice. In this study, utilizing single- and double-knockout mice, we aimed to determine whether ablation of PD-1 could restore T cell functionality in the absence of IFNAR1 signalling in LCMV-Arm-infected mice. Surprisingly, this did not re-invigorate the T cell response and instead, it converted chronic LCMV-Arm infection into a lethal disease characterized by severe lung inflammation with an infiltration of neutrophils and T cells. Depletion of CD8⁺ T cells, but not neutrophils, rescued mice from lethal disease, demonstrating that IFNAR1 is required to prevent T cell exhaustion and virus persistence in LCMV-Arm infection, and in the absence of IFNAR1, PD-L1 is required for survival. This reveals an important interplay between IFNAR1 and PD-L1 with implications for therapeutics targeting these pathways.