Abstract
Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2+ leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b+CD45+ cells. Single-cell mRNA sequencing of the entire CD11b+CD45+ population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.
Highlights
The central nervous system (CNS) was once considered entirely immune-privileged, there is growing evidence that interplay between neurons, glia, and the immune system are vital to healthy synaptic function[1]
While there are some useful expression markers to differentiate between immune cell types, when used in combination for immunohistochemistry[11] or flow cytometry[8], most transcriptomic and proteomic analyses are applied to entire populations, inherently averaging across subclasses and obfuscating cellular diversity
Sections of retina immunohistochemically stained for CD11b, a pan-myeloid cell marker, showed the presence of enlarged macrophage-like cells in and around the photoreceptors and subretinal space
Summary
The central nervous system (CNS) was once considered entirely immune-privileged, there is growing evidence that interplay between neurons, glia, and the immune system are vital to healthy synaptic function[1]. Because Arr[1] is expressed only in photoreceptors, the Arr1−/− model presents a unique opportunity to study the heterogeneity of immune responders in a time-locked manner when a specific class of neuron begins to die. Flow cytometry, and scRNAseq, we here report profound differences in the inflammatory profiles, mitotic activity, and active phagocytosis of distinct subpopulations of microglia, monocytes, and monocyte-derived macrophages within 48 hours of the onset of rod degeneration. These results reveal a greater level of phenotypic variety than previously appreciated, adding to the complexity of understanding the role of immune cells, even at short times after the onset of neurodegeneration
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