Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Temporospatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model.

Myeloid cells such as resident retinal microglia (MG) or infiltrating blood-derived macrophages (Mϕ) accumulate in areas of retinal ischemia and neovascularization (RNV) and modulate neovascular eye disease. Their temporospatial distribution and biological function in this process, however, remain unclarified. Using state-of-the-art methods, including cell-specific reporter mice and high-throughput RNA sequencing (RNA Seq), this study determined the extent of MG proliferation and Mϕ infiltration in areas with retinal ischemia and RNV in Cx3cr1CreERT2 :Rosa26-tdTomato mice and examined the transcriptional profile of MG in the mouse model of oxygen-induced retinopathy (OIR). For RNA Seq, tdTomato-positive retinal MG were sorted by flow cytometry followed by Gene ontology (GO) cluster analysis. Furthermore, intraperitoneal injections of the cell proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) were performed from postnatal day (p) 12 to p16. We found that MG is the predominant myeloid cell population while Mϕ rarely appears in areas of RNV. Thirty percent of retinal MG in areas of RNV were EdU-positive indicating a considerable local MG cell expansion. GO cluster analysis revealed an enrichment of clusters related to cell division, tubulin binding, ATPase activity, protein kinase regulatory activity, and chemokine receptor binding in MG in the OIR model compared to untreated controls. In conclusion, activated retinal MG alter their transcriptional profile, exhibit considerable proliferative ability and are by far the most frequent myeloid cell population in areas of ischemia and RNV in the OIR model thus presenting a potential target for future therapeutic approaches.

Unique microglia expression profile in developing white matter.

OBJECTIVE: Recently we demonstrated that amoeboid microglia in white matter regions are essential for proper oligodendrocyte homeostasis and myelinogenesis in the first postnatal week. Amoeboid microglia in the mouse corpus callosum change their activation profile within few days after postnatal day (P)7 with microglia of the cerebellum showing similar features. Here we expanded our previous transcriptional analysis and performed detailed bulk RNA sequencing of microglia from corpus callosum, cortex and cerebellum at P7, P10 and P42. The goal of this study was to identify a specific gene profile for both, white matter and grey matter microglia during development. RESULTS: Microglia in white matter regions display unique characteristics in the first postnatal week of murine life. In both the corpus callosum and cerebellum microglia show amoeboid morphology and a similar transcription profile during development including high expression of genes related to priming of microglia, phagocytosis and migration at P7; characteristics which are already lost at P10. Together these data verify our previous transcriptional data obtained by microarray analysis and enable a more complete view into white matter and grey matter microglia at different developmental stages.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

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

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.

A20 critically controls microglia activation and inhibits inflammasome-dependent neuroinflammation.

Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-κB) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microglia-confined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS)-like disease, due to hyperactivation of the Nlrp3 inflammasome leading to enhanced interleukin-1ß secretion and CNS inflammation. Finally, we confirm a Nlrp3 inflammasome signature and IL-1ß expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.

Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.

Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.

Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood.

Whereas microglia involvement in virtually all brain diseases is well accepted their role in the control of homeostasis in the central nervous system (CNS) is mainly thought to be the maintenance of neuronal function through the formation, refinement, and monitoring of synapses in both the developing and adult brain. Although the prenatal origin as well as the neuron-centered function of cortical microglia has recently been elucidated, much less is known about a distinct amoeboid microglia population formerly described as the "fountain of microglia" that appears only postnatally in myelinated regions such as corpus callosum and cerebellum. Using large-scale transcriptional profiling, fate mapping, and genetic targeting approaches, we identified a unique molecular signature of this microglia subset that arose from a CNS endogenous microglia pool independent from circulating myeloid cells. Microglia depletion experiments revealed an essential role of postnatal microglia for the proper development and homeostasis of oligodendrocytes and their progenitors. Our data provide new cellular and molecular insights into the myelin-supporting function of microglia in the normal CNS.

Ontogeny and homeostasis of CNS myeloid cells.

Myeloid cells in the central nervous system (CNS) represent a heterogeneous class of innate immune cells that contribute to the maintenance of tissue homeostasis differentially during development and adulthood. The subsets of CNS myeloid cells identified so far, including parenchymal microglia and non-parenchymal meningeal, perivascular and choroid-plexus macrophages, as well as disease-associated monocytes, have classically been distinguished on the basis of their surface epitope expression, localization and morphology. However, studies using cell-specific targeting, in vivo imaging, single-cell expression analysis and other sophisticated tools have now increased the depth of knowledge of this immune-cell compartment and call for reevaluation of the traditional views on the origin, fate and function of distinct CNS myeloid subsets. The concepts of CNS macrophage biology that are emerging from these new insights have broad implications for the understanding and treatment of CNS diseases.

Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation.

Recent studies have shown that tissue macrophages (MΦ) arise from embryonic progenitors of the yolk sac (YS) and fetal liver and colonize tissues before birth. Further studies have proposed that developmentally distinct tissue MΦ can be identified based on the differential expression of F4/80 and CD11b, but whether a characteristic transcriptional profile exists is largely unknown. Here, we took advantage of an inducible fate-mapping system that facilitated the identification of CD45(+)c-kit(-)CX3CR1(+)F4/80(+) (A2) progenitors of the YS as the source of F4/80(hi) but not CD11b(hi) MΦ. Large-scale transcriptional profiling of MΦ precursors from the YS stage to adulthood allowed for building computational models for F4/80(hi) tissue macrophages being direct descendants of A2 progenitors. We further identified a distinct molecular signature of F4/80(hi) and CD11b(hi) MΦ and found that Irf8 was vital for MΦ maturation. Our data provide new cellular and molecular insights into the origin and developmental pathways of tissue MΦ.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment.

Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy.

The force awakens: insights into the origin and formation of microglia.

Microglia are tissue resident macrophages of the central nervous system (CNS) that maintain homeostasis and respond to immune challenges. New genetic fate mapping tools have revealed a yolk sac origin of microglia. Once established in the CNS, microglia persist throughout the lifetime of the organism behind the blood-brain barrier and maintain themselves by self-renewal. Recent studies uncovered a broad spectrum of microglial functions that are influenced by the dynamism of brain formation and neuronal wiring. This review focuses on current findings concerning microglia origin and formation during development and discusses the factors important for microglia survival and function.

Progressive replacement of embryo-derived cardiac macrophages with age.

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

A myelin gene causative of a catatonia-depression syndrome upon aging.

Severe mental illnesses have been linked to white matter abnormalities, documented by postmortem studies. However, cause and effect have remained difficult to distinguish. CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase) is among the oligodendrocyte/myelin-associated genes most robustly reduced on mRNA and protein level in brains of schizophrenic, bipolar or major depressive patients. This suggests that CNP reduction might be critical for a more general disease process and not restricted to a single diagnostic category. We show here that reduced expression of CNP is the primary cause of a distinct behavioural phenotype, seen only upon aging as an additional 'pro-inflammatory hit'. This phenotype is strikingly similar in Cnp heterozygous mice and patients with mental disease carrying the AA genotype at CNP SNP rs2070106. The characteristic features in both species with their partial CNP 'loss-of-function' genotype are best described as 'catatonia-depression' syndrome. As a consequence of perturbed CNP expression, mice show secondary low-grade inflammation/neurodegeneration. Analogously, in man, diffusion tensor imaging points to axonal loss in the frontal corpus callosum. To conclude, subtle white matter abnormalities inducing neurodegenerative changes can cause/amplify psychiatric diseases.

Erythropoietin attenuates neurological and histological consequences of toxic demyelination in mice.

Erythropoietin (EPO) reduces symptoms of experimental autoimmune encephalomyelitis in rodents and shows neuroregenerative effects in chronic progressive multiple sclerosis. The mechanisms of action of EPO in these conditions with shared immunological etiology are still unclear. Therefore, we used a model of toxic demyelination allowing exclusion of T cell-mediated inflammation. In a double-blind (for food/injections), placebo-controlled, longitudinal four-arm design, 8-wk-old C57BL/6 mice (n = 26/group) were assigned to cuprizone-containing (0.2%) or regular food (ground chow) for 6 wks. After 3 wks, mice were injected every other day with placebo or EPO (5,000 IU/kg intraperitoneally) until the end of cuprizone feeding. Half of the mice were exposed to behavioral testing, magnetic resonance imaging (MRI) and histology immediately after treatment cessation, whereas the other half were allowed a 3-wk treatment-free recovery. Immediately after termination of cuprizone feeding, all toxin-exposed mice were compromised regarding vestibulomotor function/coordination, with EPO-treated animals performing better than placebo. Likewise, ventricular enlargement after cuprizone, as documented by MRI, was less pronounced upon EPO. After a 3-wk recovery, remarkable spontaneous improvement was observed in all mice with no measurable further benefit in the EPO group ("ceiling effect"). Histological analysis of the corpus callosum revealed attenuation by EPO of the cuprizone-induced increase in microglial numbers and amyloid precursor protein accumulations as a readout of inflammation and axonal degeneration. To conclude, EPO ameliorates neurological symptoms in the cuprizone model of demyelination, possibly by reduction of inflammation-associated axonal degeneration in white matter tracts. These findings underscore the value of future therapeutic strategies for multiple sclerosis based on EPO or EPO variants.