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.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

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.

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Hyalocytes-guardians of the vitreoretinal interface.

Originally discovered in the nineteenth century, hyalocytes are the resident macrophage cell population in the vitreous body. Despite this, a comprehensive understanding of their precise function and immunological significance has only recently emerged. In this article, we summarize recent in-depth investigations deciphering the critical role of hyalocytes in various aspects of vitreous physiology, such as the molecular biology and functions of hyalocytes during development, adult homeostasis, and disease. Hyalocytes are involved in fetal vitreous development, hyaloid vasculature regression, surveillance and metabolism of the vitreoretinal interface, synthesis and breakdown of vitreous components, and maintenance of vitreous transparency. While sharing certain resemblances with other myeloid cell populations such as retinal microglia, hyalocytes possess a distinct molecular signature and exhibit a gene expression profile tailored to the specific needs of their host tissue. In addition to inflammatory eye diseases such as uveitis, hyalocytes play important roles in conditions characterized by anomalous posterior vitreous detachment (PVD) and vitreoschisis. These can be hypercellular tractional vitreo-retinopathies, such as macular pucker, proliferative vitreo-retinopathy (PVR), and proliferative diabetic vitreo-retinopathy (PDVR), as well as paucicellular disorders such as vitreo-macular traction syndrome and macular holes. Notably, hyalocytes assume a significant role in the early pathophysiology of these disorders by promoting cell migration and proliferation, as well as subsequent membrane contraction, and vitreoretinal traction. Thus, early intervention targeting hyalocytes could potentially mitigate disease progression and prevent the development of proliferative vitreoretinal disorders altogether, by eliminating the involvement of vitreous and hyalocytes.

Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives.

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the human retina which in its most aggressive form is associated with the formation of macular neovascularization (MNV) and subretinal fibrosis leading to irreversible blindness. MNVs contain blood vessels as well as infiltrating immune cells, myofibroblasts, and excessive amounts of extracellular matrix proteins such as collagens, fibronectin, and laminin which disrupts retinal function and triggers neurodegeneration. In the mammalian retina, damaged neurons cannot be replaced by tissue regeneration, and subretinal MNV and fibrosis persist and thus fuel degeneration and visual loss. This review provides an overview of subretinal fibrosis in neovascular AMD, by summarizing its clinical manifestations, exploring the current understanding of the underlying cellular and molecular mechanisms and discussing potential therapeutic approaches to inhibit subretinal fibrosis in the future.

Transcriptional and Distributional Profiling of Microglia in Retinal Angiomatous Proliferation.

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.

The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation.

BACKGROUND: Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. METHODS: In this study, we examined interferon-regulatory factor 8 (Irf8)-deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). RESULTS: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. CONCLUSIONS: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

Transcriptomic Characterization of Human Choroidal Neovascular Membranes Identifies Calprotectin as a Novel Biomarker for Patients with Age-Related Macular Degeneration.

Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5'-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3'-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing-based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells, and reveals S100A8/A9 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration.

Immunosenescence in Choroidal Neovascularization (CNV)-Transcriptional Profiling of Naïve and CNV-Associated Retinal Myeloid Cells during Aging.

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

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.

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.

Genetic targeting of microglia.

Genetic targeting of microglia and other myeloid cells in the central nervous system (CNS) is highly desirable as they are critical effectors and regulators of changes in CNS homeostasis during development as well as in health and disease. Therefore, genetic reprogramming of microglia could constitute a central approach for potentially reducing disease burden. Previous attempts to target only microglia in vivo failed because of the similarities to their hematopoietic relatives in the circulation. However, this concept has been challenged by recent results of developmental and gene expression profiling studies which used novel molecular biological tools to unravel the origin of microglia and to define their role as specialized tissue macrophages clearly distinct from monocytes or monocyte-derived macrophages. The aim of this review is to recapitulate the history of microglia targeting approaches and finally highlight recent achievements in the field. We will discuss the pros and cons of the newly available genetic tools, their potential for future microglia research and genetic strategies.

Paracaspase MALT1 deficiency protects mice from autoimmune-mediated demyelination.

The paracaspase MALT 1 is a major player in lymphocyte activation and proliferation. MALT1 mediates Ag-induced signaling to the transcription factor NF-κB by functioning both as a scaffold protein and cysteine protease. We studied the role of MALT1 in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. MALT1-knockout mice did not develop any clinical symptoms of EAE. In addition, lymphocyte and macrophage infiltration into the spinal cord was absent in MALT1-knockout mice, as were demyelination and proinflammatory gene expression. Adoptive transfer experiments showed that MALT1 deficiency in splenocytes is sufficient for EAE resistance. Moreover, autoreactive T cell activation was severely impaired in MALT1-deficient T cells, suggesting the inability of MALT1-deficient effector T cells to induce demyelinating inflammation in the CNS. Finally, the MALT1 substrates A20 and CYLD were completely processed in wild-type T cells during EAE, which was partially impaired in MALT1-deficient T cells, suggesting a contribution of MALT1 proteolytic activity in T cell activation and EAE development. Together, our data indicate that MALT1 may be an interesting therapeutic target in the treatment of multiple sclerosis.

Pharmacological inhibition of MALT1 protease activity protects mice in a mouse model of multiple sclerosis.

BACKGROUND: The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-κB. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and shown to have promising anticancer properties in subtypes of B cell lymphoma. However, information on the therapeutic potential of small compound inhibitors that target MALT1 protease activity in autoimmunity is still lacking. METHODS: The present study aimed to elucidate whether MALT1 protease inhibitors are also useful in the treatment of lymphocyte-mediated autoimmune pathologies such as multiple sclerosis (MS). For this, we studied the therapeutic potential of a recently identified inhibitor of MALT1 protease activity, the phenothiazine derivative mepazine, in the context of experimental autoimmune encephalomyelitis (EAE), the main animal model for MS. RESULTS: We demonstrate that administration of mepazine prophylactically or after disease onset, can attenuate EAE. Importantly, while complete absence of MALT1 affects the differentiation of regulatory T (Treg) cells in vivo, the MALT1 protease inhibitor mepazine did not affect Treg development. CONCLUSIONS: Altogether, these data indicate that small molecule inhibitors of MALT1 not only hold great promise for the treatment of B cell lymphomas but also for autoimmune disorders such as MS.

Characterization of peripheral hematopoietic stem cells and monocytes in Parkinson's disease.

BACKGROUND: Emerging evidence has highlighted the pivotal role of the immune system in neurodegenerative diseases. This study investigated the impact of progressive neurodegeneration on the differentiation and development of hematopoietic stem cells in the peripheral blood of Parkinson's patients. METHODS: A colony-forming cell assay was established to study hematopoietic stem cells from venous blood of Parkinson's patients, and flow cytometry was used to analyze the expression of chemokine receptors on monocytes. RESULTS: We demonstrate that there is strong upregulation in the percentage of monocyte precursors in the peripheral blood of Parkinson's patients and asymptomatic high-risk individuals. We identify the receptor CCR2 as undergoing strong upregulation on the surface of classical monocytes in Parkinson's patients. CONCLUSIONS: The association between blood cell development and progressive cell death in the brain of Parkinson's patients should be further investigated as a potential dynamic biomarker and indicator of disease progression.

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease.

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

The Role of Osteopontin in Microglia Biology: Current Concepts and Future Perspectives.

The innate immune landscape of the central nervous system (CNS), including the brain and the retina, consists of different myeloid cell populations with distinct tasks to fulfill. Whereas the CNS borders harbor extraparenchymal CNS-associated macrophages whose main duty is to build up a defense against invading pathogens and other damaging factors from the periphery, the resident immune cells of the CNS parenchyma and the retina, microglia, are highly dynamic cells with a plethora of functions during homeostasis and disease. Therefore, microglia are constantly sensing their environment and closely interacting with surrounding cells, which is in part mediated by soluble factors. One of these factors is Osteopontin (OPN), a multifunctional protein that is produced by different cell types in the CNS, including microglia, and is upregulated in neurodegenerative and neuroinflammatory conditions. In this review, we discuss the current literature about the interaction between microglia and OPN in homeostasis and several disease entities, including multiple sclerosis (MS), Alzheimer's and cerebrovascular diseases (AD, CVD), amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD) and diabetic retinopathy (DR), in the context of the molecular pathways involved in OPN signaling shaping the function of microglia. As nearly all CNS diseases are characterized by pathological alterations in microglial cells, accompanied by the disturbance of the homeostatic microglia phenotype, the emergence of disease-associated microglia (DAM) states and their interplay with factors shaping the DAM-signature, such as OPN, is of great interest for therapeutical interventions in the future.

Hyalocyte origin, structure, and imaging.

Introduction: Hyalocytes have been recognized as resident tissue macrophages of the vitreous body since the mid-19th century. Despite this, knowledge about their origin, turnover, and dynamics is limited. Areas covered: Historically, initial studies on the origin of hyalocytes used light and electron microscopy. Modern investigations across species including rodents and humans will be described. Novel imaging is now available to study human hyalocytes in vivo. The shared ontogeny with retinal microglia and their eventual interdependence as well as differences will be discussed. Expert opinion: Owing to a common origin as myeloid cells, hyalocytes and retinal microglia have similarities, but hyalocytes appear to be distinct as resident macrophages of the vitreous body.