Streptococcus agalactiae glycolipids promote virulence by thwarting immune cell clearance.

Streptococcus agalactiae [group B Streptococcus (GBS)] is a leading cause of neonatal meningitis, with late-onset disease (LOD) occurring after gastrointestinal tract colonization in infants. Bacterial membrane lipids are essential for host-pathogen interactions, and the functions of glycolipids are yet to be fully elucidated. GBS synthesizes three major glycolipids: glucosyl-diacylglycerol (Glc-DAG), diglucosyl-DAG (Glc2-DAG), and lysyl-Glc-DAG (Lys-Glc-DAG). Here, we identify the enzyme, IagB, as responsible for biosynthesis of Glc-DAG, the precursor for the two other glycolipids in GBS. To examine the collective role of glycolipids to GBS virulence, we adapted a murine model of neonatal meningitis to simulate LOD. The GBS∆iagB mutant traversed the gut-epithelial barrier comparable to wild type but was severely attenuated in bloodstream survival, resulting in decreased bacterial loads in the brain. The GBS∆iagB mutant was more susceptible to neutrophil killing and membrane targeting by host antimicrobial peptides. This work reveals an unexplored function of GBS glycolipids with their ability to protect the bacterial cell from host antimicrobial killing.

Tumor and immune cell types interact to produce heterogeneous phenotypes of pediatric high-grade glioma.

BACKGROUND: Pediatric high-grade gliomas (PHGG) are aggressive brain tumors with 5-year survival rates ranging from <2% to 20% depending upon subtype. PHGG presents differently from patient to patient and is intratumorally heterogeneous, posing challenges in designing therapies. We hypothesized that heterogeneity occurs because PHGG comprises multiple distinct tumor and immune cell types in varying proportions, each of which may influence tumor characteristics. METHODS: We obtained 19 PHGG samples from our institution's pediatric brain tumor bank. We constructed a comprehensive transcriptomic dataset at the single-cell level using single-cell RNA-Seq (scRNA-Seq), identified known glial and immune cell types, and performed differential gene expression and gene set enrichment analysis. We conducted multi-channel immunofluorescence (IF) staining to confirm the transcriptomic results. RESULTS: Our PHGG samples included 3 principal predicted tumor cell types: astrocytes, oligodendrocyte progenitors (OPCs), and mesenchymal-like cells (Mes). These cell types differed in their gene expression profiles, pathway enrichment, and mesenchymal character. We identified a macrophage population enriched in mesenchymal and inflammatory gene expression as a possible source of mesenchymal tumor characteristics. We found evidence of T-cell exhaustion and suppression. CONCLUSIONS: PHGG comprises multiple distinct proliferating tumor cell types. Microglia-derived macrophages may drive mesenchymal gene expression in PHGG. The predicted Mes tumor cell population likely derives from OPCs. The variable tumor cell populations rely on different oncogenic pathways and are thus likely to vary in their responses to therapy.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Neural tube-associated boundary caps are a major source of mural cells in the skin.

In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature.

Perivascular fibroblasts (PVFs) are a fibroblast-like cell type that reside on large-diameter blood vessels in the adult meninges and central nervous system (CNS). PVFs contribute to fibrosis following injury but their homeostatic functions are not defined. PVFs were previously shown to be absent from most brain regions at birth and are only detected postnatally within the cerebral cortex. However, the origin, timing and cellular mechanisms of PVF development are not known. We used Col1a1-GFP and Col1a2-CreERT2 transgenic mice to track PVF development postnatally. Using lineage tracing and in vivo imaging we show that brain PVFs originate from the meninges and are first seen on parenchymal cerebrovasculature at postnatal day (P) 5. After P5, PVF coverage of the cerebrovasculature expands via local cell proliferation and migration from the meninges. Finally, we show that PVFs and perivascular macrophages develop concurrently. These findings provide the first complete timeline for PVF development in the brain, enabling future work into how PVF development is coordinated with cell types and structures in and around the perivascular spaces to support normal CNS vascular function.

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature.

Perivascular fibroblasts (PVFs) are a fibroblast-like cell type that reside on large-diameter blood vessels in the adult meninges and central nervous system (CNS). PVFs drive fibrosis following injury but their homeostatic functions are not well detailed. In mice, PVFs were previously shown to be absent from most brain regions at birth and are only detected postnatally within the cerebral cortex. However, the origin, timing, and cellular mechanisms of PVF development are not known. We used Col1a1-GFP and Col1a2-CreERT transgenic mice to track PVF developmental timing and progression in postnatal mice. Using a combination of lineage tracing and in vivo imaging we show that brain PVFs originate from the meninges and are first seen on parenchymal cerebrovasculature at postnatal day (P)5. After P5, PVF coverage of the cerebrovasculature rapidly expands via mechanisms of local cell proliferation and migration from the meninges, reaching adult levels at P14. Finally, we show that PVFs and perivascular macrophages (PVMs) develop concurrently along postnatal cerebral blood vessels, where the location and depth of PVMs and PVFs highly correlate. These findings provide the first complete timeline for PVF development in the brain, enabling future work into how PVF development is coordinated with cell types and structures in and around the perivascular spaces to support normal CNS vascular function. Summary: Brain perivascular fibroblasts migrate from their origin in the meninges and proliferate locally to fully cover penetrating vessels during postnatal mouse development.

Formation and function of the meningeal arachnoid barrier around the developing mouse brain.

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-ß-catenin signaling and that constitutively active ß-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development.

Stuck on you: Meninges cellular crosstalk in development.

The spatial and temporal development of the brain, overlying meninges (fibroblasts, vasculature and immune cells) and calvarium are highly coordinated. In particular, the timing of meningeal fibroblasts into molecularly distinct pia, arachnoid and dura subtypes coincides with key developmental events in the brain and calvarium. Further, the meninges are positioned to influence development of adjacent structures and do so via depositing basement membrane and producing molecular cues to regulate brain and calvarial development. Here, we review the current knowledge of how meninges development aligns with events in the brain and calvarium and meningeal fibroblast "crosstalk" with these structures. We summarize outstanding questions and how the use of non-mammalian models to study the meninges will substantially advance the field of meninges biology.

Retinoic acid signaling in mouse retina endothelial cells is required for early angiogenic growth.

The development of the retinal vasculature is essential to maintain health of the tissue, but the developmental mechanisms are not completely understood. The aim of this study was to investigate the cell-autonomous role of retinoic acid signaling in endothelial cells during retina vascular development. Using a temporal and cell-specific mouse model to disrupt retinoic acid signaling in endothelial cells in the postnatal retina (Pdgfbicre/+dnRAR403fl/fl mutants), we discovered that angiogenesis in the retina is significantly decreased with a reduction in retina vascularization, endothelial tip cell number and filipodia, and endothelial 'crowding' of stalk cells. Interestingly, by P15, the vasculature can overcome the early angiogenic defect and fully vascularized the retina. At P60, the vasculature is intact with no evidence of retina cell death or altered blood retinal barrier integrity. Further, we identified that the angiogenic defect seen in mutants at P6 correlates with decreased Vegfr3 expression in endothelial cells. Collectively, our work identified a previously unappreciated function for endothelial retinoic acid signaling in early retinal angiogenesis.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Techniques for visualizing fibroblast-vessel interactions in the developing and adult CNS.

Significance: Fibroblasts are found associated with blood vessels in various locations across the central nervous system (CNS): in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo; however, we still know very little regarding their functions and identity. Aim: Current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast-vessel interactions. We aimed to develop new ways to visualize CNS fibroblasts in greater detail. Approach: Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and CUBIC optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts. Results: We show that these methods can be used for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. In addition, we can combine these techniques with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays. Conclusions: We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.

A human brain vascular atlas reveals diverse mediators of Alzheimer's risk.

The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.

Emerging roles for CNS fibroblasts in health, injury and disease.

Recent transcriptomic, histological and functional studies have begun to shine light on the fibroblasts present in the meninges, choroid plexus and perivascular spaces of the brain and spinal cord. Although the origins and functions of CNS fibroblasts are still being described, it is clear that they represent a distinct cell population, or populations, that have likely been confused with other cell types on the basis of the expression of overlapping cellular markers. Recent work has revealed that fibroblasts play crucial roles in fibrotic scar formation in the CNS after injury and inflammation, which have also been attributed to other perivascular cell types such as pericytes and vascular smooth muscle cells. In this Review, we describe the current knowledge of the location and identity of CNS perivascular cell types, with a particular focus on CNS fibroblasts, including their origin, subtypes, roles in health and disease, and future areas for study.

Corrigendum: Living on the Edge of the CNS: Meninges Cell Diversity in Health and Disease.

[This corrects the article DOI: 10.3389/fncel.2021.703944.].

Not just a 'drain': venules sprout brain capillaries.

The cerebral cortex requires a dense, highly organized network of vasculature that ensures high-volume and continuous oxygen delivery to metabolically active neuronal circuits. In a recent paper, Coelho-Santos et al. used in vivo two-photon microscopy to reveal how this precise network is constructed during a short window of mouse postnatal development.

Living on the Edge of the CNS: Meninges Cell Diversity in Health and Disease.

The meninges are the fibrous covering of the central nervous system (CNS) which contain vastly heterogeneous cell types within its three layers (dura, arachnoid, and pia). The dural compartment of the meninges, closest to the skull, is predominantly composed of fibroblasts, but also includes fenestrated blood vasculature, an elaborate lymphatic system, as well as immune cells which are distinct from the CNS. Segregating the outer and inner meningeal compartments is the epithelial-like arachnoid barrier cells, connected by tight and adherens junctions, which regulate the movement of pathogens, molecules, and cells into and out of the cerebral spinal fluid (CSF) and brain parenchyma. Most proximate to the brain is the collagen and basement membrane-rich pia matter that abuts the glial limitans and has recently be shown to have regional heterogeneity within the developing mouse brain. While the meninges were historically seen as a purely structural support for the CNS and protection from trauma, the emerging view of the meninges is as an essential interface between the CNS and the periphery, critical to brain development, required for brain homeostasis, and involved in a variety of diseases. In this review, we will summarize what is known regarding the development, specification, and maturation of the meninges during homeostatic conditions and discuss the rapidly emerging evidence that specific meningeal cell compartments play differential and important roles in the pathophysiology of a myriad of diseases including: multiple sclerosis, dementia, stroke, viral/bacterial meningitis, traumatic brain injury, and cancer. We will conclude with a list of major questions and mechanisms that remain unknown, the study of which represent new, future directions for the field of meninges biology.

Single-Cell Transcriptomic Analyses of the Developing Meninges Reveal Meningeal Fibroblast Diversity and Function.

The meninges are a multilayered structure composed of fibroblasts, blood and lymphatic vessels, and immune cells. Meningeal fibroblasts secrete a variety of factors that control CNS development, yet strikingly little is known about their heterogeneity or development. Using single-cell sequencing, we report distinct transcriptional signatures for fibroblasts in the embryonic dura, arachnoid, and pia. We define new markers for meningeal layers and show conservation in human meninges. We find that embryonic meningeal fibroblasts are transcriptionally distinct between brain regions and identify a regionally localized pial subpopulation marked by the expression of µ-crystallin. Developmental analysis reveals a progressive, ventral-to-dorsal maturation of telencephalic meninges. Our studies have generated an unparalleled view of meningeal fibroblasts, providing molecular profiles of embryonic meningeal fibroblasts by layer and yielding insights into the mechanisms of meninges development and function.

Gamma Interferon Alters Junctional Integrity via Rho Kinase, Resulting in Blood-Brain Barrier Leakage in Experimental Viral Encephalitis.

Blood-brain barrier (BBB) breakdown is a hallmark of many diseases of the central nervous system (CNS). Loss of BBB integrity in CNS diseases such as viral encephalitis results in the loss of nutrient/oxygen delivery, rapid infiltration of immune cells, and brain swelling that can exacerbate neuronal injury. Despite this, the cellular and molecular mechanisms that underlie BBB breakdown in viral encephalitis are incompletely understood. We undertook a comprehensive analysis of the cellular and molecular signaling events that induce BBB breakdown in an experimental model of virus-induced encephalitis in which neonatal mice are infected with reovirus (serotype 3 strain Abney). We show that BBB leakage during reovirus infection correlates with morphological changes in the vasculature, reductions in pericytes (BBB supporting cells), and disorganization of vascular junctions. Pathway analysis on RNA sequencing from brain endothelial cells identified the activation of interferon (IFN) signaling within the brain vasculature following reovirus infection. Our in vitro and in vivo studies show that type II IFN mediated by IFN-γ, a well known antiviral signal, is a major contributor to BBB leakage during reovirus infection. We show that IFN-γ reduces barrier properties in cultured brain endothelial cells through Rho kinase (ROCK)-mediated cytoskeletal contractions, resulting in junctional disorganization and cell-cell separations. In vivo neutralization of IFN-γ during reovirus infection significantly improved BBB integrity, pericyte coverage, attenuated vascular ROCK activity, and junctional disorganization. Our work supports a model in which IFN-γ acts directly on the brain endothelium to induce BBB breakdown through a mechanism involving ROCK-induced junctional disorganization.IMPORTANCE In an experimental viral encephalitis mouse model in which mice are infected with reovirus, we show that IFN-γ induces blood-brain barrier leakage. We show that IFN-γ promotes Rho kinase activity, resulting in actin cytoskeletal contractions in the brain endothelium that lead to vascular junctional disorganization and cell-cell separations. These studies now provide insight into a previously unknown mechanism for how blood-brain barrier breakdown occurs in viral encephalitis and implicates IFN-γ-Rho kinase activity as major contributor to this phenomenon. By identifying this mechanism of blood-brain barrier breakdown, we now provide potential therapeutic targets in treating patients with viral causes of encephalitis with the hope of limiting damage to the central nervous system.

The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells.

The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs.

Retinoic acid signaling in vascular development.

Formation of the vasculature is an essential developmental process, delivering oxygen and nutrients to support cellular processes needed for tissue growth and maturation. Retinoic acid (RA) and its downstream signaling pathway is vital for normal pre- and post-natal development, playing key roles in the specification and formation of many organs and tissues. Here, we review the role of RA in blood and lymph vascular development, beginning with embryonic yolk sac vasculogenesis and remodeling and discussing RA's organ-specific roles in angiogenesis and vessel maturation. In particular, we highlight the multi-faceted role of RA signaling in CNS vascular development and acquisition of blood-brain barrier properties.