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.

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.

How many polyps need to be histologically assessed when multiple polyps are submitted for the Bowel Cancer Screening Program?

AIMS: Since 2020 there has been an increase in the number of polyps removed from patients scoped for the Bowel Cancer Screening Programme (BCSP) of England. General cellular pathology workload also continues to increase disproportionately ahead of consultant pathologist numbers in the United Kingdom. The Optical Diagnosis initiative for BCSP patients has not yet, and may not be, implemented at every hospital in England. The following study therefore aimed to determine whether only a certain number of removed polyps need to be histologically assessed to consistently guide a BCSP patient's post-polypectomy management, and whether all remaining smaller polyps beyond that number could then be discarded. METHODS: This retrospective study considered all BCSP specimens/cases submitted to the Cellular Pathology department of a large English teaching hospital from 2016 to 2024. Only cases with six or more resected polyps, for which the endoscopic report stated individual sizes, were included in the final study cohort. RESULTS: Of the 8066 BCSP cases submitted to the aforementioned department, there were six or more polyps for 345 cases. Analysis of the final study cohort of 135 cases showed that assessment of the seven largest polyps measured endoscopically was sufficient to correctly guide follow-up management of the BCSP patient as per the 2020 British Society of Gastroenterology post-polypectomy guidelines. CONCLUSIONS: When colonoscopy of a BCSP patient leads to removal of multiple polyps, only the seven largest polyps need to be assessed histologically and the remaining smaller polyps could be discarded with no impact to the patient's BCSP-related management.

Elevated temperature drives a shift from selfing to outcrossing in the insect-pollinated legume, faba bean (Vicia faba).

Climate change can threaten the reproductive success of plants, both directly, through physiological damage during increasingly extreme weather events, and indirectly, through disruption of plant-pollinator interactions. To explore how plant-pollinator interactions are modified by extreme weather, we exposed faba bean (Vicia faba) plants to elevated temperature for 5 d during flowering, simulating a heatwave. We then moved the plants to flight cages with either bumblebees or no pollinators, or to two field sites, where plants were enclosed in mesh bags or pollinated by wild insect communities. We used a morphological marker to quantify pollen movement between experimental plants. There was a substantial increase in the level of outcrossing by insect pollinators following heat stress. Proportion outcrossed seed increased from 17 % at control temperature, to 33 % following heat stress in the flight cages, and from 31 % to 80 % at one field site, but not at the other (33 % to 32 %). Abiotic stress can dramatically shift the relative contributions of cross- and self-pollination to reproduction in an insect pollinated plant. The resulting increases in gene flow have broad implications for genetic diversity and functioning of ecosystems, and may increase resilience by accelerating the selection of more stress-tolerant genotypes.

Is there sufficient Ensifer and Rhizobium species diversity in UK farmland soils to support red clover (Trifolium pratense), white clover (T. repens), lucerne (Medicago sativa) and black medic (M. lupulina)?

Rhizobia play important roles in agriculture owing to their ability to fix nitrogen through a symbiosis with legumes. The specificity of rhizobia-legume associations means that underused legume species may depend on seed inoculation with their rhizobial partners. For black medic (Medicago lupulina) and lucerne (Medicago sativa) little is known about the natural prevalence of their rhizobial partner Ensifer meliloti in UK soils, so that the need for inoculating them is unclear. We analysed the site-dependence of rhizobial seed inoculation effects on the subsequent ability of rhizobial communities to form symbioses with four legume species (Medicago lupulina, M. sativa, Trifolium repens and T. pratense). At ten organic farms across the UK, a species-diverse legume based mixture (LBM) which included these four species was grown. The LBM seed was inoculated with a mix of commercial inocula specific for clover and lucerne. At each site, soil from the LBM treatment was compared to the soil sampled prior to the sowing of the LBM (the control). From each site and each of the two treatments, a suspension of soils was applied to seedlings of the four legume species and grown in axenic conditions for six weeks. Root nodules were counted and their rhizobia isolated. PCR and sequencing of a fragment of the gyrB gene from rhizobial isolates allowed identification of strains. The number of nodules on each of the four legume species was significantly increased when inoculated with soil from the LBM treatment compared to the control. Both the proportion of plants forming nodules and the number of nodules formed varied significantly by site, with sites significantly affecting the Medicago species but not the Trifolium species. These differences in nodulation were broadly reflected in plant biomass where site and treatment interacted; at some sites there was a significant advantage from inoculation with the commercial inoculum but not at others. In particular, this study has demonstrated the commercial merit of inoculation of lucerne with compatible rhizobia.

LOS oligosaccharide modification enhances dendritic cell responses to meningococcal native outer membrane vesicles expressing a non-toxic lipid A.

Outer membrane vesicles (OMV) are released by many bacteria, and contain immunogenic antigens in addition to harmful inflammatory factors, like lipopolysaccharides. Chemically detoxified OMV have been used in vaccines against Neisseria meningitidis (Nm); however, little is known about their interaction with antigen presenting cells. In this study, we investigated the interaction of Nm OMV with human dendritic cells (DC) to gain further understanding of their biological activity. We engineered a novel serogroup B Nm that is unencapsulated (siaD), expresses pentacylated lipid A (lpxL1), hence conferring reduced toxicity, and expresses an lgtB oligosaccharide structure designed to target OMV to DC via DC-SIGN. We show that the lgtB moiety is critical for internalization of NOMV by DC. Furthermore, the lgtB moiety significantly enhances DC maturation, IL-10 and IL-23 production in the presence of a pentacylated lipid A. While different DC phenotypes were observed for each NOMV, this had little effect on Th1 and Th2 cell differentiation; however, lgtBsignificantly increased Th17 cell expansion in the presence of pentacylated lipid A. We believe that lpxL1/lgtB NOMV should be considered further as a vaccine vector, particularly considering the importance of lgtB in antigen uptake and further human studies on antigen-specific responses should be considered.

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.

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.

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.

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.

Asynchronous flowering and within-plant flowering diversity in wheat and the implications for crop resilience to heat.

BACKGROUND AND AIMS: Self-pollination dominates in wheat, with a small level of out-crossing due to flowering asynchrony and male sterility. However, the timing and synchrony of male and female flowering in wheat is a crucial determinant of seed set and may be an important factor affecting gene flow and resilience to climate change. Here, a methodology is presented for assessing the timing and synchrony of flowering in wheat, Triticum aestivum. METHODS: From the onset of flowering until the end of anthesis, the anther and stigma activity of each floret was assessed on the first five developing ears in potted plants grown under ambient conditions and originating from 'Paragon' or 'Spark-Rialto' backgrounds. At harvest maturity, seed presence, size and weight was recorded for each floret scored. KEY RESULTS AND CONCLUSIONS: The synchrony between pollen dehiscence and stigma collapse within a flower was dependent on its relative position in a spike and within a floret. Determined on the basis of synchrony within each flower, the level of pollination by pollen originating from other flowers reached approx. 30 % and did not change throughout the duration of flowering. A modelling exercise parameterized by flowering observations indicated that the temporal and spatial variability of anther activity within and between spikes may influence the relative resilience of wheat to sudden, extreme climatic events which has direct relevance to predicted future climate scenarios in the UK.

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.

Creating and Sustaining Service Industry Relationships and Families: Theorizing How Personal Workplace Relationships Both Build Community and Perpetuate Organizational Violence.

Service industry workers experience challenging labor conditions in the United States, including pay below the minimum wage, expected emotional labor, and harassment. Additionally, in part because they work long shifts in high stress environments in restaurants and bars, many build and form personal workplace relationships (PWRs). In 2021, we interviewed 38 service industry workers and managers during the COVID-19 pandemic where we examined occupational challenges they faced in the state of Texas, USA. Through our interpretive research, this essay showcases our inductive findings on how service industry workers and managers utilize communication to create and sustain PWRs. We identified how some PWRs are sustained through a unique form of occupational identification that cultivates a "service industry family", which we term familial personal workplace relationships (familial PWRs). This extends past organizational communication scholarship on family to consider occupational identification. Furthermore, our research reveals that while PWRs may build communities through care and support, they also perpetuate organizational violence, like sexual harassment and bullying.

The Effectiveness of Nutritional Education Interventions on Dietary Intake in Young Black Males: A Near-Empty Systematic Review.

The incidence of several diet and lifestyle-related diseases, previously seen only in adults, is increasing in prevalence in young people. The Black population, and particularly Black males, are at high risk of developing lifestyle-related diseases. Adolescence and young adulthood are considered a transitional period with increasing independence and responsibility, along with the development of lifelong lifestyle habits. This systematic review aimed to establish which methods and approaches to nutritional education interventions are the most effective in improving the nutritional/dietary intake in healthy young Black males. Eligibility criteria were designed using PICOS and included controlled trials of nutrition education interventions designed to improve dietary intake in healthy young Black or mixed-race males aged 14-21 years old. Medline, Cinahl and Scopus were searched in April 2021, resulting in 20,375 records being screened, and subsequently 72 full-text articles were reviewed. Risk of bias was assessed using the ROBINS-I tool. One study met the eligibility criteria. Results are presented in a narrative format as meta-analysis was not possible. This systematic review revealed a lack of evidence on the effectiveness of nutritional education interventions in this high-risk population. Limitations are noted and recommendations have been made.

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.

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.].

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.

The role of beta2 integrins and lipopolysaccharide-binding protein in the phagocytosis of dead Neisseria meningitidis.

Phagocytosis of microbial pathogens is essential for the host immune response to infection. Our previous work has shown that lipooligosaccharide (LOS) expression on the surface of Neisseria meningitidis (Nm) is essential for phagocytosis, but the receptor involved remained unclear. In this study, we show that human CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are phagocytic receptors for Nm as illustrated by the capacity of CR3- and CR4-transfected Chinese hamster ovary (CHO) cells to facilitate Nm uptake. A CR3-signalling mutant failed to internalize Nm, showing that the ability of CR3 to signal is essential for phagocytosis. Internalization of Nm by CR3-transfected CHO cells could be inhibited by the presence of CR3-specific antibodies. Furthermore, dendritic cells from leukocyte adhesion deficiency-1 patients, who have diminished expression of beta2 integrins, showed markedly reduced phagocytosis of Nm. The CR3-mediated phagocytosis required the presence of lipopolysaccharide-binding protein (LBP). Furthermore, the expression of LOS by Nm was essential for LBP binding and phagocytosis via CR3. These results reveal a critical role of CR3 and LBP in the phagocytosis of Nm and provide important insights into the initial interaction meningococci have with the immune system.

Natural Selection Towards Wild-Type in Composite Cross Populations of Winter Wheat.

Most of our crops are grown in monoculture with single genotypes grown over wide acreage. An alternative approach, where segregating populations are used as crops, is an exciting possibility, but outcomes of natural selection upon this type of crop are not well understood. We tracked allelic frequency changes in evolving composite cross populations of wheat grown over 10 generations under organic and conventional farming. At three generations, each population was genotyped with 19 SSR and 8 SNP markers. The latter were diagnostic for major functional genes. Gene diversity was constant at SSR markers but decreased over time for SNP markers. Population differentiation between the four locations could not be detected, suggesting that organic vs. non-organic crop management did not drive allele frequency changes. However, we did see changes for genes controlling plant height and phenology in all populations independently and consistently. We interpret these changes as the result of a consistent natural selection towards wild-type. Independent selection for alleles that are associated with plant height suggests that competition for light was central, resulting in the predominance of stronger intraspecific competitors, and highlighting a potential trade-off between individual and population performance.