Deletion of murine astrocytic vesicular nucleotide transporter increases anxiety and depressive-like behavior and attenuates motivation for reward.

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

STING: Stay near to STIM(1) neuroprotection.

In a recent publication in Cell, Woo et al.1 report that stimulator of interferon genes (STING) links inflammation with glutamate-driven excitotoxicity to induce ferroptosis, identifying a mechanism of inflammation-induced neurodegeneration and also a novel candidate therapeutic target for multiple sclerosis.

FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics.

Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures. We recently developed a nucleic acid cytometry platform called 'focused interrogation of cells by nucleic acid detection and sequencing' (FIND-seq), capable of isolating rare cells on the basis of RNA or DNA markers, followed by bulk or single-cell transcriptomic analysis. This platform has previously been used to characterize the splicing-dependent activation of the transcription factor XBP1 in astrocytes and HIV persistence in memory CD4 T cells from people on long-term antiretroviral therapy. Here, we outline the molecular and microfluidic steps involved in performing FIND-seq, including protocol updates that allow detection and whole transcriptome sequencing of rare HIV-infected cells that harbor genetically intact virus genomes. FIND-seq requires knowledge of microfluidics, optics and molecular biology. We expect that FIND-seq, and this comprehensive protocol, will enable mechanistic studies of rare HIV+ cells, as well as other cell subsets that were previously difficult to recover and sequence.

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Astrocyte control of brain metastasis.

Developing therapeutics to improve metastatic brain cancer prognosis is hampered by limited experimental systems that recapitulate the brain tumor microenvironment. In this issue of Developmental Cell, Ishibashi et al. describe a glial-cancer cell co-culture system that enabled the identification of a targetable, astrocyte-driven mechanism of brain metastasis.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology.

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

Actions taken and barriers encountered by professionals working with adults with intellectual disabilities who experience grief: a qualitative approach.

Experience of grief has increased among people with intellectual disability because of their longer life expectancy. Professionals supporting this population are often critical of the lack of adequate tools for dealing with this situation. The objective of this study was to identify the strategies and barriers that these professionals are confronted with when dealing people with intellectual disability who are going through the grieving process. A qualitative study was conducted involving 20 professionals working with people with intellectual disability. Four themes were extracted using thematic analysis: Exclusion of clients from end-of-life and grief processes, Strategies to support the client's grief process, Emotional and personal difficulties faced by the professionals, and Coping and regulation of the professional's grief process. Barriers identified by these professionals include not having the specific skills to support clients in their grief and the emotional impact of the death of a client.

Tapinarof validates the aryl hydrocarbon receptor as a therapeutic target: A clinical review.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that has wide-ranging roles, including regulation of inflammation and homeostasis. AhR is not a cell surface receptor; rather, it exists in a cytoplasmic complex that responds to a wide variety of structurally dissimilar endogenous, microbial, and environmental ligands. The ubiquitous expression of AhR, its ability to be activated by a wide range of ligands, and its capacity to act as a master regulator for gene expression and homeostasis make it a promising new therapeutic target. Clinical trials of tapinarof cream have now validated AhR agonism as a therapeutic approach that can deliver significant efficacy for treating inflammatory skin diseases, including psoriasis and atopic dermatitis. Tapinarof 1% cream is a first-in-class, nonsteroidal, topical, AhR agonist with a pharmacokinetic profile that results in localized exposure at sites of disease, avoiding systemic safety concerns, drug interactions, or off-target effects. Psoriasis and atopic dermatitis both involve epidermal inflammation, cellular immune responses, dysregulation of skin barrier protein expression, and oxidative stress. On the basis of the clinical effectiveness of tapinarof cream for treating inflammatory skin diseases, we review how targeting AhR may offer a significant opportunity in other conditions that share key aspects of pathogenesis, including asthma, inflammatory bowel disease, eosinophilic esophagitis, ophthalmic, and nervous system diseases.

Therapeutic induction of antigen-specific immune tolerance.

The development of therapeutic approaches for the induction of robust, long-lasting and antigen-specific immune tolerance remains an important unmet clinical need for the management of autoimmunity, allergy, organ transplantation and gene therapy. Recent breakthroughs in our understanding of immune tolerance mechanisms have opened new research avenues and therapeutic opportunities in this area. Here, we review mechanisms of immune tolerance and novel methods for its therapeutic induction.

Neuroinflammation: An astrocyte perspective.

Astrocytes are abundant glial cells in the central nervous system (CNS) that play active roles in health and disease. Recent technologies have uncovered the functional heterogeneity of astrocytes and their extensive interactions with other cell types in the CNS. In this Review, we highlight the intricate interactions between astrocytes, other CNS-resident cells, and CNS-infiltrating cells as well as their potential therapeutic value in the context of inflammation and neurodegeneration.

PD-L1 positive astrocytes attenuate inflammatory functions of PD-1 positive microglia in models of autoimmune neuroinflammation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disorder of the central nervous system (CNS). Current therapies mainly target inflammatory processes during acute stages, but effective treatments for progressive MS are limited. In this context, astrocytes have gained increasing attention as they have the capacity to drive, but also suppress tissue-degeneration. Here we show that astrocytes upregulate the immunomodulatory checkpoint molecule PD-L1 during acute autoimmune CNS inflammation in response to aryl hydrocarbon receptor and interferon signaling. Using CRISPR-Cas9 genetic perturbation in combination with small-molecule and antibody-mediated inhibition of PD-L1 and PD-1 both in vivo and in vitro, we demonstrate that astrocytic PD-L1 and its interaction with microglial PD-1 is required for the attenuation of autoimmune CNS inflammation in acute and progressive stages in a mouse model of MS. Our findings suggest the glial PD-L1/PD-1 axis as a potential therapeutic target for both acute and progressive MS stages.

Astrocytes on steroids binge on synapses to cope with stress.

Many mechanisms by which stress mediates its effects within the central nervous system still remain unknown. Byun, Kim, Kim et al. find that early-life stress triggers corticosterone release to drive astrocyte-dependent synapse elimination and altered behavior. Thus, this work defines a steroid-sensitive astrocyte transcriptional circuit controlling behavior, highlighting how the study of CNS immunoregulation may shed light on behavior.

Regulation of the programmed cell death protein 1/programmed cell death ligand 1 axis in relapsing-remitting multiple sclerosis.

The programmed cell death protein 1/programmed cell death ligand 1 axis plays an important role in the adaptive immune system and has influence on neoplastic and inflammatory diseases, while its role in multiple sclerosis is unclear. Here, we aimed to analyse expression patterns of programmed cell death protein 1 and programmed cell death ligand 1 on peripheral blood mononuclear cells and their soluble variants in multiple sclerosis patients and controls, to determine their correlation with clinical disability and disease activity. In a cross-sectional study, we performed in-depth flow cytometric immunophenotyping of peripheral blood mononuclear cells and analysed soluble programmed cell death protein 1 and programmed cell death ligand 1 serum levels in patients with relapsing-remitting multiple sclerosis and controls. In comparison to control subjects, relapsing-remitting multiple sclerosis patients displayed distinct cellular programmed cell death protein 1/programmed cell death ligand 1 expression patterns in immune cell subsets and increased soluble programmed cell death ligand 1 levels, which correlated with clinical measures of disability and MRI activity over time. This study extends our knowledge of how programmed cell death protein 1 and programmed cell death ligand 1 are expressed in the membranes of patients with relapsing-remitting multiple sclerosis and describes for the first time the elevation of soluble programmed cell death ligand 1 in the blood of multiple sclerosis patients. The distinct expression pattern of membrane-bound programmed cell death protein 1 and programmed cell death ligand 1 and the correlation between soluble programmed cell death ligand 1, membrane-bound programmed cell death ligand 1, disease and clinical factors may offer therapeutic potential in the setting of multiple sclerosis and might improve future diagnosis and clinical decision-making.

Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

B-cell-specific checkpoint molecules that regulate anti-tumour immunity.

The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.