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Brain cancers carry bleak prognoses, with therapeutic advances helping only a minority of patients over the past decade. The brain tumour microenvironment (TME) is highly immunosuppressive and differs from that of other malignancies as a result of the glial, neural and immune cell populations that constitute it. Until recently, the study of the brain TME was limited by the lack of methods to de-convolute this complex system at the single-cell level. However, novel technical approaches have begun to reveal the immunosuppressive and tumour-promoting properties of distinct glial and myeloid cell populations in the TME, identifying new therapeutic opportunities. Here, we discuss the immune modulatory functions of microglia, monocyte-derived macrophages and astrocytes in brain metastases and glioma, highlighting their disease-associated heterogeneity and drawing from the insights gained by studying these malignancies and other neurological disorders. Lastly, we consider potential approaches for the therapeutic modulation of the brain TME.This review discusses the immunosuppressive and tumour-promoting properties of microglia, monocyte-derived macrophage and astrocyte subsets in the brain tumour microenvironment, identifying new therapeutic opportunities for primary brain tumours and brain metastases.

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells 1 , 2 . Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders 3 , 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. Lactate produced by dendritic cells (DCs) suppresses T-cell-mediated autoimmunity through a mechanism in which lactate activates HIF-1α–NDUFA4L2 signalling in DCs and thereby limits DC-mediated pro-inflammatory responses such as the development of encephalitogenic T cells.

Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD) 1 —a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity 2 . However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR–NF-κB–C/EBPβ signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases. The herbicide propyzamide increases inflammation in the small and large intestine, and the AHR–NF-κB–C/EBPβ signalling axis—which operates in T cells and dendritic cells to promote intestinal inflammation—is targeted by propyzamide.

Cell–cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell–cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell–cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR–Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell–cell interactions. Key points SPEAC-seq uses CRISPR screening, cell coculture in droplets and microfluidic sorting to enable forward genetic screens of regulators of cell–cell communication. The procedure provides a detailed experimental workflow and discusses technical aspects. SPEAC-seq enables the defined pairing of interaction partners, is adaptable to different CRISPR screening modalities, is scalable for unbiased genome-wide discovery of functional regulators and can be adapted with few modifications to study a variety of cell types. This protocol presents a method that combines genome-wide CRISPR libraries with cell coculture in droplets to study functional regulators of cell–cell communication.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis 1 , 2 , 3 , 4 , 5 , 6 , 7 – 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. In an experimental autoimmune encephalomyelitis model in mice, a subset of astrocytes retains an epigenetically regulated memory of past inflammation, causing exacerbated inflammation upon subsequent rechallenge.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system 1 . Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis 2 , 3 . However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis 4 . Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR–Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers. The pathogenic function of XBP1-expressing astrocytes in experimental autoimmune encephalomyelitis and multiple sclerosis have been studied using FIND-seq, a new method combining microfluidics cytometry, PCR-based detection of nucleic acids and cell sorting for in-depth single-cell transcriptomics analyses of rare cells.

Neuroimmune interactions—signals transmitted between immune and brain cells—regulate many aspects of tissue physiology 1 , including responses to psychological stress 2 , 3 , 4 – 5 , which can predispose individuals to develop neuropsychiatric diseases 6 , 7 , 8 – 9 . Still, the interactions between haematopoietic and brain-resident cells that influence complex behaviours are poorly understood. Here, we use a combination of genomic and behavioural screens to show that astrocytes in the amygdala limit stress-induced fear behaviour through epidermal growth factor receptor (EGFR). Mechanistically, EGFR expression in amygdala astrocytes inhibits a stress-induced, pro-inflammatory signal-transduction cascade that facilitates neuron–glial crosstalk and stress-induced fear behaviour through the orphan nuclear receptor NR2F2 in amygdala neurons. In turn, decreased EGFR signalling and fear behaviour are associated with the recruitment of meningeal monocytes during chronic stress. This set of neuroimmune interactions is therapeutically targetable through the administration of psychedelic compounds, which reversed the accumulation of monocytes in the brain meninges along with fear behaviour. Together with validation in clinical samples, these data suggest that psychedelics can be used to target neuroimmune interactions relevant to neuropsychiatric disorders and potentially other inflammatory diseases. Inflammatory monocytes in the brain meninges promote stress-induced fear behaviour, and the pathways involved can be modulated using psychedelic compounds.

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) . 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 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 ; 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.

Astrocytes are multifunctional cells of the central nervous system (CNS), are intimately linked into the immune modulatory processes that govern homeostasis and neuroinflammation, and display significant functional heterogeneity. The discovery of specific astrocyte subsets associated with various neurological diseases, including Multiple Sclerosis (MS), has prompted the investigation of astrocyte subsets within the brain tumor microenvironment (TME). As bulk transcriptomic analyses fail to address this question, there is need for high-resolution data to deconvolute complex astrocyte reactive programs found in the TME. To this end, we established a syngeneic model of glioblastoma (GBM) by orthotopic implantation of the GL261 cell line in transgenic C57BL6 mice expressing a fluorescent reporter under the control of the pan-astrocyte marker Aldh1l1. Aldh1l1-expressing astrocytes harvested 15 days after GL261 orthotopic tumor implantation were analyzed by droplet-based single-cell RNA sequencing (DropSeq). We identified 14 distinct astrocyte subsets based on transcriptional similarities, and at least 4 subsets that were specifically enriched in the TME. The most highly expanded astrocyte subset (cluster 0) presented a mixed phenotype with upregulation of both pro- and anti-inflammatory pathways, along with loss of homeostatic neurosupportive functions. We observed strong evidence of interferon-γ (IFNγ) signaling, leading us to hypothesize that cluster 0 astrocytes could represent an analogous subset to the regulatory IFNγ-induced TRAIL+ astrocyte population observed in a model of the chronic autoinflammatory disease MS. The cluster 0 signature was similar to that of the previously reported T cell-killing TRAIL+ astrocytes. In vivo, we observed increased TRAIL expression on astrocytes in the TME, along with evidence of transcriptionally activating STAT1 phosphorylation. To validate our findings in mice, we performed single-nucleus RNA sequencing from 10 GBM patients and detected increased expression of IFNγ–TRAIL axis components, although cell type identification was not yet possible. By use of an independent cohort of human GBM, we validated the presence of an astrocyte subset associated with early recurrence that showed signs of IFNγ signaling. With this study, we aim to shed light on astrocyte subpopulations in the GBM microenvironment and to uncover regulatory pathways, such as immunosuppressive IFNγ signaling, that may represent a therapeutic target.