Multiple sclerosis is a chronic inflammatory disease of the CNS 1 . Astrocytes contribute to the pathogenesis of multiple sclerosis 2 , but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-(IFN) produced by meningeal natural killer (NK) cells, in which IFN expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFN+ NK cells that are licensed by the microbiome.

Graphical Abstract Highlights d A multidisciplinary approach to study effects of environmental factors on astrocytes d Linuron boosts IRE1a-XBP1 signaling in astrocytes via Sigmar1 d Astrocytic IRE1a-XBP1 signaling promotes CNS inflammation in EAE d IRE1a-XBP1 signaling is activated in astrocytes in multiple sclerosis

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Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L -kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4 + T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3 + regulatory T cells and the production of immunosuppressive transforming growth factor β in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.

Endotoxin tolerance aims at opposing hyperinflammatory responses to lipopolysaccharide (LPS) exposure. The aryl hydrocarbon receptor (AhR) participates in protection against LPS-mediated tissue damage, as it plays a necessary role in restraining the proinflammatory action of IL-1β and TNF-α while fostering the expression of protective TGF-β. TGF-β, in turn, promotes durable expression of the immune regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 degrades L-tryptophan to L-kynurenine-an activating ligand for AhR-thus establishing a feed-forward loop. In this study, we further demonstrate that L-kynurenine also promotes the dissociation of the Src kinase-AhR cytosolic complex, leading to the activation of both genomic and non-genomic events in conventional dendritic cells (cDCs) primed with LPS. Specifically, the Src kinase, by phosphorylating the downstream target IDO1, triggers IDO1's signaling ability, which results in enhanced production of TGF-β, an event key to establishing full endotoxin tolerance. We demonstrated that exogenous L-kynurenine can substitute for the effects of continued or repeated LPS exposure and that the AhR-Src-IDO1 axis represents a critical step for the transition from endotoxin susceptibility to tolerance. Moreover, much like fully endotoxin-tolerant dendritic cells (DCs) (i.e., treated twice with LPS in vitro), DCs-treated once with LPS in vitro and then with kynurenine-confer resistance on naïve recipients to an otherwise lethal LPS challenge. This may have clinical implications under conditions in which pharmacologically induced onset of endotoxin tolerance is a therapeutically desirable event.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates numerous cellular responses. Originally investigated in toxicology because of its ability to bind environmental contaminants, AhR has attracted enormous attention in the field of immunology in the last 20 years. In addition, the discovery of endogenous and plant-derived ligands points to AhR also having a crucial role in normal cell physiology. Thus, AhR is emerging as a promiscuous receptor that can mediate either toxic or physiologic effects upon sensing multiple exogenous and endogenous molecules. Within this scenario, several factors appear to contribute to the outcome of gene transcriptional regulation by AhR, including the nature of the ligand as such and its further metabolism by AhR-induced enzymes, the local tissue microenvironment, and the presence of coregulators or specific transcription factors in the cell. Here, we review the current knowledge on the array of transcription factors and coregulators that, by interacting with AhR, tune its transcriptional activity in response to endogenous and exogenous ligands.

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