Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.

Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment.

Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy.

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.

Innate immune memory in the brain shapes neurological disease hallmarks.

Innate immune memory is a vital mechanism of myeloid cell plasticity that occurs in response to environmental stimuli and alters subsequent immune responses. Two types of immunological imprinting can be distinguished-training and tolerance. These are epigenetically mediated and enhance or suppress subsequent inflammation, respectively. Whether immune memory occurs in tissue-resident macrophages in vivo and how it may affect pathology remains largely unknown. Here we demonstrate that peripherally applied inflammatory stimuli induce acute immune training and tolerance in the brain and lead to differential epigenetic reprogramming of brain-resident macrophages (microglia) that persists for at least six months. Strikingly, in a mouse model of Alzheimer's pathology, immune training exacerbates cerebral ß-amyloidosis and immune tolerance alleviates it; similarly, peripheral immune stimulation modifies pathological features after stroke. Our results identify immune memory in the brain as an important modifier of neuropathology.

Microglial control of astrocytes in response to microbial metabolites.

Microglia and astrocytes modulate inflammation and neurodegeneration in the central nervous system (CNS)1-3. Microglia modulate pro-inflammatory and neurotoxic activities in astrocytes, but the mechanisms involved are not completely understood4,5. Here we report that TGFα and VEGF-B produced by microglia regulate the pathogenic activities of astrocytes in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Microglia-derived TGFα acts via the ErbB1 receptor in astrocytes to limit their pathogenic activities and EAE development. Conversely, microglial VEGF-B triggers FLT-1 signalling in astrocytes and worsens EAE. VEGF-B and TGFα also participate in the microglial control of human astrocytes. Furthermore, expression of TGFα and VEGF-B in CD14+ cells correlates with the multiple sclerosis lesion stage. Finally, metabolites of dietary tryptophan produced by the commensal flora control microglial activation and TGFα and VEGF-B production, modulating the transcriptional program of astrocytes and CNS inflammation through a mechanism mediated by the aryl hydrocarbon receptor. In summary, we identified positive and negative regulators that mediate the microglial control of astrocytes. Moreover, these findings define a pathway through which microbial metabolites limit pathogenic activities of microglia and astrocytes, and suppress CNS inflammation. This pathway may guide new therapies for multiple sclerosis and other neurological disorders.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Anaplastic ganglioglioma-A diagnosis comprising several distinct tumour types.

AIMS: Anaplastic ganglioglioma is a rare tumour, and diagnosis has been based on histological criteria. The 5th edition of the World Health Organization Classification of Tumours of the Central Nervous System (CNS WHO) does not list anaplastic ganglioglioma as a distinct diagnosis due to lack of molecular data in previous publications. We retrospectively compiled a cohort of 54 histologically diagnosed anaplastic gangliogliomas to explore whether the molecular profiles of these tumours represent a separate type or resolve into other entities. METHODS: Samples were subjected to histological review, desoxyribonucleic acid (DNA) methylation profiling and next-generation sequencing. Morphological and molecular data were summarised to an integrated diagnosis. RESULTS: The majority of tumours designated as anaplastic gangliogliomas resolved into other CNS WHO diagnoses, most commonly pleomorphic xanthoastrocytoma (16/54), glioblastoma, isocitrate dehydrogenase protein (IDH) wild type and diffuse paediatric-type high-grade glioma, H3 wild type and IDH wild type (11 and 2/54), followed by low-grade glial or glioneuronal tumours including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumour and diffuse leptomeningeal glioneuronal tumour (5/54), IDH mutant astrocytoma (4/54) and others (6/54). A subset of tumours (10/54) was not assignable to a CNS WHO diagnosis, and common molecular profiles pointing to a separate entity were not evident. CONCLUSIONS: In summary, we show that tumours histologically diagnosed as anaplastic ganglioglioma comprise a wide spectrum of CNS WHO tumour types with different prognostic and therapeutic implications. We therefore suggest assigning this designation with caution and recommend comprehensive molecular workup.

Oligosarcomas, IDH-mutant are distinct and aggressive.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Endogenous retroviruses are associated with hippocampus-based memory impairment.

Retrotransposons compose a staggering 40% of the mammalian genome. Among them, endogenous retroviruses (ERV) represent sequences that closely resemble the proviruses created from exogenous retroviral infection. ERVs make up 8 to 10% of human and mouse genomes and range from evolutionarily ancient sequences to recent acquisitions. Studies in Drosophila have provided a causal link between genomic retroviral elements and cognitive decline; however, in mammals, the role of ERVs in learning and memory remains unclear. Here we studied 2 independent murine models for ERV activation: muMT strain (lacking B cells and antibody production) and intracerebroventricular injection of streptozotocin (ICVI-STZ). We conducted behavioral assessments (contextual fear memory and spatial learning), as well as gene and protein analysis (RNA sequencing, PCR, immunohistochemistry, and western blot assays). Mice lacking mitochondrial antiviral-signaling protein (MAVS) and mice lacking stimulator of IFN genes protein (STING), 2 downstream sensors of ERV activation, provided confirmation of ERV impact. We found that muMT mice and ICVI-STZ mice induced hippocampal ERV activation, as shown by increased gene and protein expression of the Gag sequence of the transposable element intracisternal A-particle. ERV activation was accompanied by significant hippocampus-related memory impairment in both models. Notably, the deficiency of the MAVS pathway was protective against ICVI-STZ-induced cognitive pathology. Overall, our results demonstrate that ERV activation is associated with cognitive impairment in mice. Moreover, they provide a molecular target for strategies aimed at attenuating retroviral element sensing, via MAVS, to treat dementia and neuropsychiatric disorders.

Molecular characterisation of sporadic endolymphatic sac tumours and comparison to von Hippel-Lindau disease-related tumours.

AIMS: Although inactivation of the von Hippel-Lindau gene (VHL) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (ELSTs), the genetic background of sporadic ELST is largely unknown. The aim of this study was to determine the prevalence of VHL mutations in sporadic ELSTs and compare their characteristics to VHL-disease-related tumours. METHODS: Genetic and epigenetic alterations were compared between 11 sporadic and 11 VHL-disease-related ELSTs by targeted sequencing and DNA methylation analysis. RESULTS: VHL mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic ELSTs (82%). No other cancer-related genetic pathway was altered except for TERT promoter mutations in two sporadic ELST and one VHL-disease-related ELST (15%). Loss of heterozygosity of chromosome 3 was found in 6/10 (60%) VHL-disease-related and 10/11 (91%) sporadic ELSTs resulting in biallelic VHL inactivation in 8/10 (73%) sporadic ELSTs. DNA methylation profiling did not reveal differences between sporadic and VHL-disease-related ELSTs but reliably distinguished ELST from morphological mimics of the cerebellopontine angle. VHL patients were significantly younger at disease onset compared to sporadic ELSTs (29 vs. 52 years, p < 0.0001, Fisher's exact test). VHL-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). CONCLUSION: Biallelic inactivation of VHL is the main mechanism underlying ELSTs, but unknown mechanisms beyond VHL may rarely be involved in the pathogenesis of sporadic ELSTs.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.