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.

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.

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.

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.

1,2-13C2-Glucose Tracing Approach to Assess Metabolic Alterations of Human Monocytes under Neuroinflammatory Conditions.

Neuroinflammation is one of the common features in most neurological diseases including multiple sclerosis (MScl) and neurodegenerative diseases such as Alzheimer's disease (AD). It is associated with local brain inflammation, microglial activation, and infiltration of peripheral immune cells into cerebrospinal fluid (CSF) and the central nervous system (CNS). It has been shown that the diversity of phenotypic changes in monocytes in CSF relates to neuroinflammation. It remains to be investigated whether these phenotypic changes are associated with functional or metabolic alteration, which may give a hint to their function or changes in cell states, e.g., cell activation. In this article, we investigate whether major metabolic pathways of blood monocytes alter after exposure to CSF of healthy individuals or patients with AD or MScl. Our findings show a significant alteration of the metabolism of monocytes treated with CSF from patients and healthy donors, including higher production of citric acid and glutamine, suggesting a more active glycolysis and tricarboxylic acid (TCA) cycle and reduced production of glycine and serine. These alterations suggest metabolic reprogramming of monocytes, possibly related to the change of compartment (from blood to CSF) and/or disease-related. Moreover, the levels of serine differ between AD and MScl, suggesting different phenotypic alterations between diseases.

Immunomodulatory Effects of New Phenanthrene Derivatives from Dendrobium crumenatum.

Three new phenanthrene derivatives (1, 2, 4), one new fluorenone (3), and four known compounds (5-8) were isolated from the ethyl acetate extract of Dendrobium crumenatum Sw. stems using column chromatography. The chemical structures were elucidated by analysis of spectroscopic data. The absolute configuration of 4 was determined by electronic circular dichroism calculation. We also evaluated the immunomodulatory effects of compounds isolated from D. crumenatum in human peripheral blood mononuclear cells from healthy individuals and those from patients with multiple sclerosis in vitro. Dendrocrumenol B (2) and dendrocrumenol D (4) showed strong immunomodulatory effects on both CD3+ T cells and CD14+ monocytes. Compounds 2 and 4 could reduce IL-2 and TNF production in T cells and monocytes that were treated with phorbol-12-myristate-13-acetate and ionomycin (PMA/Iono). Deep immune profiling using high-dimensional single-cell mass cytometry could confirm immunomodulatory effects of 4, quantified by the reduction of activated T cell population under PMA/Iono stimulation, in comparison to the stimulated T cells without treatment.

Phenotypic comparison of human alveolar macrophages before and after in vivo rhinovirus 16 challenge.

We used mass cytometry to extensively characterize bronchoalveolar lavage macrophages before and two days after in vivo rhinovirus 16 infection in a heterogeneous population of healthy and asthma/COPD subjects. Multivariate partial least squares discriminant analysis revealed distinct clusters of alveolar macrophages before versus after the virus, suggesting changes in overall phenotype.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

SARS-CoV-2 mRNA vaccinations fail to elicit humoral and cellular immune responses in patients with multiple sclerosis receiving fingolimod.

BACKGROUND: SARS-CoV-2 mRNA vaccination of healthy individuals is highly immunogenic and protective against severe COVID-19. However, there are limited data on how disease-modifying therapies (DMTs) alter SARS-CoV-2 mRNA vaccine immunogenicity in patients with autoimmune diseases. METHODS: As part of a prospective cohort study, we investigated the induction, stability and boosting of vaccine-specific antibodies, B cells and T cells in patients with multiple sclerosis (MS) on different DMTs after homologous primary, secondary and booster SARS-CoV-2 mRNA vaccinations. Of 126 patients with MS analysed, 105 received either anti-CD20-based B cell depletion (aCD20-BCD), fingolimod, interferon-ß, dimethyl fumarate, glatiramer acetate, teriflunomide or natalizumab, and 21 were untreated MS patients for comparison. RESULTS: In contrast to all other MS patients, and even after booster, most aCD20-BCD- and fingolimod-treated patients showed no to markedly reduced anti-S1 IgG, serum neutralising activity and a lack of receptor binding domain-specific and S2-specific B cells. Patients receiving fingolimod additionally lacked spike-reactive CD4+ T cell responses. The duration of fingolimod treatment, rather than peripheral blood B and T cell counts prior to vaccination, determined whether a humoral immune response was elicited. CONCLUSIONS: The lack of immunogenicity under long-term fingolimod treatment demonstrates that functional immune responses require not only immune cells themselves, but also access of these cells to the site of inoculation and their unimpeded movement. The absence of humoral and T cell responses suggests that fingolimod-treated patients with MS are at risk for severe SARS-CoV-2 infections despite booster vaccinations, which is highly relevant for clinical decision-making and adapted protective measures, particularly considering additional recently approved sphingosine-1-phosphate receptor antagonists for MS treatment.

Berlin Registry of Neuroimmunological entities (BERLimmun): protocol of a prospective observational study.

BACKGROUND: Large-scale disease overarching longitudinal data are rare in the field of neuroimmunology. However, such data could aid early disease stratification, understanding disease etiology and ultimately improve treatment decisions. The Berlin Registry of Neuroimmunological Entities (BERLimmun) is a longitudinal prospective observational study, which aims to identify diagnostic, disease activity and prognostic markers and to elucidate the underlying pathobiology of neuroimmunological diseases. METHODS: BERLimmun is a single-center prospective observational study of planned 650 patients with neuroimmunological disease entity (e.g. but not confined to: multiple sclerosis, isolated syndromes, neuromyelitis optica spectrum disorders) and 85 healthy participants with 15 years of follow-up. The protocol comprises annual in-person visits with multimodal standardized assessments of medical history, rater-based disability staging, patient-report of lifestyle, diet, general health and disease specific symptoms, tests of motor, cognitive and visual functions, structural imaging of the neuroaxis and retina and extensive sampling of biological specimen. DISCUSSION: The BERLimmun database allows to investigate multiple key aspects of neuroimmunological diseases, such as immunological differences between diagnoses or compared to healthy participants, interrelations between findings of functional impairment and structural change, trajectories of change for different biomarkers over time and, importantly, to study determinants of the long-term disease course. BERLimmun opens an opportunity to a better understanding and distinction of neuroimmunological diseases.

Lentiviral delivery of human erythropoietin attenuates hippocampal atrophy and improves cognition in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a trinucleotide (CAG) repeat expansion in the huntingtin gene (HTT). The R6/2 transgenic mouse model of HD expresses exon 1 of the human HTT gene with approximately 150 CAG repeats. R6/2 mice develop progressive behavioural abnormalities, impaired neurogenesis, and atrophy of several brain regions. In recent years, erythropoietin (EPO) has been shown to confer neuroprotection and enhance neurogenesis, rendering it a promising molecule to attenuate HD symptoms. In this study, the therapeutic potential of EPO was evaluated in female R6/2 transgenic mice. A single bilateral injection of a lentivirus encoding human EPO (LV-hEPO) was performed into the lateral ventricles of R6/2 mice at disease onset (8 weeks of age). Control groups were either untreated or injected with a lentivirus encoding green fluorescent protein (LV-GFP). Thirty days after virus administration, hEPO mRNA and protein were present in injected R6/2 brains. Compared to control R6/2 mice, LV-hEPO-treated R6/2 mice exhibited reduced hippocampal atrophy, increased neuroblast branching towards the dentate granular cell layer, and improved spatial cognition. Our results suggest that LV-hEPO administration may be a promising strategy to reduce cognitive impairment in HD.

A characterization of the molecular phenotype and inflammatory response of schizophrenia patient-derived microglia-like cells.

Different lines of evidence support a causal role for microglia in the pathogenesis of schizophrenia. However, how schizophrenia patient-derived microglia are affected at the phenotypic and functional level is still largely unknown. We used a recently described model to induce patient-derived microglia-like cells and used this to analyze changes in the molecular phenotype and function of myeloid cells in schizophrenia. We isolated monocytes from twenty recent-onset schizophrenia patients and twenty non-psychiatric controls. We cultured the cells towards an induced microglia-like phenotype (iMG), analyzed the phenotype of the cells by RNA sequencing and mass cytometry, and their response to LPS. Mass cytometry showed a high heterogeneity of iMG in cells derived from patients as well as controls. The prevalence of two iMG clusters was significantly higher in schizophrenia patients (adjusted p-value < 0.001). These subsets are characterized by expression of ApoE, Ccr2, CD18, CD44, and CD95, as well as IRF8, P2Y12, Cx3cr1 and HLA-DR. In addition, we found that patient-derived iMG show an enhanced response to LPS, with increased secretion of TNF-α. Further studies are needed to replicate these findings, to determine whether similar subclusters are present in schizophrenia patients in vivo, and to address how these subclusters are related to the increased response to LPS, as well as other microglial functions.

Myeloid cell-based therapies in neurological disorders: How far have we come?

The pathogenesis of neurological disorders such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) is multifactorial and incompletely understood. The development of therapies for these disorders of the central nervous system (CNS) is thus far very challenging. Neuroinflammation is one of the processes that contribute to the pathogenesis of CNS diseases, and therefore represents an important therapeutic target. Myeloid cells derived from the bone marrow are ideal candidates for cell therapy in the CNS as they are capable of targeting the brain and providing neuroprotective and anti-inflammatory effects. In this review, experimental and clinical evidence for the therapeutic potential of myeloid cells in neurological disorders will be discussed. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon ß-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.

Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke.

BACKGROUND AND PURPOSE: Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive. METHODS: The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor-deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity. RESULTS: Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor-deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung. CONCLUSIONS: Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.

Diverse modulatory effects of bibenzyls from Dendrobium species on human immune cell responses under inflammatory conditions.

Dendrobium plants are widely used in traditional Chinese medicine. Their secondary metabolites such as bibenzyls and phenanthrenes show various pharmacological benefits such as immunomodulation and inhibitory effects on cancer cell growth. However, our previous study also showed that some of these promising compounds (i.e., gigantol and cypripedin) also induced the expression of inflammatory cytokines including TNF in human monocytes, and thus raising concerns about the use of these compounds in clinical application. Furthermore, the effects of these compounds on other immune cell populations, apart from monocytes, remain to be investigated. In this study, we evaluated immunomodulatory effects of seven known bibenzyl compounds purified from Dendrobium species in human peripheral blood mononuclear cells (PBMCs) that were stimulated with lipopolysaccharide (LPS). Firstly, using flow cytometry, moscatilin (3) and crepidatin (4) showed the most promising dose-dependent immunomodulatory effects among all seven bibenzyls, determined by significant reduction of TNF expression in LPS-stimulated CD14+ monocytes. Only crepidatin at the concentration of 20 µM showed a significant cytotoxicity, i.e., an increased cell death in late apoptotic state. In addition, deep immune profiling using high-dimensional single-cell mass cytometry (CyTOF) revealed broad effects of Dendrobium compounds on diverse immune cell types. Our findings suggest that to precisely evaluate therapeutic as well as adverse effects of active natural compounds, a multi-parameter immune profiling targeting diverse immune cell population is required.

Protocol to characterize immune cell subpopulations in cerebrospinal fluid of patients with neuroinflammatory diseases using mass cytometry.

Phenotypic and compositional changes of immune cells in cerebrospinal fluid (CSF) can be used as biomarkers to help diagnose and track disease activity for neuroinflammatory and neurodegenerative diseases. Here, we present a workflow to perform high-dimensional immune profiling at single-cell resolution using cytometry by time-of-flight (CyTOF) on cells isolated from the CSF of patients with neuroinflammation. We describe steps for sample collection and preparation, barcoding to allow for multiplexing, and downstream data analysis using R. For complete details on the use and execution of this protocol, please refer to Fernández-Zapata et al.1.

New Phenolic Glycosides from Coelogyne fuscescens Lindl. var. brunnea and Their Cytotoxicity against Human Breast Cancer Cells.

The phytochemical investigation of the whole plants of Coelogyne fuscescens Lindl. var. brunnea led to the discovery of three new phenolic glycosides, i.e., coelofusides A-C (1-3) and 12 known compounds (4-15). For the first time, we reported the nuclear magnetic resonance (NMR) data of 4-O-(6'-O-glucosyl-4″-hydroxybenzoyl)-4-hydroxybenzyl alcohol (4) in this study. The identification of the structures of newly discovered compounds was done through the analysis of their spectroscopic data [NMR, mass spectrometry, ultraviolet, Fourier transform infrared, optical rotation, and circular dichroism (CD)]. In comparison to anticancer drugs (i.e., etoposide and carboplatin), we evaluated anticancer potential of the isolated compounds on two different breast cancer cell lines, namely, T47D and MDA-MB-231. Human fibroblast HaCaT cells were used as the control cells. After a 48 h incubation, flavidin (8), coelonin (10), 3,4-dihydroxybenzaldehyde (11), and oxoflavidin (12) showed significant cytotoxic effects against breast cancer cells. Among them, oxoflavidin (12) exhibited the most potent cytotoxicity on MDA-MB-231 with an IC50 value of 26.26 ± 4.33 µM. In the nuclear staining assay, oxoflavidin induced apoptosis after 48 h in both T47D and MDA-MB-231 cells in a dose-dependent manner. Furthermore, oxoflavidin upregulated the expression of apoptotic genes, such as p53, Bax, poly(ADP-ribose) polymerase, caspase-3, and caspase-9 genes while significantly decreasing antiapoptotic protein (Bcl-2) expression levels.

Multiomic spatial landscape of innate immune cells at human central nervous system borders.

The innate immune compartment of the human central nervous system (CNS) is highly diverse and includes several immune-cell populations such as macrophages that are frequent in the brain parenchyma (microglia) and less numerous at the brain interfaces as CNS-associated macrophages (CAMs). Due to their scantiness and particular location, little is known about the presence of temporally and spatially restricted CAM subclasses during development, health and perturbation. Here we combined single-cell RNA sequencing, time-of-flight mass cytometry and single-cell spatial transcriptomics with fate mapping and advanced immunohistochemistry to comprehensively characterize the immune system at human CNS interfaces with over 356,000 analyzed transcriptomes from 102 individuals. We also provide a comprehensive analysis of resident and engrafted myeloid cells in the brains of 15 individuals with peripheral blood stem cell transplantation, revealing compartment-specific engraftment rates across different CNS interfaces. Integrated multiomic and high-resolution spatial transcriptome analysis of anatomically dissected glioblastoma samples shows regionally distinct myeloid cell-type distributions driven by hypoxia. Notably, the glioblastoma-associated hypoxia response was distinct from the physiological hypoxia response in fetal microglia and CAMs. Our results highlight myeloid diversity at the interfaces of the human CNS with the periphery and provide insights into the complexities of the human brain's immune system.