Protocol of a prospective multicenter study on comorbidity impact on multiple sclerosis and antibody-mediated diseases of the central nervous system (COMMIT).

Comorbidities in patients with multiple sclerosis (MS) and antibody-mediated diseases of the central nervous system (CNS) including neuromyelitis optica spectrum disorder (NMOSD), and myelin oligodendrocyte glycoprotein (MOG)-antibody-associated disease (MOGAD) are common and may influence the course of their neurological disease. Comorbidity may contribute to neuronal injury and therefore limit recovery from attacks, accelerate disease progression, and increase disability. This study aims to explore the impact of comorbidity, particularly vascular comorbidity, and related risk factors on clinical and paraclinical parameters of MS, NMOSD and MOGAD. We propose COMMIT, a prospective multicenter study with longitudinal follow-up of patients with MS, NMOSD, and MOGAD, with or without comorbidities, as well as healthy subjects as controls. Subjects will be stratified by age, sex and ethnicity. In consecutive samples we will analyze levels of inflammation and neurodegeneration markers in both fluid and cellular compartments of the peripheral blood and cerebrospinal fluid (CSF) using multiple state-of-the-art technologies, including untargeted proteomics and targeted ultrasensitive ELISA assays and quantitative reverse transcription polymerase chain reaction (RT-qPCR) as well as high-dimensional single-cell technologies i.e., mass cytometry and single-cell RNA sequencing. Algorithm-based data analyses will be used to unravel the relationship between these markers, optical coherence tomography (OCT) and magnetic resonance imaging (MRI), and clinical outcomes including frequency and severity of relapses, long-term disability, and quality of life. The goal is to evaluate the impact of comorbidities on MS, NMOSD, and MOGAD which may lead to development of treatment approaches to improve outcomes of inflammatory demyelinating diseases of the CNS.

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.

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.

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.

Proteomic and transcriptomic profiling of brainstem, cerebellum and olfactory tissues in early- and late-phase COVID-19.

Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19.

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.

Associations of myeloid cells with cellular and humoral responses following vaccinations in patients with neuroimmunological diseases.

Disease-modifying therapies (DMTs) are widely used in neuroimmunological diseases such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Although these treatments are known to predispose patients to infections and affect their responses to vaccination, little is known about the impact of DMTs on the myeloid cell compartment. In this study, we use mass cytometry to examine DMT-associated changes in the innate immune system in untreated and treated patients with MS (n = 39) or NMOSD (n = 23). We also investigated the association between changes in myeloid cell phenotypes and longitudinal responsiveness to homologous primary, secondary, and tertiary SARS-CoV-2 mRNA vaccinations. Multiple DMT-associated myeloid cell clusters, in particular CD64+HLADRlow granulocytes, showed significant correlations with B and T cell responses induced by vaccination. Our findings suggest the potential role of myeloid cells in cellular and humoral responses following vaccination in DMT-treated patients with neuroimmunological 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.

Inhibitory Effect of Isopanduratin A on Adipogenesis: A Study of Possible Mechanisms.

The root of Boesenbergia rotunda, a culinary plant commonly known as fingerroot, has previously been reported to possess anti-obesity activity, with four flavonoids identified as active principles, including pinostrobin, panduratin A, cardamonin, and isopanduratin A. However, the molecular mechanisms underlying the antiadipogenic potential of isopanduratin A remain unknown. In this study, isopanduratin A at non-cytotoxic concentrations (1-10 µM) significantly suppressed lipid accumulation in murine (3T3-L1) and human (PCS-210-010) adipocytes in a dose-dependent manner. Downregulation of adipogenic effectors (FAS, PLIN1, LPL, and adiponectin) and adipogenic transcription factors (SREBP-1c, PPARγ, and C/EBPα) occurred in differentiated 3T3-L1 cells treated with varying concentrations of isopanduratin A. The compound deactivated the upstream regulatory signals of AKT/GSK3ß and MAPKs (ERK, JNK, and p38) but stimulated the AMPK-ACC pathway. The inhibitory trend of isopanduratin A was also observed with the proliferation of 3T3-L1 cells. The compound also paused the passage of 3T3-L1 cells by inducing cell cycle arrest at the G0/G1 phase, supported by altered levels of cyclins D1 and D3 and CDK2. Impaired p-ERK/ERK signaling might be responsible for the delay in mitotic clonal expansion. These findings revealed that isopanduratin A is a strong adipogenic suppressor with multi-target mechanisms and contributes significantly to anti-obesogenic activity. These results suggest the potential of fingerroot as a functional food for weight control and obesity prevention.

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.

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.

Differential compartmentalization of myeloid cell phenotypes and responses towards the CNS in Alzheimer's disease.

Myeloid cells are suggested as an important player in Alzheimer´s disease (AD). However, its continuum of phenotypic and functional changes across different body compartments and their use as a biomarker in AD remains elusive. Here, we perform multiple state-of-the-art analyses to phenotypically and metabolically characterize immune cells between peripheral blood (n = 117), cerebrospinal fluid (CSF, n = 117), choroid plexus (CP, n = 13) and brain parenchyma (n = 13). We find that CSF cells increase expression of markers involved in inflammation, phagocytosis, and metabolism. Changes in phenotype of myeloid cells from AD patients are more pronounced in CP and brain parenchyma and upon in vitro stimulation, suggesting that AD-myeloid cells are more vulnerable to environmental changes. Our findings underscore the importance of myeloid cells in AD and the detailed characterization across body compartments may serve as a resource for future studies focusing on the assessment of these cells as biomarkers in AD.

Isotopic tracing of glucose metabolites in human monocytes to assess changes in inflammatory conditions.

Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.

Store-operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses. Using mass cytometry (CyTOF) to analyze the immune cell composition in the lamina propria (LP) of patients with ulcerative colitis (UC) and Crohn's disease (CD), we observed an enrichment of CD4+ effector T cells producing IL-17A and TNF, CD8+ T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store-operated Ca2+ entry (SOCE), which results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI and STIM proteins. We observed that the pharmacologic inhibition of SOCE attenuated the production of proinflammatory cytokines including IL-2, IL-4, IL-6, IL-17A, TNF, and IFNγ by human colonic T cells and ILCs, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, but had no effect on the viability, differentiation, and function of intestinal epithelial cells. T cell-specific deletion of CRAC channel genes in mice showed that Orai1, Stim1, and Stim2-deficient T cells have quantitatively distinct defects in SOCE, which correlate with gradually more pronounced impairment of cytokine production by Th1 and Th17 cells and the severity of IBD. Moreover, the pharmacologic inhibition of SOCE with a selective CRAC channel inhibitor attenuated IBD severity and colitogenic T cell function in mice. Our data indicate that SOCE inhibition may be a suitable new approach for the treatment of IBD.

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.

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.

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.

Human small intestinal infection by SARS-CoV-2 is characterized by a mucosal infiltration with activated CD8+ T cells.

The SARS-CoV-2 pandemic has so far claimed over three and a half million lives worldwide. Though the SARS-CoV-2 mediated disease COVID-19 has first been characterized by an infection of the upper airways and the lung, recent evidence suggests a complex disease including gastrointestinal symptoms. Even if a direct viral tropism of intestinal cells has recently been demonstrated, it remains unclear, whether gastrointestinal symptoms are caused by direct infection of the gastrointestinal tract by SARS-CoV-2 or whether they are a consequence of a systemic immune activation and subsequent modulation of the mucosal immune system. To better understand the cause of intestinal symptoms we analyzed biopsies of the small intestine from SARS-CoV-2 infected individuals. Applying qRT-PCR and immunohistochemistry, we detected SARS-CoV-2 RNA and nucleocapsid protein in duodenal mucosa. In addition, applying imaging mass cytometry and immunohistochemistry, we identified histomorphological changes of the epithelium, which were characterized by an accumulation of activated intraepithelial CD8+ T cells as well as epithelial apoptosis and subsequent regenerative proliferation in the small intestine of COVID-19 patients. In summary, our findings indicate that intraepithelial CD8+ T cells are activated upon infection of intestinal epithelial cells with SARS-CoV-2, providing one possible explanation for gastrointestinal symptoms associated with COVID-19.

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.