Elucidation of neuronal and glial cell phenotypes and functions utilizing mass spectrometry techniques

Microglia and brain macrophages are increasingly evidenced as key regulators in CNS development, homeostasis and pathology. A better understanding of the cellular dynamics and mechanisms that regulate microglial homeostasis and function will provide the means to manipulate these cells for therapeutic purposes. In the first section of the study, the cellular complexity of CNS myeloid compartment were unraveled using an adoptive transfer experiment with gene-modified bone marrow cells, bone marrow transplantation in a mouse model of facial nerve axotomy or high-throughput techniques such as single-cell RNA sequencing (scRNA-Seq). These findings demonstrated that, followed CNS conditioning, bone marrow-derived cells are recruited to the CNS including the retina, preferentially to the lesioned sites of the CNS. These infiltrating bone marrow-derived macrophages stably integrated into the CNS myeloid cell compartment of the lesioned brain. Similarly, the results obtained from scRNA-Seq revealed spatial and temporal microglial heterogeneity in both mouse and human brain. In diseased brain, the composition of microglial sub-populations was altered, and their microglial signatures could be rapidly changed during neurodegeneration (such as facial nerve axotomy) and/or neuroinflammation (such as multiple sclerosis). In the second part of the study, microglial heterogeneity was investigated in human post-mortem brain tissue and fresh brain biopsies at the single-cell protein level. Again, these results highlight the cellular complexity of the CNS myeloid compartment including the microglia subpopulations described in the first section, which complemented the transcriptomic signatures revealed by scRNA-Seq. Moreover, the findings translated mouse microglial phenotypes to the human system, emphasizing the translational potential of the methodology for further investigation in clinical applications. Besides single-cell phenotypic and functional characterization by mass cytometry, functions and metabolomics of CNS cells can be assessed using mass spectrometry. For this approach, the human neuroblastoma cell line SH-SY5Y was used for the establishment of the methodology. The study demonstrated that the SHSY5Y cell line was capable of synthesizing targeted metabolites (in this case “morphine”). Briefly, SH-SY5Y cells were cultured in the presence of 13C-, 2H- or 18O-labelled precursors of the morphine biosynthetic pathway. SH-SY5Y cells de novo incorporate the stable isotope-labelledprecursors into endogenously synthesized morphine. The finding unequivocally proved the capacity of de novo morphine synthesis of human neuroblastoma cells. This established methodology can be applied for future metabolomic study of CNS cells including microglial cells

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

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

Immune modulatory effect of a novel 4,5-dihydroxy-3,3´,4´-trimethoxybibenzyl from Dendrobium lindleyi

Dendrobium bibenzyls and phenanthrenes such as chrysotoxine, cypripedin, gigantol and moscatilin have been reported to show promising inhibitory effects on lung cancer growth and metastasis in ex vivo human cell line models, suggesting their potential for clinical application in patients with lung cancer. However, it remains to be determined whether these therapeutic effects can be also seen in primary human cells and/or in vivo. In this study, we comparatively investigated the immune modulatory effects of bibenzyls and phenanthrenes, including a novel Dendrobium bibenzyl derivative, in primary human monocytes. All compounds were isolated and purified from a Thai orchid Dendrobium lindleyi Steud, a new source of therapeutic compounds with promising potential of tissue culture production. We detected increased frequencies of TNF- and IL-6-expressing monocytes after treatment with gigantol and cypripedin, whereas chrysotoxine and moscatilin did not alter the expression of these cytokines in monocytes. Interestingly, the new 4,5-dihydroxy-3,3′,4′-trimethoxybibenzyl derivative showed dose-dependent immune modulatory effects in lipopolysaccharide (LPS)-treated CD14lo and CD14hi monocytes. Together, our findings show immune modulatory effects of the new bibenzyl derivative from Dendrobium lindleyi on different monocyte sub-populations. However, therapeutic consequences of these different monocyte populations on human diseases including cancer remain to be investigated

Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPβhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68- dendritic cells. Unlike in Crohn's disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS

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

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.</p

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

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 CD(4+) effector T cells producing IL-17A and TNF, CD(8+) T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store-operated Ca(2+) entry (SOCE), which results from the opening of Ca(2+) release-activated Ca(2+) (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