Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To ...overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.
The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors ...mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1-9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-alpha, IL-6, CXCL-10, and IL-10, and the expression of IFN-beta. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-beta, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.
Objective
The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO‐IgGs) directed against the aquaporin‐4 (AQP4) water channel located on astrocyte foot processes in the ...perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO‐IgGs and their role in disease pathogenesis.
Methods
Fluorescence‐activated cell sorting and single‐cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE).
Results
Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen‐targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4‐specific rAbs recognized conformational epitopes and mediated both AQP4‐directed antibody‐dependent cellular cytotoxicity and complement‐mediated lysis. When administered to rats with EAE, an AQP4‐specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition.
Interpretation
Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4‐specfic Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease. Ann Neurol 2009;66:617–629
Astrocytes have important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-Is) in ...astrocytes during experimental CNS autoimmunity and also in CNS lesions from patients with multiple sclerosis (MS). IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and the suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered interferon (IFN)-β are partly mediated by AHR. Dietary tryptophan is metabolized by the gut microbiota into AHR agonists that have an effect on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-aldehyde, or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AHR agonists were decreased. These findings suggest that IFN-Is produced in the CNS function in combination with metabolites derived from dietary tryptophan by the gut flora to activate AHR signaling in astrocytes and suppress CNS inflammation.
Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes ...from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14
+
cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.
Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum‐free ...media with brain‐derived growth factors (colony stimulating factor 1 receptor CSF1R ligands and TGF‐β1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia (hMGL). Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of hMGL in culture, with observations that diverge from those previously made in rodent microglia.
Main Points
Fetal bovine serum as culture supplement helps maintain microglia signature genes.
Transcriptomic differences induced by culture media alter critical immune activities.
Human microglia in culture survive independently of CSF1R signaling.
Astrocytes in multiple sclerosis Ludwin, Samuel K; Rao, Vijayaraghava TS; Moore, Craig S ...
Multiple Sclerosis Journal,
08/2016, Volume:
22, Issue:
9
Book Review, Journal Article
Peer reviewed
Recent experimental and clinical studies on astrocytes are unraveling the capabilities of these multi-functional cells in normal homeostasis, and in central nervous system (CNS) disease. This review ...focuses on understanding their behavior in all aspects of the initiation, evolution, and resolution of the multiple sclerosis (MS) lesion. Astrocytes display remarkable flexibility and variability of their physical structure and biochemical output, each aspect finely tuned to the specific stage and location of the disease, participating in both pathogenic and beneficial changes seen in acute and progressive forms. As examples, chemo-attractive or repulsive molecules may facilitate the entry of destructive immune cells but may also aid in the recruitment of oligodendrocyte precursors, essential for repair. Pro-inflammatory cytokines may attack pathogenic cells and also destroy normal oligodendrocytes, myelin, and axons. Protective trophic factors may also open the blood–brain barrier and modulate the extracellular matrix to favor recruitment and persistence of CNS-specific immune cells. A chronic glial scar may confer structural support following tissue loss and inhibit ingress of further noxious insults and also inhibit migration of reparative cells and molecules into the damaged tissue. Continual study into these processes offers the therapeutic opportunities to enhance the beneficial capabilities of these cells while limiting their destructive effects.
Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that ...under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.
Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response ...that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS.
The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.
Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection ...followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.
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