BackgroundBrain inflammation plays a key role in neurological disease. Although much research has been conducted investigating inflammatory events in animal models, potential differences in human brain versus rodent models makes it imperative that we also study these phenomena in human cells and tissue.MethodsPrimary human brain cell cultures were generated from biopsy tissue of patients undergoing surgery for drug-resistant epilepsy. Cells were treated with pro-inflammatory compounds IFNγ, TNFα, IL-1β, and LPS, and chemokines IP-10 and MCP-1 were measured by immunocytochemistry, western blot, and qRT-PCR. Microarray analysis was also performed on late passage cultures treated with vehicle or IFNγ and IL-1β.ResultsEarly passage human brain cell cultures were a mixture of microglia, astrocytes, fibroblasts and pericytes. Later passage cultures contained proliferating fibroblasts and pericytes only. Under basal culture conditions all cell types showed cytoplasmic NFκB indicating that they were in a non-activated state. Expression of IP-10 and MCP-1 were significantly increased in response to pro-inflammatory stimuli. The two chemokines were expressed in mixed cultures as well as cultures of fibroblasts and pericytes only. The expression of IP-10 and MCP-1 were regulated at the mRNA and protein level, and both were secreted into cell culture media. NFκB nuclear translocation was also detected in response to pro-inflammatory cues (except IFNγ) in all cell types. Microarray analysis of brain pericytes also revealed widespread changes in gene expression in response to the combination of IFNγ and IL-1β treatment including interleukins, chemokines, cellular adhesion molecules and much more.ConclusionsAdult human brain cells are sensitive to cytokine challenge. As expected ‘classical’ brain immune cells, such as microglia and astrocytes, responded to cytokine challenge but of even more interest, brain pericytes also responded to such challenge with a rich repertoire of gene expression. Immune activation of brain pericytes may play an important role in communicating inflammatory signals to and within the brain interior and may also be involved in blood brain barrier (BBB) disruption . Targeting brain pericytes, as well as microglia and astrocytes, may provide novel opportunities for reducing brain inflammation and maintaining BBB function and brain homeostasis in human brain disease.
Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.
Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.
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