Project description:Microglia isolated from glioma patients gain anti-tumor activities upon poly (I:C) stimulation. Expression profiles of human tumor-infiltrating microglia/macrophages before (untreated) and after treatment with poly (I:C) for 48h (induced). Tumor-infiltrating microglia/macrophages were isolated from freshly excised brain tumors
Project description:Microglia, brain resident macrophages, require instruction from the central nervous system microenvironment to maintain their identity, morphology, and to regulate inflammatory responses. We investigated the heterogeneity of response of microglia to the presence of neurons and astrocytes by performing single-cell sequencing of microglia in both monoculture, and in coculture with neurons and astrocytes.
Project description:The immune functions of microglia in the brain are compromised in the presence of tumors that have the ability to “hijack” their pro-inflammatory nature by sending out signals that promote cancer progression. To analyze the tumor-supportive microglia phenotype, human microglia cells (HMC3) were treated with anti-inflammatory cytokines in serum-deprived cell culture media, and analyzed by LC-MS/MS using an Orbitrap QE instrument.
Project description:The immune functions of microglia in the brain are compromised in the presence of tumors that have the ability to “hijack” their pro-inflammatory nature by sending out signals that promote cancer progression. To analyze the tumor-supportive microglia phenotype, human microglia cells (HMC3) were treated with anti-inflammatory cytokines in serum-enriched cell culture media, and analyzed by LC-MS/MS using an Orbitrap QE instrument.
Project description:The aim of the experiment was to identify differences in gene expression in the microglia due to the presence of a brain tumor, at 14 days after a stereotactic injection of 5 x 10E4 rat C6 glioma cells into the right striatum of the rat. The control (naive) animals were left untreated. The brain hemispheres from the control animals, or the tumor-bearing hemispheres were dissected, gently dissociated into cells and the microglia (CD11b+CD45low cells) were sorted out.
Project description:Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence-activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6-week-old mice to compare the transcriptomes of FACS-sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, M-bM-^HM-<50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations. Wildtype and GFP expressing microglia from mouse brainstems were flow sorted or captured by laser microdissection. Differences between the two isolation methods were verified and further examined in neurodegenerative disease models.
Project description:Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood. Several maternal environmental factors, such as aberrant microbiome, immune activation, and poor nutrition, can influence prenatal brain development. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that after maternal immune activation (MIA) microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was concomitant with changes in the chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single-cell RNA sequencing revealed that MIA does not induce a distinct subpopulation but rather decreases the contribution to inflammatory microglia states. Prenatal replacement of MIA microglia with physiological infiltration of naïve microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment impacts the long-term microglia reactivity and proper striatal circuit development.
Project description:Determination of the mechanism by which microglia regulate growth of brain tumor initiating cells (BTICs) and differentiation. Results identify the factors involved in the regulation and provide mechanistic basis. We subjected brain tumor initiating cells to microarray to determine the genes involved in BTICs growth and differentiation when exposed to microglia conditioned medium (MCM) for 6h. BTICs were grown in MCM or control medium for 6h, and media was removed and then the cells were subjected for RNA extraction and hybridization on Affymatrix microarrays. Three (3) control and four (4) MCM samples were generated. The overall objective was to collect the RNA from the MCM exposed differentiated BTICs.
Project description:Microglia become activated by disturbances in the homeostasis of their local microenvironment. While there are varying degrees of microglia activation, a pro-inflammatory reactive state induced by exposure to stimuli like LPS and IFN-γ. In this study, we identified a total of 5497 proteins in whole cell proteome and 4938 proteins for secretome of activated BV-2 mouse microglia cell line with LPS or IFN-γ using improved shotgun proteomic approach. From the differentially expressed proteins in stimulated microglia, we were able to classify pathways related immune-inflammatory response or metabolism. Moreover, we performed longitudinal quantification of secreted proteins during the detrimental activation of microglia which releases neurotoxic molecules mediated neuronal cell loss in the brain region.