AffyExon1 genearray expression profile of adult mouse microglia and splenic Ly6C monocytes
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ABSTRACT: Gene profiling of CNS-derived microglia vs splenic CD11b+Ly6C+ monocyte subsets deom adult mice Gene array identified 1572 genes that were enriched in microglia vs. 611 monocyte enriched genes with a greater than 5-fold difference (P<0.001). Gene profiling of CD11b+CD45Low microglia isolated from the CNS and CD11b+Ly6C+ monocyte subsets isolated from the spleen of naM-CM-/ve adult mice.
Project description:Microglia isolation from adult mouse brains remains difficult in comparison to microglia isolation from brains from newborn mouse brains as connective tissue, extracellular matrix materials and thicker myelin sheaths substantially influence traditional isolation protocols used with newborn mouse brains. Since the introduction of magnetic activated cell sorting (MACS), it has been reported that it is possible to also obtain microglia cells from adult mouse brains with a very good purity. However, most studies only used flow cytometry and/or qPCR to determine microglia enrichment and enrichment of traditional microglia marker proteins. Therefore we wanted to characterize in an unbiased way the proteomes of with MACS isolated microglia cells (CD11b positive cells) in comparison to all non-CD11b positive cells (termed non-target cell fraction (NTCF)), which consist of primarily astrocytes, neurons and oligodendrocytes. Therefore, we isolated from three adult mouse brains via MACS CD11b positive cells as well as the remaining CD11b negative cells (NTCF) to characterize the proteome of both cell fractions. The obtained proteomes were compared to a dataset of proteomic signatures for all CNS cell types determined by Sharma et al., (2015, doi: 10.1038/nn.4160) to determine the amount of enrichment of microglia-specific proteins.
Project description:Administration of G-CSF mobilizes a unique population of CD11b+Ly6C+CD34+mature monocytes that can inhibit GVHD in murine models of BMT via an iNOS-dependent mechanism. The transcriptional profiles of flow sorted lineage-CD11b+CD34+ cells from G-CSF treated mice were compared with conventional splenic Ly6C+ and Ly6C- monocytes, progenitor cells and cultured myeloid-derived suppressor cells. Further comparisons were made with lineage-CD11b+CD34+ cells from G-CSF treated mice that had been grown in culture or that were derived from iNOS ko mice. We used microarrays to detail the global programme of gene expression underlying diffrenetiation of each of these cell types Lin-CD11b+CD34+ populations were isolated directly from the spleens of G-CSF-treated C57BL/6 mice or iNOS ko mice. In untreated C57BL/6 mice, Lin-CD11b+CD115+Ly6C+ and Lin-CD11b+CD115+Ly6C- monocytes were isolated from the spleen and Lin-CD117+CD115+CD135-Ly6C+CD11b- common monocyte progenitors were isolated from the bone marrow. Myeloid-derived suppressor cells (Ly6C+CD11b+ cells derived from G-CSF, GM-CSF and IL-13 cultured C57BL/6 bone marrow) were also isolated and compared with the above populations. Lin-CD11b+CD34+ spleen cells derived from G-CSF-treated C57BL/6 mice were cultured for 3 days in Flt3 ligand and SCF and then compared to the original input population.
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:Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear.results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b+/Ly6C+ monocyte/macrophage populations in the pathophysiology of disease after AKI. we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b+Â cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies Macrophage populations were sorted by Flow Cytometry into low and intermediate populations by Itgam(Cd11b) and Ly6c markers. The cells obtained in 5 weeks sham, 5 weeks IR, 9 day sham, and 9 day IR with 6 samples per group (3 int and 3 low). Cells were sorted in 350ul of RLT lysing buffer and kept at -80c until RNA extraction.Sample amplification, fragmentation, hybridization,washing and scanning were performed according to validated Affymetrix protocol in a CLIA certified lab.
Project description:We developed a lipid-siRNA conjugate, termed EG18, that potentiates prolonged gene silencing throughout the CNS after injection into CSF. To help inform viable therapeutic targets, we used scRNAseq to determine cell-specific gene silencing. Here, we used an siRNA sequencing targeting Ppib and inject 15 nmol of either Ppib-EG18 or a non-targeting control (NTC)-EG18 into the lateral ventricles of adult mice. After 1 month, brains were dissociated into single cells and bead-sorted into Cd11b+ and Cd11b- populations. The Cd11b+ dataset contains microglia and macrophages, while the Cd11b- sample comprised a sampling of other isolated perivascular and parenchymal populations. scRNAseq revealed gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for disease-modifying therapies.
Project description:We investigated the innate immune system in the SOD1 ALS model. We found that splenic Ly6CHi monocytes were activated and their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We found a decrease in resident microglia in the spinal cord with disease progression. Two months prior to disease onset, splenic Ly6CHi monocytes had an M1 signature which included increased CCR2. At one month prior to disease onset, microglia expressed increased CCL2 and other chemotaxis-associated molecules. Microglia derived from the spinal cord of SOD1 mice recruited Ly6C+ monocytes to the CNS. Treatment with anti-Ly6C mAb modulated the Ly6CHi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss and extended survival. In humans with ALS, CD14+/CD16- monocytes (analogue of Ly6CHi monocytes) exhibited an ALS specific microRNA inflammatory signature similar to that observed in the SOD1 mouse providing a direct link between the animal model and the human disease. Thus, the SOD1-like profile of monocytes in ALS subjects may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach. This study used the NanoString nCounter hybridization system and nCounter miRNA expression assays to identify and quantitate miRNAs in blood CD14+CD16- monocytes from ALS, MS and HC subjects Total RNA was isolated from FACS sorted CD14+CD16- blood-derived monocytes from sporadic ALS (n=8), MS (n=8) and HC (n=8) subjects. RNA was profiled using the NanoString nCounter miRNA expression assay
Project description:Previous reports have defined three subsets of mouse NK cells on the basis of the expression of CD27 and CD11b. The developmental relationship between these subsets was unclear. To address this issue, we evaluated the overall proximity between mouse NK cell subsets defined by CD27 and CD11b expression using pangenomic gene expression profiling. The results suggest that CD27+CD11b-, CD27+CD11b+ and CD27-CD11b+ correspond to three different intermediates stages of NK cell development. Experiment Overall Design: Spleen cells from RAG-/- mice have been isolated and stained with anti-NK1.1, anti CD27 and anti CD11b antibodies. NK1.1+ cells were sorted into CD27+ CD11b-, CD27+ CD11b+ and CD27- CD11b+ subsets by flow cytometry. There are two independent replicates for each sample. Total RNA was extracted with the RNeasy microkit (Qiagen) and gene expression profiles were performed according to manufacturer instructions (Affymetrix mouse 430 2.0).
Project description:Background Proteomic characterization of microglia has been limited by low yield and contamination by non-microglial proteins by magnetic-activated cell sorting (MACS) enrichment strategies. To determine whether a fluorescent-activated cell sorting (FACS)-based strategy overcomes these limitations, we compared microglial proteomes of MACS and FACS-based purified CD11b+ microglia in order to identify core sets of microglial proteins in adult mouse brain tissue. Results Quantitative multiplex proteomics by tandem mass tag mass spectrometry (TMT-MS) of MACS-enriched (N = 5) and FACS-purified (N = 5) adult wild-type CD11b+ microglia identified 1,791 proteins, of which 953 were differentially expressed indicating significant differences between both approaches. While the FACS-purified microglia proteome was enriched with cytosolic, endoplasmic reticulum and ribosomal proteins involved in protein metabolism and immune system functions, the MACS-enriched microglia proteome was enriched with proteins related to mitochondrial function and synaptic transmission. As compared to MACS, the FACS microglial proteome showed strong enrichment for canonical microglial proteins while neuron, astrocyte, and oligodendrocyte proteins were decreased. Interestingly, we observed enrichment of endothelial specific proteins in the FACS microglia proteome. By comparing FACS-purified microglia proteomes with transcriptomes, we observed highly concordant as well as highly discordant proteins that were abundant at the protein level but low at the transcript level. Conclusions We demonstrate that TMT-MS proteomics of FACS-purified adult microglia is superior to column-based enrichment approaches, resulting in purer and highly-enriched microglial proteomes. We also define core sets of highly-abundant adult microglial proteins including Moesin (Msn) that can guide future studies.
Project description:We investigated the innate immune system in the SOD1 ALS model. We found that splenic Ly6CHi monocytes were activated and their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We found a decrease in resident microglia in the spinal cord with disease progression. Two months prior to disease onset, splenic Ly6CHi monocytes had an M1 signature which included increased CCR2. At one month prior to disease onset, microglia expressed increased CCL2 and other chemotaxis-associated molecules. Microglia derived from the spinal cord of SOD1 mice recruited Ly6C+ monocytes to the CNS. Treatment with anti-Ly6C mAb modulated the Ly6CHi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss and extended survival. In humans with ALS, CD14+/CD16- monocytes (analogue of Ly6CHi monocytes) exhibited an ALS specific microRNA inflammatory signature similar to that observed in the SOD1 mouse providing a direct link between the animal model and the human disease. Thus, the SOD1-like profile of monocytes in ALS subjects may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach This study used the NanoString nCounter hybridization system and the Nanostring GX Human Immunology and Nanostring Human Inflammation assays to identify and quantitate immune-related genes in blood CD14+CD16- monocytes from ALS, MS and HC subjects Total RNA was isolated from FACS sorted CD14+CD16- blood-derived monocytes from sporadic sALS (n=10), fALS (n=4) and HC (n=10) subjects. RNA was profiled using the Nanostring GX Human Immunology and Nanostring Human Inflammation assays
Project description:Type I interferons (IFN-I) are crucial for effective antimicrobial defence in the central nervous system (CNS) but also can cause severe neurological disease (termed cerebral interferonopathy) as exemplified by Aicardi-Goutières Syndrome and chronic viral infection. In the CNS, microglia and astrocytes have essential roles in host responses to infection and injury, with both cell types responding to IFN-I. However, the extent to which the IFN-I responses of these cells differ, if at all, is still unknown. Here we determined the global transcriptional responses of astrocytes and microglia to the IFN-I, IFN-alpha. MGCs were prepared from 2–4 day-old C57BL/6 mice. Purified primary astrocytes were obtained from the MGCs by magnetic activated cell sorting using anti-CD11b beads. Microglia were obtained from mixed glial cell cultures by mechanical shaking for 4 h. After treating astrocytes and microglia with IFN-alpha for 12 h, microarray using Affymetrix mouse genome array 430 2.0 array was performed on total RNA extracted from these cells. We found that under basal conditions, each cell type has a unique gene expression pattern reflective of its developmental origin and biological function. Following stimulation with IFN-alpha for 12 h, astrocytes and microglia also displayed a common core response that was characterized by the increased expression of genes required for pathogen detection and elimination. Microglia had a more extensive and diverse response to IFN-alpha with twice the number of genes upregulated (282 vs. 141 genes) when compared with astrocytes. Validation of the findings in vivo further suggested that astrocytes and microglia play important but distinct roles in the development of IFN-alpha-driven cerebral interferonopathies.