Project description:Objective: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome. Methods: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM. Results: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype. Significance: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention. Total RNA was isolated from three independently-generated sets of flow sorted bone marrow monocytes (CD11b+ CD45high CD115+ Ly6G- cells) and brainstem microglia (CD11b+ CD45low CD115low Ly6G- cells) for Illumina RNA-Seq, and two additional pools were subsequently generated and submitted for Affymetrix Mouse Exon 1.0ST microarray. Two of the RNA-Seq samples were additionally analyzed by the microarray, for a total of 6 samples (3 monocyte, 3 microglia) in each platform. Data outputs were analyzed by two analysis methods each (RNA-Seq data: ALEXA-Seq and Cufflinks; microarray data: Partek and Aroma). All four lists were merged into a new high-confidence gene list of transcripts that were significantly differentially expressed (DE) in three out of the four analyses. In this dataset, we includeRNA-Seq data obtained from flow sorted mouse bone marrow monocytes and brainstem microglia.

Project description:Objective: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome. Methods: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM. Results: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype. Significance: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention. Total RNA was isolated from three independently-generated sets of flow sorted bone marrow monocytes (CD11b+ CD45high CD115+ Ly6G- cells) and brainstem microglia (CD11b+ CD45low CD115low Ly6G- cells) for Illumina RNA-Seq, and two additional pools were subsequently generated and submitted for Affymetrix Mouse Exon 1.0ST microarray. Two of the RNA-Seq samples were additionally analyzed by the microarray, for a total of 6 samples (3 monocyte, 3 microglia) in each platform. Data outputs were analyzed by two analysis methods each (RNA-Seq data: ALEXA-Seq and Cufflinks; microarray data: Partek and Aroma). All four lists were merged into a new high-confidence gene list of transcripts that were significantly differentially expressed (DE) in three out of the four analyses. In this dataset, we include exon expression data obtained from flow sorted mouse bone marrow monocytes and brainstem microglia.

Project description:Macrophages in the dura mater are substantial contributors to the immune defense of the brain, however, their site-specific origin and function in bacterial infections of the central nervous system are incompletely understood. In a natural model of streptococcal meningoencephalitis, where bacteria systemically spread via the bloodstream to the brain , we found streptococci to be largely restricted in the meninges . Further sporadic spread of bacteria to the underlying brain parenchyma caused a region-restricted microglia activation. Invasion of monocytes, but not granulocytes into brain and leptomeninges correlated to the disease severity. Inflammation in the dura was accompanied by activation and loss of dural macrophages, and by the rapid engraftment of highly activated monocytes. In addition, monocyte progenitors in the skull marrow underwent drastic changes and acquired a more immature phenotype likely due to infection-induced emergency myelopoiesis. Notably, while dural monocytes were derived from adjacent skull marrow in a CCR2-independent fashion, the high demand for dural monocytes in streptococcal meningoencephalitis required intact CCR2 signalling and involved the long bone marrow, indicating heterogeneity in monocyte recruitment. Furthermore, meningoencephalitis increased monocyte progeny from monocyte-dendritic cell progenitors compared to the homeostatic, granulocyte-monocyte progenitor-dominated origin. Accordingly, monocytes in the dura, recruited from distinct reservoirs depending on disease-inherent needs, are intertwined with the disease course and may thus offer opportunities for therapeutic interventions. Macrophages in the dura mater are substantial contributors to the immune defense of the brain, however, their site-specific origin and function in bacterial infections of the central nervous system are incompletely understood. In a natural model of streptococcal meningoencephalitis, where bacteria systemically spread via the bloodstream to the brain , we found streptococci to be largely restricted in the meninges . Further sporadic spread of bacteria to the underlying brain parenchyma caused a region-restricted microglia activation. Invasion of monocytes, but not granulocytes into brain and leptomeninges correlated to the disease severity. Inflammation in the dura was accompanied by activation and loss of dural macrophages, and by the rapid engraftment of highly activated monocytes. In addition, monocyte progenitors in the skull marrow underwent drastic changes and acquired a more immature phenotype likely due to infection-induced emergency myelopoiesis. Notably, while dural monocytes were derived from adjacent skull marrow in a CCR2-independent fashion, the high demand for dural monocytes in streptococcal meningoencephalitis required intact CCR2 signalling and involved the long bone marrow, indicating heterogeneity in monocyte recruitment. Furthermore, meningoencephalitis increased monocyte progeny from monocyte-dendritic cell progenitors compared to the homeostatic, granulocyte-monocyte progenitor-dominated origin. Accordingly, monocytes in the dura, recruited from distinct reservoirs depending on disease-inherent needs, are intertwined with the disease course and may thus offer opportunities for therapeutic interventions.

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Mus musculus

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