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Identification of state-specific proteomic and transcriptomic signatures of microglia-derived extracellular vesicles

ABSTRACT: We performed quantitative label-free quantitative mass spectrometry (LFQ-MS) on 16 cell lysates and 16 EV lysates derived from BV2 microglia. These included four groups of BV2 cells (n=4/group) that were treated (72 hours) with either LPS (100ng/mL) to polarize to a pro-inflammatory state, IL-10 (50ng/mL) to polarize to a protective state, TGFβ (50ng/mL) to polarize to a homeostatic state or untreated controls

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Suspension Culture, Cell Culture

SUBMITTER: Juliet Santiago

LAB HEAD: Srikant Rangaraju

PROVIDER: PXD043959 | Pride | 2023-11-21

REPOSITORIES: Pride

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			Action	DRS
	CTL_1_CELL.raw	Raw
	CTL_1_EV.raw	Raw
	CTL_2_CELL.raw	Raw
	CTL_2_EV.raw	Raw
	CTL_3_CELL.raw	Raw

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Publications

Identification of State-Specific Proteomic and Transcriptomic Signatures of Microglia-Derived Extracellular Vesicles.

Santiago Juliet V JV Natu Aditya A Ramelow Christina C CC Rayaprolu Sruti S Xiao Hailian H Kumar Vishnu V Kumar Prateek P Seyfried Nicholas T NT Rangaraju Srikant S

Molecular & cellular proteomics : MCP 20231111 12

Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impact EV composition ...[more]

PMID: 37952696

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The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model

Project description:In the context of studying the role of the COP9 signalosome (CSN) in neuroinflammation and ischemic neuronal damage, we studied the effect of the cullin NEDDylation state-modifying drugs MLN4924 and CSN5i-3 in BV2 microglial cells, an immortalized murine cell line featuring many of the characteristics of primary microglia. Owing to its potent inhibitory effect on the NEDDylation cascade, MLN4924 exhibits a CSN5-like anti-inflammatory activity. Csn5i-3 is a small molecule inhibitor that specifically binds to CSN5, while it resides in the CSN holocomplex and blocks its deNEDDylase activity, thus leading to an accumulation of NEDDylated cullins. We performed untargeted mass spectrometry-based proteomics of the cell lysates after treating BV2 cells with these drugs under basal culture stress conditions. The proteomic analysis revealed that MLN4924 and CSN5i-3 substantially altered the microglial proteome.

2023-10-24 | PXD044650 | Pride

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

Project description:Different brain cell types play distinct roles in brain development and disease via cellular mechanisms. Molecular characterization of these cellular mechanisms using cell type-specific approaches, particularly at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain pose several technical barriers. To overcome these, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. We generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under resting conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes, and the LPS effect was partly captured by analysis of the TurboID-NES-labeled proteome of BV2 cells.

2023-03-11 | PXD036744 | Pride

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

Project description:The wild type (WT) and TLR4 -/- pulmonary fibroblasts were treated with phosphate-buffered saline (PBS), 1 µg/mL recombinant mouse (rm) CIRP, 2 ng/mL rmTGF-ß1 (R&D Systems), or rmCIRP plus rmTGF-ß1. The cell lysates collected at 24 hours were used for high throughput mRNA sequencing.

2021-07-15 | GSE178255 | GEO

Human airway epithelial cells co-treated with IL-4 and IL-13 for seven days.

Project description:Nasal basal epithelial cells from 6 healthy adult donors were grown in air-liquid interface (ALI) cultures for fourteen days. Cells were then treated with 10 ng/mL of IL-4 and 10 ng/mL of IL-13 for seven additional days. Next, cell lysates were used for low-input RNA-seq library preparation (Smart-seq2) and sequencing.

2024-05-29 | GSE268072 | GEO

RNA Sequencing of BV2 cells stimulated by sTREM-1

Project description:Purpose: The goals of this study are to compare the transcriptome of BV2 cells stimulated with or without sTREM-1, explore its effect on the phenotype and function of BV2 cells, and explore potential signaling pathways. Methods: BV2 cells were incubated with or without sTREM-1 recombinant protein (1ug/mL) for 6 h, then the mRNA profiles were generated by deep sequencing, in triplicate, using Illumina GAIIx. Results: Sequencing libraries were generated using the NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) and sequenced on an Illumina platform. Paired-end reads were then generated and evaluated for quality control. Clean raw data with high quality were aligned to the reference genome using Spliced Transcripts Alignment to a Reference (STAR) software. For quantification, FeatureCouts was used to count the read numbers mapped of each gene, 17047 transcripts were identified. Differential expression analysis between two groups was performed using the Edge R package. Genes with an adjusted RNAs with P < 0.05 and |log2FC | > 1 were identified as differentially expressed. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to sTREM-1 function. Gene ontologies (GO) Analyses were conducted to assess biological processes, molecular functions, and cellular components. Conclusions: Our study represents the detailed analysis of BV2 cells transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that BV2 cells are activated after sTREM-1 recombinant protein stimulation, and the phagocytic function is enhanced.

2022-05-04 | GSE185830 | GEO

RNA-seq of BV2 cultured microglia following high glucose and CD200Fc treatments

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2023-10-30 | GSE238008 | GEO

Transcriptome analysis of BV2 cells in response to LPS stimulation

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2016-04-18 | E-MTAB-3450 | biostudies-arrayexpress

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2020-09-21 | E-MTAB-9108 | biostudies-arrayexpress

Histone acetylation H2BK20ac marks cell-state specific active regulatory elements

Project description:Characterisation of different histone modifications is crucial to understand gene regulation. In order to study the most predictive histone modification for active enhancers we created unbiased set of enhancers and used machine learning approach. Our approach revealed an unconventional histone modification H2BK20ac as most efficient marker of active enhancers. H2BK20ac also showed superior coverage of tissue specific active enhancers in complex invivo samples. Adding H2BK20ac to set of conventional histone modifications lead to identification of new chromatin state which could be active enhancers. H2BK20ac tends to occur only at cell-type specific active promoters and showed higher specificity for related disease mutations than H3K27ac and other histone modifications. Using transient state of BV2 microglia cells after lipopolysaccharide based activation, we found that H2BK20ac also marks cell-state specific cis-regulatory elements. Further analysis using inhibition of TGF-beta pathway in BV2 cells and LPS stimulation, revealed differential patterns of H2BK20ac and H3K27ac at genome locations associated with opposite roles response to stmulation. Our study about H2BK20ac hints about a new mechanism of regulation of cell-type specificity and a distinct mode of action of pathways to maintain balance between cell-responses. Chip-seq of H2BK20ac and other histone modifcation was performed in 3 cell types and embryonic mouse forebrain. The sensitivity for active enhancers was compared for different histone modification ChIP-seq.The level of H2BK20ac at promoters and enhancers was assesed for relationship to cell-type specific expression. H2BK20ac signals were also analysed during cell-state transition when microgila are stimulated by LPS

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