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      Transcriptome sequencing reveals that LPS-triggered transcriptional responses in established microglia BV2 cell lines are poorly representative of primary microglia

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          Abstract

          Background

          Microglia are resident myeloid cells in the CNS that are activated by infection, neuronal injury, and inflammation. Established BV2 microglial cell lines have been the primary in vitro models used to study neuroinflammation for more than a decade because they reduce the requirement of continuously maintaining cell preparations and animal experimentation models. However, doubt has recently been raised regarding the value of BV2 cell lines as a model system.

          Methods

          We used triplicate RNA sequencing (RNA-seq) to investigate the molecular signature of primary and BV2 microglial cell lines using two transcriptomic techniques: global transcriptomic biological triplicate RNA-seq and quantitative real-time PCR. We analyzed differentially expressed genes (DEGs) to identify transcription factor (TF) motifs (−950 to +50 bp of the 5′ upstream promoters) and epigenetic mechanisms.

          Results

          Sequencing assessment and quality evaluation revealed that primary microglia have a distinct transcriptomic signature and express a unique cluster of transcripts in response to lipopolysaccharide. This microglial signature was not observed in BV2 microglial cell lines. Importantly, we observed that previously unidentified TFs (i.e., IRF2, IRF5, IRF8, STAT1, STAT2, and STAT5A) and the epigenetic regulators KDM1A, NSD3, and SETDB2 were significantly and selectively expressed in primary microglia (PM). Although transcriptomic alterations known to occur in BV2 microglial cell lines were identified in PM, we also observed several novel transcriptomic alterations in PM that are not frequently observed in BV2 microglial cell lines.

          Conclusions

          Collectively, these unprecedented findings demonstrate that established BV2 microglial cell lines are probably a poor representation of PM, and we establish a resource for future studies of neuroinflammation.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12974-016-0644-1) contains supplementary material, which is available to authorized users.

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          Most cited references43

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          The transcriptional landscape of the yeast genome defined by RNA sequencing.

          The identification of untranslated regions, introns, and coding regions within an organism remains challenging. We developed a quantitative sequencing-based method called RNA-Seq for mapping transcribed regions, in which complementary DNA fragments are subjected to high-throughput sequencing and mapped to the genome. We applied RNA-Seq to generate a high-resolution transcriptome map of the yeast genome and demonstrated that most (74.5%) of the nonrepetitive sequence of the yeast genome is transcribed. We confirmed many known and predicted introns and demonstrated that others are not actively used. Alternative initiation codons and upstream open reading frames also were identified for many yeast genes. We also found unexpected 3'-end heterogeneity and the presence of many overlapping genes. These results indicate that the yeast transcriptome is more complex than previously appreciated.
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            MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway.

            MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45(low), major histocompatibility complex (MHC) class II(low) phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo. These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.
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              Microglia and neuroinflammation: a pathological perspective

              Microglia make up the innate immune system of the central nervous system and are key cellular mediators of neuroinflammatory processes. Their role in central nervous system diseases, including infections, is discussed in terms of a participation in both acute and chronic neuroinflammatory responses. Specific reference is made also to their involvement in Alzheimer's disease where microglial cell activation is thought to be critically important in the neurodegenerative process.
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                Author and article information

                Contributors
                amitabhdas.kn@gmail.com
                eio3492@gmail.com
                arifpharmju@gmail.com
                yths21@gmail.com
                jincchai@gmail.com
                yslee@hanyang.ac.kr
                kspark73@gmail.com
                khjung2@gmail.com
                82-31-400-5513 , ygchai@hanyang.ac.kr
                Journal
                J Neuroinflammation
                J Neuroinflammation
                Journal of Neuroinflammation
                BioMed Central (London )
                1742-2094
                11 July 2016
                11 July 2016
                2016
                : 13
                Affiliations
                [ ]Institute of Natural Science and Technology, Hanyang University, Ansan, 15588 Republic of Korea
                [ ]Department of Molecular and Life Sciences, Hanyang University, Ansan, 15588 Republic of Korea
                [ ]Department of Bionanotechnology, Hanyang University, Seoul, 04673 Republic of Korea
                Article
                644
                10.1186/s12974-016-0644-1
                4940985
                27400875
                ca5c15a9-dee8-4b8a-ab27-b8384d0405fc
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003725, National Research Foundation of Korea;
                Award ID: 2011-0030049
                Award ID: 2013R1A1A3011026
                Award Recipient :
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                Research
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                © The Author(s) 2016

                Neurosciences

                gene regulation, innate immunity, transcription factors, microglia, rna sequencing

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