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

Das A, Kim SH, Arifuzzaman S, Yoon T, Chai JC, Lee YS, Park KS, Jung KH, Chai YG - J Neuroinflammation (2016)

Bottom Line: 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.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).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Natural Science and Technology, Hanyang University, Ansan, 15588, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

Differences in the expression of selected TF families between BV2 cell lines and PM. a Heat map representation showing the commonly expressed TF families between BV2 cell lines and PM cells after 2- and 4-h LPS stimulation. b Heat map of the TF families that were unique to PM cells, which showed a distinct signature following 2- and 4-h LPS stimulation compared to BV2 cell lines. c UCSC Browser images representing normalized RNA-seq read densities. d Transcript abundance (in read count) was evaluated using RNA-seq in 2- and 4-h LPS-induced BV2 cell lines and PM cells. e Confirmation of differentially expressed TFs was performed using quantitative reverse transcription-polymerase chain reaction. The genes that were common and unique to PM cells compared to BV2 cell lines are shown. Gene expression was normalized to GAPDH transcript levels. *P < 0.01 and **P < 0.001 compared to the control. The data represent three biologically independent experiments. f Patterns of TF motif enrichment within the promoters of the indicated genes in 4-h LPS-induced PM cells. g The activity of highly connected positive regulators of the inflammatory genes IRF1, IRF2, STAT1, and STAT2 led to the activation of this network, as assessed using the IPA molecule activity predictor in LPS-induced PM cells. h Results of the GO term analysis using DAVID. The genes that were regulated by STAT1 and IRF1 in response to LPS in PM cells are shown
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Fig5: Differences in the expression of selected TF families between BV2 cell lines and PM. a Heat map representation showing the commonly expressed TF families between BV2 cell lines and PM cells after 2- and 4-h LPS stimulation. b Heat map of the TF families that were unique to PM cells, which showed a distinct signature following 2- and 4-h LPS stimulation compared to BV2 cell lines. c UCSC Browser images representing normalized RNA-seq read densities. d Transcript abundance (in read count) was evaluated using RNA-seq in 2- and 4-h LPS-induced BV2 cell lines and PM cells. e Confirmation of differentially expressed TFs was performed using quantitative reverse transcription-polymerase chain reaction. The genes that were common and unique to PM cells compared to BV2 cell lines are shown. Gene expression was normalized to GAPDH transcript levels. *P < 0.01 and **P < 0.001 compared to the control. The data represent three biologically independent experiments. f Patterns of TF motif enrichment within the promoters of the indicated genes in 4-h LPS-induced PM cells. g The activity of highly connected positive regulators of the inflammatory genes IRF1, IRF2, STAT1, and STAT2 led to the activation of this network, as assessed using the IPA molecule activity predictor in LPS-induced PM cells. h Results of the GO term analysis using DAVID. The genes that were regulated by STAT1 and IRF1 in response to LPS in PM cells are shown

Mentions: To further investigate common and unique characteristics between LPS-treated PM or the BV2 cell lines, we again used RNA-seq data to compare the transcriptome of BV2 cell lines with that of PM. In a similar approach (see the “Methods” section), we compared the transcripts in LPS-treated BV2 cell lines with those of PM. Differential expression analysis clearly revealed that LPS elicited the induction of a unique gene set in response to stimulation with this TLR ligand at the 2- and 4-h time point in BV2 cell lines and PM cells (Fig. 2d; Additional file 3: Figure S3) suggesting a substantial number of dissimilarities between the two cell types. PM cells up-regulated 220 for 2 h and 682 genes for 4 h that are not common to the BV2 cell lines. In contrast, BV2 cell lines up-regulated 63 for 2 h and 35 genes for 4 h that are not common to the PM cells (Fig. 2d; Additional file 3: Figure S3). The unique gene set is presented at the 2- and 4-h time point in BV2 cell lines in Additional file 5: Table S1. However, PM and the BV2 cell lines also had similarities in their transcriptomes. Of the up-regulated genes, BV2 cell lines and PM shared 142 genes for 2 h and 264 genes for 4 h following LPS treatment (Fig. 2d; Additional file 3: Figure S3). Importantly, this technology allowed us to identify several specific gene families involved in immune responses that were uniquely altered in LPS-treated PM cells. We found that LPS elicited the induction of unique 10 cytokines, 9 chemokines, 13 (interferon (IFN))-regulated genes (IRGs), 9 TFs, 3 epigenetic regulators, and 11 undetected transcripts in response to stimulation with this TLR ligand at the 4-h time point in PM cells (Figs. 4b, 5b, and 6f). The following inflammatory response- and immune response-related genes were markedly affected only in PM: cytokines/chemokines (CCL6, CCL8, CX3CL1, CXCL1, CXCL3, CXCL9, CXCL11, CXCL16, IL12B, IL18BOS, IL18BP, IL19, IL23A, IL27, IRAK1BP1, SOCS1, TNFSF11A, and TNFSF15), IRGs (GBP2B, GBP4, GBP9, GBP10, GBP11, IFI44I, IFIH1, IFNB1, etc.), TFs (IRF2, IRF5, IRF8, STAT5A, etc.), epigenetic regulators (KDM1A, NSD3, and SETDB2), and undetected transcripts (CLEC4A1, CLEC7, CLEC7A, GPR18, MMP3, MMP9, MMP12, etc.). These data suggest that following LPS treatment, PM express a unique set of genes, distinct from that of BV2 cell lines, which may offer potential targets for further investigations into microglia biology.


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

Das A, Kim SH, Arifuzzaman S, Yoon T, Chai JC, Lee YS, Park KS, Jung KH, Chai YG - J Neuroinflammation (2016)

Differences in the expression of selected TF families between BV2 cell lines and PM. a Heat map representation showing the commonly expressed TF families between BV2 cell lines and PM cells after 2- and 4-h LPS stimulation. b Heat map of the TF families that were unique to PM cells, which showed a distinct signature following 2- and 4-h LPS stimulation compared to BV2 cell lines. c UCSC Browser images representing normalized RNA-seq read densities. d Transcript abundance (in read count) was evaluated using RNA-seq in 2- and 4-h LPS-induced BV2 cell lines and PM cells. e Confirmation of differentially expressed TFs was performed using quantitative reverse transcription-polymerase chain reaction. The genes that were common and unique to PM cells compared to BV2 cell lines are shown. Gene expression was normalized to GAPDH transcript levels. *P < 0.01 and **P < 0.001 compared to the control. The data represent three biologically independent experiments. f Patterns of TF motif enrichment within the promoters of the indicated genes in 4-h LPS-induced PM cells. g The activity of highly connected positive regulators of the inflammatory genes IRF1, IRF2, STAT1, and STAT2 led to the activation of this network, as assessed using the IPA molecule activity predictor in LPS-induced PM cells. h Results of the GO term analysis using DAVID. The genes that were regulated by STAT1 and IRF1 in response to LPS in PM cells are shown
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Fig5: Differences in the expression of selected TF families between BV2 cell lines and PM. a Heat map representation showing the commonly expressed TF families between BV2 cell lines and PM cells after 2- and 4-h LPS stimulation. b Heat map of the TF families that were unique to PM cells, which showed a distinct signature following 2- and 4-h LPS stimulation compared to BV2 cell lines. c UCSC Browser images representing normalized RNA-seq read densities. d Transcript abundance (in read count) was evaluated using RNA-seq in 2- and 4-h LPS-induced BV2 cell lines and PM cells. e Confirmation of differentially expressed TFs was performed using quantitative reverse transcription-polymerase chain reaction. The genes that were common and unique to PM cells compared to BV2 cell lines are shown. Gene expression was normalized to GAPDH transcript levels. *P < 0.01 and **P < 0.001 compared to the control. The data represent three biologically independent experiments. f Patterns of TF motif enrichment within the promoters of the indicated genes in 4-h LPS-induced PM cells. g The activity of highly connected positive regulators of the inflammatory genes IRF1, IRF2, STAT1, and STAT2 led to the activation of this network, as assessed using the IPA molecule activity predictor in LPS-induced PM cells. h Results of the GO term analysis using DAVID. The genes that were regulated by STAT1 and IRF1 in response to LPS in PM cells are shown
Mentions: To further investigate common and unique characteristics between LPS-treated PM or the BV2 cell lines, we again used RNA-seq data to compare the transcriptome of BV2 cell lines with that of PM. In a similar approach (see the “Methods” section), we compared the transcripts in LPS-treated BV2 cell lines with those of PM. Differential expression analysis clearly revealed that LPS elicited the induction of a unique gene set in response to stimulation with this TLR ligand at the 2- and 4-h time point in BV2 cell lines and PM cells (Fig. 2d; Additional file 3: Figure S3) suggesting a substantial number of dissimilarities between the two cell types. PM cells up-regulated 220 for 2 h and 682 genes for 4 h that are not common to the BV2 cell lines. In contrast, BV2 cell lines up-regulated 63 for 2 h and 35 genes for 4 h that are not common to the PM cells (Fig. 2d; Additional file 3: Figure S3). The unique gene set is presented at the 2- and 4-h time point in BV2 cell lines in Additional file 5: Table S1. However, PM and the BV2 cell lines also had similarities in their transcriptomes. Of the up-regulated genes, BV2 cell lines and PM shared 142 genes for 2 h and 264 genes for 4 h following LPS treatment (Fig. 2d; Additional file 3: Figure S3). Importantly, this technology allowed us to identify several specific gene families involved in immune responses that were uniquely altered in LPS-treated PM cells. We found that LPS elicited the induction of unique 10 cytokines, 9 chemokines, 13 (interferon (IFN))-regulated genes (IRGs), 9 TFs, 3 epigenetic regulators, and 11 undetected transcripts in response to stimulation with this TLR ligand at the 4-h time point in PM cells (Figs. 4b, 5b, and 6f). The following inflammatory response- and immune response-related genes were markedly affected only in PM: cytokines/chemokines (CCL6, CCL8, CX3CL1, CXCL1, CXCL3, CXCL9, CXCL11, CXCL16, IL12B, IL18BOS, IL18BP, IL19, IL23A, IL27, IRAK1BP1, SOCS1, TNFSF11A, and TNFSF15), IRGs (GBP2B, GBP4, GBP9, GBP10, GBP11, IFI44I, IFIH1, IFNB1, etc.), TFs (IRF2, IRF5, IRF8, STAT5A, etc.), epigenetic regulators (KDM1A, NSD3, and SETDB2), and undetected transcripts (CLEC4A1, CLEC7, CLEC7A, GPR18, MMP3, MMP9, MMP12, etc.). These data suggest that following LPS treatment, PM express a unique set of genes, distinct from that of BV2 cell lines, which may offer potential targets for further investigations into microglia biology.

Bottom Line: 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.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).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Natural Science and Technology, Hanyang University, Ansan, 15588, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus