Limits...
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

Induction of inflammatory response-related genes following TLR4 activation. a Quantitative real-time reverse transcriptase-PCR analysis of the expression of inflammatory genes in PM stimulated with LPS (10 ng/ml). The expression of inflammatory genes was significantly up-regulated at the indicated times in cells treated with LPS (10 ng/ml) compared to untreated cells. b BV2 cell lines or PM were stimulated with different doses of LPS (10–100 ng/ml) for 2 and 4 h before analysis of inflammatory response-related genes by quantitative real-time reverse transcriptase-PCR analysis. Gene expression was normalized to GAPDH transcript levels. The data represent three biologically independent experiments. The values are the mean ± SD of triplicate wells. *P < 0.01 and **P < 0.001 compared to the control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940985&req=5

Fig1: Induction of inflammatory response-related genes following TLR4 activation. a Quantitative real-time reverse transcriptase-PCR analysis of the expression of inflammatory genes in PM stimulated with LPS (10 ng/ml). The expression of inflammatory genes was significantly up-regulated at the indicated times in cells treated with LPS (10 ng/ml) compared to untreated cells. b BV2 cell lines or PM were stimulated with different doses of LPS (10–100 ng/ml) for 2 and 4 h before analysis of inflammatory response-related genes by quantitative real-time reverse transcriptase-PCR analysis. Gene expression was normalized to GAPDH transcript levels. The data represent three biologically independent experiments. The values are the mean ± SD of triplicate wells. *P < 0.01 and **P < 0.001 compared to the control

Mentions: To determine the proper time course responses, we performed an expression analysis in TLR4-stimulated versus control PM. LPS (10 ng/ml) caused a transient up-regulation of key inflammatory response-related genes, peaking at 2 and 4 h for TNF-α, CXCL10, IL1A, IL1B, CCL4, and CCL5 (Fig. 1a). In response to stimulation by different doses of LPS (10–100 ng/ml), BV2 cell lines and PM caused significant up-regulation of key inflammatory response-related genes at 2 and 4 h. The fold induction in the increase of TNF-α and IL1B in response to different doses of LPS (10–100 ng/ml) was similar in both cell types at 2 and 4 h (Fig. 1b). We hence used the lower doses for subsequent analyses. Notably, morphological analysis shows that at 4 h, LPS-treated BV2 cell lines and PM show similar morphology and responses as compared to control cells (Additional file 1: Figure S1A).Fig. 1


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)

Induction of inflammatory response-related genes following TLR4 activation. a Quantitative real-time reverse transcriptase-PCR analysis of the expression of inflammatory genes in PM stimulated with LPS (10 ng/ml). The expression of inflammatory genes was significantly up-regulated at the indicated times in cells treated with LPS (10 ng/ml) compared to untreated cells. b BV2 cell lines or PM were stimulated with different doses of LPS (10–100 ng/ml) for 2 and 4 h before analysis of inflammatory response-related genes by quantitative real-time reverse transcriptase-PCR analysis. Gene expression was normalized to GAPDH transcript levels. The data represent three biologically independent experiments. The values are the mean ± SD of triplicate wells. *P < 0.01 and **P < 0.001 compared to the control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940985&req=5

Fig1: Induction of inflammatory response-related genes following TLR4 activation. a Quantitative real-time reverse transcriptase-PCR analysis of the expression of inflammatory genes in PM stimulated with LPS (10 ng/ml). The expression of inflammatory genes was significantly up-regulated at the indicated times in cells treated with LPS (10 ng/ml) compared to untreated cells. b BV2 cell lines or PM were stimulated with different doses of LPS (10–100 ng/ml) for 2 and 4 h before analysis of inflammatory response-related genes by quantitative real-time reverse transcriptase-PCR analysis. Gene expression was normalized to GAPDH transcript levels. The data represent three biologically independent experiments. The values are the mean ± SD of triplicate wells. *P < 0.01 and **P < 0.001 compared to the control
Mentions: To determine the proper time course responses, we performed an expression analysis in TLR4-stimulated versus control PM. LPS (10 ng/ml) caused a transient up-regulation of key inflammatory response-related genes, peaking at 2 and 4 h for TNF-α, CXCL10, IL1A, IL1B, CCL4, and CCL5 (Fig. 1a). In response to stimulation by different doses of LPS (10–100 ng/ml), BV2 cell lines and PM caused significant up-regulation of key inflammatory response-related genes at 2 and 4 h. The fold induction in the increase of TNF-α and IL1B in response to different doses of LPS (10–100 ng/ml) was similar in both cell types at 2 and 4 h (Fig. 1b). We hence used the lower doses for subsequent analyses. Notably, morphological analysis shows that at 4 h, LPS-treated BV2 cell lines and PM show similar morphology and responses as compared to control cells (Additional file 1: Figure S1A).Fig. 1

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